Balanced X-Autosome Translocation in Infertile Woman: Report of Two Cases

Balanced X-Autosome Translocation in Infertile Woman: Report of Two Cases

*Habib S,1 Islam SS2

Abstract
Individuals with apparently balanced translocations, often, show no clinical findings. X-chromosomal translocations involving different autosomes have been reported. The phenotypic manifestations of these translocations depend on several factors. X-autosome translocations can also affect fertility where chromosomal changes result in inactivation of genes governing reproduction. This report is described two cases of phenotypically normal Bangladeshi women with the complaint of primary infertility associated with secondary amenorrhea and streak ovaries. Chromosomal analysis revealed an apparently balanced reciprocal translocation involving the long arm of the X chromosome (q2) with the short arm of chromosome 1(p3) and the long arm of chromosome 19(q13) in all the cells with the karyotype 46,X,t(X:1)(q22:p32) and 46,X,t(X:19)(q22:q13.1). Studies examining X-chromosome deletions have predicted that Xq aberrations within the Xq13–Xq27 region can result in gonadal failure. Reciprocal translocations between autosomes and gonosomes contribute significantly to primary infertility.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):151-156]

Keywords: Balanced X-autosome translocation, phenotype, Primary infertility

Introduction

X-autosome translocations are rare and associated with different phenotypes. There are balanced and unbalanced X-autosome translocation. Balanced type is usually associated with normal phenotype whereas the unbalanced one is with various congenital anomalies. Phenotypic effects of balanced X-autosome translocations in females: a retrospective survey of 104 cases reported from UK laboratories by Water JJ et al.1 Their cases were: multiple congenital abnormalities and/or developmental delay (MCA/DD): 26 (42%); gonadal dysfunction: 22 (35%); phenotypically normal with or without recurrent miscarriage (NRM): 9 (15%); recognized X-linked syndrome: 5 (8%). X chromosome translocations are frequently associated with primary or secondary amenorrhea. In this report, the clinical, biochemical and cytogenetic aspects of two healthy infertile women with balanced X-autosome translocation between chromosome X and two different autosomes: chromosome 1 and 19 were presented.

 

  1. *Dr. Saequa Habib MBBS MD (Pathology) Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University. saequa20@yahoo.com
  2. SM Shahedul Islam, B Sc, M Sc (Biochemistry & Molecular Biology) Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University.

 

*For correspondence

 

Case Presentation

Case 1

A 27 year-old female with the complaint of primary infertility, was referred to the department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh for cytogenetic evaluation. She was born to a nonconsanguineous parent and her mother had no menstrual problem. Her siblings were healthy. Her menarche was at 13 years of age but she had irregular menstruation with 4-8 months interval. She was married for 7 years. Her husband lived abroad. Her physical examination revealed normal height and weight and showed normal intelligence. On per vaginal examination small uterus was found with no abnormality of the external genitalia. Her luteinizing hormone and TSH level were normal but follicle stimulating hormone and anti-Mullerian hormone were at postmenopausal level. Ultrasound examination of the pelvis revealed normal uterus and streak ovaries.

Cytogenetic analysis of the peripheral blood lymphocytes was carried out according to the standard karyotyping technique. Peripheral blood lymphocytes were stimulated with phytohemagglutinin and harvested at 72 hours with colchicine. Hypotonic treatment was given to the cells and then they were fixed with Carnoy’s fixative. Standard GTG banding was done.2 Karyotype analysis of 100 metaphases revealed a pattern of 46, X, t(X;1)(q22; p32), suggestive of a balanced sex autosome translocation involving the long arm of chromosome X and short arm of chromosome 1 (Figure 1). ISCN guidelines for the chromosomal nomenclature (2016) were followed for the karyotype analysis and analysed by using Leica DM6000 B Motorized microscope and Leica Cytovision software.3 Parental and siblings karyotypes were not done.

Case 2

A 32 year-old female was referred with the complaint of primary infertility. She had two sisters and they had children. She had delayed puberty with menarche at the age of 15 years. After then menstruation occurred only after taking pills. She was married for 16 years. Her height was 5 feet 3 inch and weight was 60 kg. She was graduated and was a health worker. She had complaint of decrease libido. On per vaginal examination small uterus was found with no abnormality of the external genitalia. Her luteinizing hormone, thyroid stimulating hormone, prolactin and testosterone level were normal but follicle stimulating hormone was at postmenopausal level. Ultrasound examination of the pelvis revealed hypoplastic uterus and streak ovaries.

Cytogenetic analysis of the peripheral blood lymphocytes was carried out and standard GTG banding was done.2 Karyotype analysis of 100 metaphases revealed 46, X, t(X;19)(q22; p13.1), suggestive of a balanced sex autosome translocation involving the long arm of chromosome X and long arm of chromosome 19 (Figure 2).

Table  I: Previously reported cases of balanced X; 1 and X; 19 translocations

Symptoms Karyotypes Data by
Developmental delay 46, X, t (X; 1)(p22.1;p31) de novo Waters JJ et al1 (2001)
Recurrent miscarriages 46, X, t (X; 1)(p22.1;p32) de novo
Learning difficulties 46, X, t (X; 1)(p11.4;p36.3) de novo
Mother: abnormal scan 46, X, t (X; 1)(q26;p22) de novo
Multiple congenital anomalies/developmental delay 46, X, t (X; 1)(q26;p22) mat
Primary amenorrhea 46X: t (X; 1) (q21;p32) de novo Venkateshwari A et al9 (2015)

 

Primary amenorrhea 46,X,t(X; 1)( q22;p13) Razavi Z and   Momtaz HE10 (2017)
Primary amenorrhea 46,X,t(X;19)(q28;p13.1) Shetty DL et al11 (2014)

 

 

 

 

 

 

 

 

 

Figure 1. Photomicrograph of a karyotype showing translocation between chromosome X and chromosome 1 [46, X, t(X; 1) (q22; p32)] (Giemsa stain).

 

 

 

 

 

 

 

 

 

Figure 2. Photomicrograph of a karyotype showing translocation between chromosome X and chromosome 19 [46, X, t(X; 19) (q22; p13.1)] (Giemsa stain).

Discussion

Chromosomal conditions involving the sex chromosomes often affect sex determination (whether a person has the sexual characteristics of a male or a female), sexual development, and the ability to have children (fertility). The signs and symptoms of these conditions vary widely and range from mild to severe. They can be caused by missing or extra copies of the sex chromosomes or by structural changes in the chromosomes.

X-autosome translocations are rare, being estimated to occur in about 1/30,000 live births.4   In cases of balanced X-autosome translocation in female carriers, the normal X chromosome is usually inactivated, leaving the derivative X chromosome in the active state. The present cases revealed sex autosome translocation in phenotypically normal female with secondary amenorrhea. Cytogenetic analysis revealed 46, X, t (X; 1) (q22; p32) and 46, X, t(X; 19) (q22; p13.1) karyotype indicating its possible association with irregular menstruation and abnormal hormone level. Most carriers of an X-autosome translocation are phenotypically normal.5,6,1 In female carriers, gonadal dysgenesis may occur, and ∼9% may have multiple anomalies and/or mental retardation.7 Since the 2 copies of the X chromosome are necessary for ovarian development and integrity, the gonadal dysgenesis with infertility in our patient can be attributed to the partial loss of Xq, which contains various genes necessary for a normal ovarian reproductive function. MG Mattei et al concluded that  in X-autosome translocation 50% women will be sterile.8 There are reported cases of balanced X; 1 and X;19 translocations with different clinical manifestations including infertility showed in Table I.

Balanced X-autosome translocations show exchange between long arm segments of an X chromosome to an autosome with larger number of breakpoints. Infertility because of gonadal dysgenesis is common among those women in whom the breakpoint in the derivative X-chromosome involves the critical region Xq13–q26.1,5,6 X-autosome translocation causing gonadal dysgenesis with bilateral streak gonads as well as aberrant ovarian and sex development has been demonstrated by numerous studies.12, 13 Translocations involving the long arms of the X-chromosome and several autosomes (1–4, 6–9, 11, 12, 14, 15, 17, 19, 21, and 22), resulting in various degrees of gonad dysfunction, have also been reported.14

X-autosomal translocations are generally of maternal in origin or may arise in de novo.6 Fertility effects of a balanced X-autosome translocation vary depending on the sex of the carrier, the position of the translocation breakpoints and the pattern of X-inactivation.5,6,7 In the reported cases X autosome translocation may be de novo as their siblings had no menstrual abnormality. To conclude that balanced X-autosome translocation can be a cause secondary amenorrhea associated with infertility and should be investigated by cytogenetic analysis followed by genetic counseling.

References

  1. Waters JJ, Campbell PL, Crocker AJ, Campbell CM. Phenotypic effects of balanced X-autosome translocations in females: a retrospective survey of 104 cases reported from UK laboratories. Hum Genet 2001; 108 (4): 318– 27.
  2. Verma RS and Babu A. Human chromosome: manual of basic techniques.1st New York, USA. 1989: pp 4-44, 152-165.
  3. ISCN: an international system for human cytogenomic nomenclature .In Jean McGowan-Jordan, Annet Simons, Michael Schmid eds. Cytogenetic and Genome Research. New York, Karger. 2016: Vol. 149, No. 1-2.
  4. Sharp AJ, Spotswood HT, Robinson DO, Turner BM, Jacobs PA. Molecular and cytogenetic analysis of the spreading of X inactivation in X;autosome translocations. Hum Mol Genet. 2002; 11 (25): 3145– 56.
  5. Madan, K. Balanced structural changes involving the human X: effect on sexual phenotype.  Genet.1983; 63: 216–221.
  6. Kalz‐Füller B, Sleegers E, Schwanitz G, Schubert R. Characterisation, phenotypic manifestations and X‐inactivation pattern in 14 patients with X‐autosome translocations.  Genet.1999; 55: 362–366.
  7. Schmidt, M. and Du Sart, D.) Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced X‐autosome translocations: a review of 122 cases. J. Med. Genet1992; 42:161–169.
  8. Mattei MG, Mattei JF, Ayme S Giraud F. X-Autosome translocation: cytogenetic characteristics and their consequences. Hum Genet 1982; 61:295-309.
  9. Venkateshwari A, Srilekha A, Veena K, Sujatha M, Jyothy A. A Rare De Novo Balanced 1X; 1 Translocation in an Indian Female with Primary Amenorrhea. J Reprod Infertil. 2015; 16: 171–3.
  10. Razavi Z and Momtaz HE. Balanced Reciprocal Translocation t(X; 1) in a Girl with Tall Stature and Primary Amenorrhea Iran J Med Sci. 2017 Mar; 42(2): 210–214.
  11. Shetty DL, Kadam AP, Koppaka NT, Dalvi RC, Chavan DS, Das BR, Mandava S. X-autosome translocations in amenorrhoea: a report of a three way translocation from Indian Population. Gynecol Endocrinol, 2014 Early Online: 1–5.
  12. Mohandas T, Geller RL, Gerald P, Yen H. Cytogenetic and molecular studies on a recombinant human X chromosome: implications for the spreading of X chromosome inactivation. In Proceedings of the National Academy of Sciences of the United States of America, 1987;84(14):4954–4958.
  13. Carpenter NJ, Say B, Browning D. Gonadal dysgenesis in a patient with an X; 3 translocation: case report and review. Journal of Medical Genetics, 1980; 17(3): 216–221.
  14. Madan KP, Hompes GA, Schoemaker J, Ford CE. X-autosome translocation with a breakpoint in Xq22 in a fertile woman and her 47, XXX infertile daughter. Human Genetics, 1981; 59(4):290–296.

Overexpression/amplification of Her-2/neu in malignant tumors-A Short Review

Overexpression/amplification of Her-2/neu in malignant tumors-A Short Review

*Kabir E

Abstract

Her-2/neu overexpression has been shown to play a significant role in development and progression of many malignant tumors of body, mostly affects tumors of epithelial origin. Her-2 is a protein involved in normal cell growth. However, HER2/neu may be made in larger than normal amounts by some types of cancer cells.This may cause cancer cells to grow more quickly and spread to other parts of the body. Checking the amount of HER2/neu on some types of cancer cells may help plan of treatment.  Amplification and/or overexpression of HER-2/neu in human tumor tissue has been found  in  breast, ovarian, endometrial, colon, gastric or gastroesophageal junction, urothelial, bladder, salivary duct and cervix cancer. The degree of overexpression correlates with tumor progression, resistance to chemotherapy and a poor prognosis. Testing for this overexpression/amplification in tumor and its recurrence is very important. Immunohistochemistry and FISH are two important modern techniques which can identify overexpression of this protein in formalin fixed paraffin embedded tissue.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):145-150]

Keywords: Her-2/neu overexpression, Immunohistochemistry of Her-2/neu, FISH, parrafin embedded tissue

*Professor Enamul Kabir, Professor of Pathology, Sir Salimullah Medical College, Dhaka, Bangladesh. enamulkabir1213@gmail.com

Introduction

EGFR was the first discovered epidermal growth factor receptor.1 HER2 is so named because it has a similar structure to human epidermal growth factor receptor, or HER1. Neu is so named because it was derived from a rodent glioblastoma cell line, a type of neural tumor. ErbB-2 was named for its similarity to ErbB (avian erythroblastosis oncogene B), the oncogene later found to code for EGFR.2 ERBB2, a known proto-oncogene, is located at the long arm of human chromosome 17 (17q12).2

Growth factor binding results in receptor dimerization, subsequent tyrosine kinase activity and autophosphorylation of specific tyrosine residues. After that event downstream activation of signal transduction cascades occur and  MAPK, Akt and JNK pathways become activated. It  leads to DNA synthesis, cell proliferation, and differentiation. EGFR, also known as ErbB-1, and the three related receptors of the ErbB family: ErbB-2, ErbB-3, and ErbB-4. ErbB-2 is also known as HER2 in humans and neu in rodents. The HER-2/neu oncogene was first identified as a dominant transforming gene in chemically induced adrenal neuroblastomas of neonatal mice and was referred to as neu.3,4 Subsequently, three groups independently identified the human homologue of this gene.5,6 Sequence analysis of the gene demonstrated a close relation ship to the human epidermal growth factor receptor (HER-i) or c-erbB oncogene.5,6 Because of the similarities with HER-i, this gene was considered to code for a membrane receptor.5,6

Pathophysiology

Normal cells express 40,000 to 100,000 EGFR receptors, cancer cells may express up to 2,000,000 receptors.1 Stimulation of overexpressed EGFR receptors may cause  cancer by inducing cancer-cell proliferation while simultaneously blocking apoptosis. Cancer cells cause  invasion and metastasis by activating invasion and metastasis of hyperproliferative cells and by stimulating tumor-induced neovascularization.1

Amplification and/or overexpression of HER-2/neu in human tumor tissue has been associated with a poor prognosis in   cancers of breast,7 ovary,8 endometrium,9 colon,10 gastric or gastroesophageal junction,11 urothelial,12 bladder,12 salivary duct13 and cervix.14 Amplification, also known as the over-expression of the ERBB2 gene, occurs in approximately 15-30% of breast cancers, 7-34% of patients with gastric cancer and in 30% of salivary duct carcinomas.2 The degree of overexpression correlates with tumor progression, resistance to chemotherapy and a poor prognosis.1

However, regarding involvement of  HER2 protein 3+ expression in nonepithelial malignancies, it was very rare, often non-existent, in malignancies of non-epithelial origin. Out of 965  malignant melanoma cases, only one showed HER2 3+ expression. In 1,211 sarcomas of soft tissues and 1,136 neuroendocrine tumors, none exhibited 3+ HER2 protein expression. No HER2 3+ expression was detected in gastrointestinal stromal tumors (GIST), small cell lung cancers (SCLC), kidney cancers, and glioblastomas.15,16

Tests for Her-2

Immunohistochemistry and in situ hybridization (ISH, FISH) are the recommended methods for determining Her2 status for treatment with Her-2-targeted therapy. Neither method is 100% sensitive or specific.Updated ASCO-CAP (2013) guidelines have resulted in increased proportion of patients being eligible for Her2-targeted therapy. Her2-positive cases are not a homogeneous group – borderline positive cases may not be as responsive to Her-2-targeted therapy. Challenges in Her-2 laboratory testing include polysomy / co-amplification, and genetic heterogeneity.17,18

Immunohistochemistry (IHC) and HER-2 in situ hybridization are the most commonly used techniques for Her-2 expression. IHC can be done on formalin-fixed, paraffin-embedded tissue . Tests are usually performed on biopsy samples obtained by either fine-needle aspiration, core needle biopsy, vacuum-assisted breast biopsy, or surgical excision. Immunohistochemistry is used to measure the amount of HER2 protein present in the sample. The sample is given a score based on the cell membrane staining pattern. Specimens with equivocal IHC results should then be validated using fluorescence in situ hybridisation(FISH).2 HER-2 scoring was reported per American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines published in 2007 and updated in 2013.   IHC test was considered positive (IHC+) when IHC3+ was obtained above the guidelines defined thresholds; an IHC test was considered negative (IHC-) when IHC 2+ (equivocal), IHC 1+, or IHC 0 was obtained.15,17,18,19

HER2 in situ hybridization technique

In a study 37,992 samples  were analyzed by IHC and 21,642 samples were also examined with ISH. FISH was used for evaluation of the HER2 amplification status. FISH was performed with a probe specific for HER2 (17q11.2-q12 region) and a probe for the pericentromeric region of chromosome 17. Interphase nuclei were examined and the ratio of HER2 signals to chromosome 17 centromere signals were evaluated to indicate amplification status of this gene.15. HER2/CEP17 ratio higher than 2.2 was considered amplified (ISH+), and HER2/CEP17 ratio between 1.8 and 2.2 (equivocal) in FISH or HER2/CEP17 ratio <1.8 in FISH was considered non-amplified (ISH-). 15,17

 

HER2 amplification was also evaluated by CISH. The HER2 and chromosome 17 probes are detected using two color ISH in formalin-fixed, paraffin-embedded human cancer tissue specimens following staining on automated slide stainer, and visualized by light microscopy. Consistent with the CISH package insert, HER2/CEP17 ratio higher than 2.0 was considered amplified (ISH+); HER2/CEP17 ratio <2.0 in CISH was considered non-amplified (ISH-). 11,670 patients had IHC and FISH; 9972 patient, IHC and CISH. IHC test was considered positive (IHC+) when IHC 3+ was obtained. ISH test was considered positive (ISH+) when the HER2/CEP17 ratio was >2.2 (by FISH) or 2.0 (by CISH).15, 17,18,19

The extracellular domain of HER2 can be shed from the surface of tumour cells and enter the circulation. Measurement of serum HER2 by enzyme-linked immunosorbent assay (ELISA) offers a far less invasive method of determining HER2 status than a biopsy and consequently has been extensively investigated. Results so far have suggested that changes in serum HER2 concentrations may be useful in predicting response to trastuzumab therapy.2

Clinical Significance

The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is erbB-2, and the other members being epidermal growth factor receptor, erbB-3 (neuregulin-binding; lacks kinase domain), and erbB-4. All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. Notably, no ligands for HER2 have yet been identified.2

HER2 has been firmly established in preclinical and clinical settings. Among all four HER family proteins, HER2 has the strongest catalytic kinase activity and functions as the most active signaling complex of the HER family after dimerization with other HER family members. 20,21 Overexpression of HER2 in breast cancer leads to increased homodimerization (HER2:HER2) and heterodimerization (e.g., HER2:HER3), which initiates a strong pro-tumorigenic signaling cascade.22 Overexpression of HER2 protein drives malignant transformation in cell culture and transgenic mouse models.23,24 The anti-HER2 antibody trastuzumab represents an effective, targeted therapy with significant efficacy in treatment of HER2-positive breast and gastric cancer.25,26

Regarding the mechanism of activation of EGFR, It starts by ligand binding at the extracellular domain which results in homo and heterodimerization, leading to phosphorylation, activation of downstream signaling pathways which upregulate expression of genes, proliferation and angiogenesis. Abnormalities in the expression of EGFR play an essential role in the development of different types of cancer. HER2 is the preferred heterodimerization partner for EGFR.; this biological characteristic together with structural homology has played a key role in the development of dual synthetic inhibitors against EGFR/HER2. 27

Overactivation of the ErbB protein family, which is comprised of 4 receptor tyrosine kinase members, can drive the development and progression of a wide variety of malignancies, including colorectal, head and neck, and certain non-small cell lung cancers (NSCLCs). As a result, agents that target a specific member of the ErbB family have been developed for the treatment of cancer.28

Her2 targeted therapy include Herceptin (trastuzumab) and Others: pertuzumab (Perjeta), T-DM1 (Kadcyla), and lapatinib (Tykerb). Recent data shows that a combination of pertuzumab, trastuzumab, and docetaxel (PTD) improved progression free survival compared to patients who had only trastuzumab and docetaxel (TD).29,30

Recent development  of HER2 mutation

Apart from gene amplification ,somatic HER2 (encoded by ERBB2) mutations, , have been reported recurrently in the literature. Mutations in HER2 are clustered in the extracellular, transmembrane and kinase domains. HER2 mutations have been found in non-small-cell lung cancers (NSCLC) and can direct treatment31. Also, HER-2 mutations are infrequent in a wide variety of cancers  but targetable. As for example In breast cancers, activating mutations were identified as follows: G309A, D769H/Y, V777L, P780ins, V842I, and R896C,32 L755S was associated with lapatinib resistance. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. Recently, phase II SUMMIT trial, which is a HER2 mutant basket trial, showed mutation status can contribute to response to neratinib regardless of tumor type 33.

Conclusion

Checking the amount of HER2/neu on some types of cancer cells may help plan treatment. It’s overexpression is mostly confined in malignant tumors of epithelial origin. Immunohistochemistry and FISH are two preferred techniques for identification of its overexpression/amplification. Targetting of Her-2/neu gene with proper drug is important for both treatment and recurrence of many cancer.

 

References

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  20. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, Kofler M, Jorissen RN, Nice EC, Burgess AW, Ward CW. The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation, poised to interact with other ErbB receptors. Mol Cell. 2003 Feb; 11(2):495-50.
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  22. Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A. 2003 Jul 22; 100(15):8933-8.
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  25. Spector NL, Blackwell KL. Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol.2009 Dec 1;27(34):5838-47. doi: 10.1200/JCO.2009.22.1507. Epub 2009 Nov 2.
  26. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N.Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005 Oct 20; 353(16):1673-84.
  27. Bello M, Saldaña-Rivero L, Correa-Basurto J, García B, Sánchez-Espinosa VA. Structural and energetic basis for the molecular recognition of dual synthetic vs. natural inhibitors of EGFR/HER2. Int J Biol Macromol. 2018 May;111:569-586. doi: 10.1016/j.ijbiomac.2017.12.162. Epub 2018 Jan 9.
  28. Roskoski R Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res. 2014 Jan;79:34-74. doi: 10.1016/j.phrs.2013.11.002. Epub 2013 Nov 20.
  29. CLEOPATRA trial. Most recent: Swain et al, NEJM 2015 Feb 19;372(8):724-34.
  30. NeoSphere trial. Gianni et al, Lancet Oncol 2012 Jan;13(1):25-32.
  31. Mazières J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, et al. (June 2013). Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. Journal of Clinical Oncology. 31 (16): 1997–2003. doi:1200/JCO.2012.45.6095PMID2361010
  32. Bose R, Kavuri SM, Searleman AC, et al. Activating HER2 mutations in HER2 gene amplification negative breast cancer. Cancer Discov 2013;3:224-37.
  33. Hyman DM, Piha-Paul SA, Won H, et al. HER kinase inhibition in patients with HER2- and HER3-mutant cancers. Nature 2018;554:189-94.

Role of Cytopathology in Eyelid Growth with Histopathological Correlation

Role of Cytopathology in Eyelid Growth with Histopathological Correlation

*Paul R,1 Kundu UK,2 Kabir E,3 Islam MN4

Abstract
The purpose of this study was to evaluate diagnostic accuracy of cytopathology in  different eyelid lesions using fine needle aspiration cytology (FNAC) & scrape cytology with histopathological correlation. Accurate diagnosis of eyelid tumors is necessary to guide ophthalmologists to design optimal management. Fine needle aspiration from 85 eyelid growth and histopathological correlation were studied. Immunohistochemical analysis were done in few cases.  A majority of the patients (43 out of 85) were in the 26-50 age group (53.49% male vs 46.51% female). Mean age was 43.22±17.42 (range 19 – 90 years) years. Of the malignant lesions, basal cell carcinoma were highest (12 in number, 36.36%) followed by sebaceous gland carcinoma and squamous cell carcinoma. Less common  malignant tumor were Non-Hodgkin’s lymphoma. Among benign neoplastic lesions, nevi were most common (14 in number, 43.75%) followed by haemangioma and squamous papilloma. Other less common  benign tumors were fibroepithelial polyp, adenoma, lipoma and neurofibroma. Most common benign cystic lesions of eyelid are cyst (10 in number, 50%) of moll/Hydrocystoma/Sudoriferous cyst, followed by Dermoid cyst, Epidermal inclusion cyst and Sebaceous cyst. Present study revealed that accuracy of cytopathological diagnosis of malignant eyelid growths were 97.65%. Cytopathology had a high diagnostic accuracy rate. Aspiration cytology was cost effective and offers rapid diagnosis with minimal discomfort to the patient.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):134-144]

Key wards: Eye lid growth, Cytopathology, Histopathology, Correlation

Introduction
Eyelid growth is a common cause to be presented to ophthalmologists1 and many of them can be treated as day care service. At the same time some of the tumors demand emergency surgical intervention and thus early referral.2 Introduction of cytopathology prior to excision biopsy would contribute to early diagnosis and management plan.3 Pathologic conditions affecting the eyelid may be inflammatory or neoplastic. Neoplastic lesion may be benign or malignant.1 It becomes difficult to decide clinically either it is a true neoplasm or an inflammatory lesion. In all such cases, cytopathology (FNAC / scrape cytology) proved to be very useful in quickly determining the nature of the lesion and also deciding the mode of treatment.4 FNAC has a high diagnostic accuracy rate, if the aspirated material is sufficient for microscopical examination and if it is properly interpreted.5 Aspiration cytology is also cost effective and offers rapid diagnosis, with minimal discomfort to the patient..6

  1. *Dr. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka. ritapaul16@gmail.com
  2. Utpal Kumar Kundu, Assistant Professor (Eye), Mugda Medical College, Dhaka.
  3. Professor Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Professor Md. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.

*For correspondence

Method

This was a cross sectional study conducted at the Departmentof Pathology of Sir Salimullah Medical College, Dhaka and National Institute of Ophthalmology and Hospital, Dhaka, Bangladesh from January 2012 to December 2013. 85 adult patients with eye lid growths of both sexes were recruited. Purposive sampling technique was used. FNAC was done using 22 Guage needle without anaesthesia and smears were stained with Papanicolaou’s stain. Biopsy was taken by clinician and diagnosis was confirmed by histopathological examination. Inclusion criteria were adult patients with eyelid growth. Exclusion criteria included patients who were clinically diagnosed to  have inflammatory eyelid lesions, patients belonging to less than 18 years of age, and very tiny growth <0.5cm in diameter.

FNA Features of different Eye-lid lesions

Smears from nevus showed single and small clusters of cells with rounded or oval nuclei and indistinct cytoplasm.7 Smears from haemangioma showed only blood, with a few cases showing an occasional cluster of endothelial cells. Smears from squamous papilloma showed degenerated squamous epithelial cells along with mature superficial squamous cells. Smears from neurofibroma showed cohesive spindle-shaped cells within fibrillary mesenchymal background material. Smears from hidrocystoma / Sudoriferous cyst / Cyst of Moll showed foamy macrophages in the background of proteinecious material. Smears from epidermal inclusion cyst showed high cellularity with numerous nucleated squamous cells and anucleated squames in a background of keratinous debris.8 Smears from dermoid Cyst showed anucleated and nucleated squamous epithelium and keratin debris.9 Smears from chalazion showed a polymorphic picture with neutrophils, plasma cells and macrophages. The granulomas are more of histiocytic cells with abundant vacuolated cytoplasm; the backround is generally dirty with nuclear debris and fat spaces.10 Smears from molluscum contagiosum showed Molluscum bodies, in the enlarged superficial cells of the epidermis.11 Molluscum bodies, also called Henderson-Patterson bodies, were large, round cytoplasmic inclusions (within the enlarged cells of epidermis), which push the nucleus to the periphery.12  Smears from Rhinosporidiosis showed many scattered basophilic rhinosporidial endospores and rhinosporidial spores in a background of amorphous eosinophilic material.13 Smears from basal cell carcinoma showed tightly cohesive small clusters of uniform hyper­chromatic basaloid cells with high nuclear-cytoplasmic ratio and absence of cytoplasmic vacuolation. Peripheral palisading of nuclei may be evident in some clusters. Squamous, sebaceous and adenoid differentiation may be seen and pigmented variant may be seen.14 Smears from sebaceous gland carcinoma showed large pale cells and vacuolated cytoplasm however another type is poorly differentiated cells with dark and irregular nuclei. The smears of squamous cell carcinoma showed markedly enlarged hyper-chromatic nuclei of variable size and keratinization.10 Smear from cutaneous melanoma showed atypical dispersed population of cells with abundant cytoplasm, eccentric uniform hyperchromatic nuclei, internuclear inclusions in the background of melanin pigment.15 In Non-Hodgkin’s lymphoma, cytology smears showed a monotonous population of lymphocytes with round nuclei having coarse granular chromatin. Histopathological examination of biopsied tissue confirmed the diagnosis16 and in a few cases by the help of immuno-histochemical analysis using specific antibodies.

Results

Age distribution of the patients presented with eyelid growths showed almost half (50.59%) of the patients comprised of middle age group (26-50 years) with a little more than one-fourth (30.59%) above 50 years of age. Gender distribution of the patients presented with eyelid growths showed slightly male preponderance (50.59%) and female constituted 49.41%. Mean age was 43.22±17.42 (range 19 – 90 years) years.

Among benign neoplastic lesions, nevus was most common, followed by vascular lesion and squamous papilloma. Others were adenoma, lipoma and hamartoma. Correlation with cytopathological and histopathological examination were done. Cytopathologically diagnosed fourteen (14) cases of Nevus were confirmed by histopathological examination. Cytologically diagnosed Vascular Lesion in eight (8) cases were histologically confirmed in seven (7). The other case was histologically diagnosed as Hamartoma. Five (5) cases of squamous papilloma corresponded cytologically and histologically. One (1) case of cytologically diagnosed lipoma was also confirmed histologically. Both (2) cases of histologically diagnosed Fibroepithelial Polyp were cytologically diagnosed as benign Mesenchymal lesion. One (1) case of histologically diagnosed Neurofibroma was cytologically diagnosed as a malignant peripheral nerve sheeth tumor (False positive; Table III).

Cytologically diagnosed Cyst of Moll/ Hydrocystoma/ Sudoriferous Cyst corresponded histologically in all ten (10) cases. Five (5) cases of cytologically diagnosed Dermoid Cyst were confirmed histologically. Cytologically diagnosed three (3) cases of Epidermal Inclusion Cyst corresponded histologically. Two (2) cases of cytologically diagnosed Sebaceous Cyst were later on confirmed histologically (Table IV). Among the malignant lesions encountered during the present study, Basal cell carcinoma was the most common malignancy, followed by Sebaceous Gland Carcinoma and Squamous Cell Carcinoma. Non-Hodgkin’s lymphoma (NHL) was found to be less common. Cytological diagnosis was confirmed by histopathological examination in the present study. Eleven (11) cases of cytologically diagnosed Basal Cell Carcinoma were confirmed histologically. One (1) case of cytologically diagnosed Nevus (False negative) was histologically diagnosed as Basal Cell Carcinoma. Sebaceous Gland Carcinoma corresponded cytologically and histologically in all nine (9) cases. Nine (9) cases of cytologically diagnosed Squamous Cell Carcinoma were also confirmed histologically. Lymphoma corresponded cytologically and histologically in both (2) cases, which was later on confirmed by immuno-histochemical study as Non-Hodgkin’s lymphoma of ‘B’ cell origin. One (1) case of cytologically diagnosed Small Cell Tumor was confirmed histologically (Table V).

Comparison of diagnosis between cytopathology with histopathology among the malignant eyelid growths

Among 85 eyelid growths, 32 cases were cytopathologically and histopathologically true positive for malignant lesions. The comparison between cytopathology and histopathology were statistically highly significant (p < 0.0001; Table VI). Out of 85 (100%) patients of eyelid growths 32 (37.64%) were positive for malignancy, 51(60.00%) were negative for malignancy, 01(1.18%) was false positive(1.18%) and 1 (1.18%) was false negative (Table VII). The validity of cytopathology to diagnose malignant eyelid growths, Sensitivity, Specificity, PPV, NPV and Accuracy were 96.97%, 98.08%, 96.97%, 98.08% and 97.65% respectively (Table VIII).

Out of a total 33 histologically confirmed cases of malignant tumor, thirteen (13) cases were ulcerated. From these ulcerated lesions, samples were collected by scraping. Out of these, nine (9) cases were Basal Cell Carcinoma, three (3) cases were Squamous Cell Carcinoma and one (1) case was found to be Sebaceous (Meibomian) Gland Carcinoma.

Table I: Distribution of different eyelid lesions with Cytological and Histological Diagnosis (n=85)

Eyelid Growth Cytological Diagnosis Histological Diagnosis
Nevus 15 (17.65) 14 (16.47)
Vascular Lesion 8 (9.41) 7 (8.23)
Squamous Papilloma 5 (5.88) 5 (5.88)
Adenoma 1 (1.18) 1 (1.18)
Lipoma 1 (1.18) 1 (1.18)
Hamartoma 0 (0.00) 1 (1.18)
Benign Mesenchymal Lesion 2 (2.36) 0 (0.00)
Fibroepithelial Polyp 0 (0.00) 2 (2.36)
Neurofibroma 0 (0.00) 1 (1.18)
Cyst Of Moll/ Hydrocystoma/ Sudoriferous Cyst 10 (11.76) 10 (11.76)
Dermoid Cyst 5 (5.88) 5 (5.88)
Epidermal Inclusion Cyst 3 (3.52) 3 (3.52)
Sebaceous Cyst 2 (2.36) 2 (2.36)
Basal Cell Carcinoma 11 (12.94) 12 (14.12)
Sebaceous Gland Carcinoma 9 (10.58) 9 (10.58)
Squamous Cell Carcinoma 9 (10.58) 9 (10.58)
Lymphoma 2 (2.36) 2 (2.36)
Small Cell Tumor 1 (1.18) 1 (1.18)
Malignant Peripheral Nerve Sheath Tumor (MPNST) 1(1.18) 0 (0.00)
Total 85 (100) 85 (100)

Table II: Age distribution of the patients of all types of eyelid lesions with percentage

(n=85)

Age Frequency Percentage
Up to 25 16 18.82
26 – 50 43 50.59
Above 50 26 30.59
Total 85 100.0

Table III: Distribution of different benign neoplastic eyelid growths

Final Histological Diagnosis  

Cytological Diagnosis

Total (Final Histology) Nevus Haemangioma Squamous Papilloma Adenoma Lipoma Hamartoma Benign Mesenchymal Lesion Fibroepithelial Polyp Neurofibroma

 

MPNST
Nevus 14 14 0 0 0 0 0 0 0 0 0
Haemangioma 7 0 7 0 0 0 0 0 0 0 0
Squamous Papilloma 5 0 0 5 0 0 0 0 0 0 0
Adenoma 1 0 0 0 1 0 0 0 0 0 0
Lipoma 1 0 0 0 0 1 0 0 0 0 0
Hamartoma 1 0 1 0 0 0 0 0 0 0 0
Fibroepithelial Polyp

 

2 0 0 0 0 0 0 2 0 0 0
Neurofibroma 1 0 0 0 0 0 0 0 0 0 1
Total = 32 Total= 32

Table IV: Distribution of different benign cystic eyelid lesions

Final Histological

Diagnosis

Cytological Diagnosis
Total Final Histology Cyst of Moll Dermoid Cyst Epidermal Inclusion Cyst Sebaceous Cyst
Cyst of Moll 10 10 0 0 0
Dermoid Cyst

 

5 0 5 0 0
Epidermal Inclusion Cyst

 

3 0 0 3 0
Sebaceous Cyst 2 0 0 0 2
Tolal 20 Total = 20

 

Table V: Distribution of malignant eyelid lesions (total of 85 cases each)

Final

Histological

Diagnosis

Cytological Diagnosis
Total Final Histology Basal Cell Carcinoma Sebaceous Gland Carcinoma Squamous Cell Carcinoma Lymphoma Small Cell Tumor Nevus
Basal Cell Carcinoma

 

12 11 0 0 0 0 1
Sebaceous Gland Carcinoma

 

9 0 9 0 0 0 0
Squamous Cell Carcinoma

 

9 0 0 9 0 0 0
Lymphoma 2 0 0 0 2 0 0
Small Cell Tumor 1 0 0 0 0 1 0
Total = 33 Total = 33

 

TableVI: Comparison of diagnosis between Cytopathology with Histopathology among the Malignant Eyelid Growths

 

Cytopathology Histopathology Total
Malignant Benign
Malignant 32 (37.64) 1 (1.18%) 33 (100.0%)
Benign 1 (1.18%)  51 (60.00%) 52 (100.0%)
Total 33 (38.82%)   52 (61.18%)  85 (100.0%)

* p value < 0.0001

 

Table VII: Assessment of diagnostic accuracy of cytopathology of eyelid growths

 

Cytopathological Diagnosis No. of cases Percentage
Positive for Malignancy 32 37.64%
Negative for Malignancy 51 60.00%
False Positive 1 1.18%
False Negative 1 1.18%
Total 85 100%

 

Table VIII: Cytopathological validity of different malignant eyelid growths

Sensitivity Specificity Positive Predictive Negative Predictive Value Accuracy
96.97% 98.08% 96.97% 98.08% 97.65%

Figure 1. (A) Case 1. Basal cell carcinoma, (B) Photomicrograph of cytology smear of basal cell carcinoma showing tightly cohesive small clusters of uniform hyper­chromatic basaloid cells (Pap’s, x200), (C) Photomicrograph of Basal cell carcinoma showing atypical basaloid cell with retraction artifact (H&E, x400)

Figure 2. (A) Case 2.  Squamous cell carcinoma, (B) Photomicrograph of cytology smear of Squamous cell carcinoma showing enlarged hyper-chromatic nuclei of variable size and keratinization (Pap’s, x400), (C) Photomicrograph of Squamous cell carcinoma (Grade-I) showing atypical squamous cell invading deeply into the dermis. It also shows squamous pearl. (H&E, x200)

Figure 3. (A) Case 3. Sebaceous (meibomian) gland carcinoma, (B) Photomicrograph of cytology smear of sebaceous (meibomian) gland carcinoma showing atypical tumor cells arranged in clusters and singly with foamy eosinophilic cytoplasm (Pap’s, x400), (C) Photomicrograph of sebaceous (meibomian) gland carcinoma showing atypical tumor cells and necrosis (H&E, X200).

Figure 4. A. Case 4. Non-Hodgkin’s Lymphoma, B. Photomicrograph of cytology smear of Non-Hodgkin’s Lymphoma showing monomorphous population of atypical lymphoid cells, scanty cytoplasm with clumped chromatin. (Pap’s x200), C. Photomicrograph of Non-Hodgkin’s Lymphoma showing  hypercellular proliferations. Most of the tumor cells are monotonous in appearance, having large nuclei with condensed chromatin. (H&E, x400), D. Photomicrograph of IHC study showing lymphoid cells with positive staining for LCA. (IHC, x400), E. Photomicrograph of IHC study showing scattered lymphoid cells with positive staining for CD3. (IHC, x400), F. Photomicrograph of IHC study showing  majority of atypical  lymphoid cells with positive staining for CD20 (IHC, x400) Conclusion: Immunostaining results favor the diagnosis of Non-Hodgkin’s Lymphoma of “ B” cell origin.

Figure 5. A. Case 5.  Hydrocystoma. B. Photomicrograph of cytology smear of benign cystic lesion showing foamy macrophages in the background of proteinecious material (Pap’s, x200), C. Photomicrograph of hydrocystoma showing cyst wall lined by a double layer of columnar cells with eosinophilic cytoplasm and prominent papillary projections. (H&E x400)

Figure 6. A. Case. Nevus, B. Photomicrograph of cytology smear of nevus showing single and small clusters cells with rounded or oval nuclei and indistinct cytoplasm (Pap’s x200). C. Photomicrograph of nevus showing nests of round cells in the underlining dermis. (H&E, x200)

Discussion

The present study was conducted with an aim to assess the cytopathological and histopathological correlation of different types of eyelid growths. It was a hospital based cross sectional study which enrolled 85 clinically suspected eyelid growths. Out of them 52 (61.18%) were benign and 33 (38.82%) were malignant. A recent study by Mondal and Dutta,8 Fine needle aspirates from 80 eyelid swellings were studied.  Forty eight cases of benign and 32 cases of malignant lesions were diagnosed by FNAC.

Mean age in the present study was 43.22 years (SD ±17.42) (range 19 – 90 years). Pombejara et al.17 reported mean age of presentation 52.4 years ± SD 21.8 years in Thailand. Most benign growths were within the age group 26-50 years and malignant eyelid lesions were in patients above 51 years of age.Mondal and Dutta8 studied 80 eyelid lesions by FNAC, in which 32 cases were malignant. In that study most common malignant lesion was basal cell carcinoma (12 cases, 15%) followed by sebaceous gland carcinoma (nine cases, 11.25%) and squamous cell carcinoma (eight cases, 10%). In the present study, malignancy were 38.82% (33 out of 85), and the most frequent malignant tumor was basal cell carcinoma (12 out of 33, 36.37%) followed by sebaceous gland carcinoma (09, 27.27%), squamous cell carcinoma (09, 27.27%), Non-Hodgkin’s lymphoma (2, 6.06%) and small cell carcinoma (1,3.03%).

Among benign lesions, in the present study, nevus was most common in 14 cases (43.75%), followed by haemangioma 7 cases (21.88%) and squamous papilloma in 5 cases (15.64%). Other less common lesions were fibroepithelial polyp, adenoma, lipoma, neurofibroma and hamartoma. In the present study, among benign cystic lesions (20 cases) of eyelid, sudoriferous cyst was most common in 10 cases (50.00%) followed by dermoid cyst in 5 cases (25.00%), epidermal inclusion cysts in 3 cases (15.00%), and sebaceous cysts in 2 cases (10.00%). In a recent study by Toshida et al.,18 the most frequent diagnosis among 106 benign lesions were nevus in 23 cases (21.7%). The second common was squamous cell papilloma in 18 cases (17.0%), followed by seborrheic keratosis in 14 cases (13.2%). Less common causes were epidermal cyst in 10 cases (9.4%) and dermoid cyst in 7 cases (6.6%).

 

Ulcerated skin of eye-lid can be scraped safely and it is recommended to combine FNAC with scrape cytology for any ulcerated lesions of eyelid skin and conjunctiva (Rai, 2007). In the present study, out of a total 33 histologically confirmed malignant tumors, thirteen (13) ulcerated cases were taken by scraping. Of these, nine (09) were Basal Cell Carcinoma, three (3) were Squamous Cell Carcinoma and one (1) was found to be Sebaceous (Meibomian) Gland Carcinoma.

In recent studies by Mondal and Dutta 8 and  Arora et al.5 showed accuracy of cytological diagnosis of eyelid growths were 83.87% and 89.4% respectively. In the present study, accuracy of cytological diagnosis of malignant eyelid growths was 97.65%. These comparisons are clearly emphasizing need for cytopathology and histopathology of all surgically removed specimens.19,20 The present study also compared cytopathological and histopathological diagnosis of all specimens.When comparing cytopathology with histopathology of the clinically suspected malignant eyelid growths, the comparison between cytopathology and histopathology was statistically highly significant (p<0.0001).In the present study, Sensitivity, Specificity and Accuracy of cytopathology to diagnose malignant eyelid growths were 96.97%, 98.08% and 97.65% respectively.

References

  1. Abdi U, Tyagi N, Maheshwari V, Gogi R and Tyagi SP. Tumors of eyelid: A clinicopathologic study. J Indian Med Assoc, 1996;94: 405-9.
  2. Cook BE Jr. and Bartley GB. Epidemiologic characteristics and clinical course of patients with malignant eyelid tumors in an incidence cohort in Olmsted county, Minnesota. Ophthalmology, 1999; 106: 46-50.
  3. Hughes MO. A Pictorial Anatomy of the Human Eye/Anophthalmic Socket: A Review for Ocularists. The Journal of Ophthalmic Prosthetics, 2004; 51-53.
  4. Jakobiec FA, Bonanno PA and Sigelman J. Conjunctival adnexal cysts and dermoids. Arch Ophthalmol, 1978;96: 1404-1409.
  5. Arora R, Rewari R and Betheri SM. Fine needle aspiration cytology of eyelid tumors. Acta cytol, 1990; 34(2): 227-32.
  6. Dey P, Radhika S, Rajwanshi A, Ray R, Nijhawan R and Das A. Fine needle aspiration biopsy of orbital and eyelid lesions. Acta Cytol, 1993; 37: 903-7.
  7. Brooks Christine, Scope, Alon, Braun, Ralph, P,Marghoob and Ashfaq A. Dermoscopy of nevi and melanoma in childhood. Expert Review of Dermatology, 2011; 6 (1): 19–34.
  8. Mondal SK and Dutta Cytohistological study of eyelid lesions and pitfalls in fine needle aspiration cytology. J Cytol, 2008; 25(4):133-7.
  9. Baschinsky D, Hameed A, Keyhani-Rofagha S. Fine-needle aspiration cytological features of dermoid cyst of the parotid gland: a report of two cases.diagncytopathol, 1999; 20(6): 387-8.
  10. Vemuganti GK and Rai NN. Neoplastic lesions of eyelids, eyeball and orbit. J Cytol, 2007; 24: 30-36.
  11. Brown ST, Nalley JF, Kraus SJ. Molluscum contagiosum. Sex Transm Dis. Jul-Sep, 1981; 8(3), pp227-34.
  12. Stulberg DLHutchinson AG.Molluscum contagiosum and warts. Am Fam Physician.  2003; 67(6): 1233-40
  13. Pathak D and Neelaiah S. Disseminated cutaneous rhinosporidiosis: diagnosis by fine needle aspiration cytology. Acta Cytol, 2006; 50: 111-2.
  14. Baron K, Curling OM, Paridaens AD and Hungerford JL. The role of cytology in the diagnosis of peri-ocular basal cell carcinomas.OphthalPlastReconstr Surg, 1996; 12: 190-4.
  15. Saqi A, McGrath CM, Skovronsky D and Yu GH. Cytomorphologic features of fine-needle aspiration of metastatic and recurrent melanoma.Diagn Cytopathol, 2002; 27: 286-290.
  16. Pugh WC, Manning JT and Butller JJ. Paraimmunoblastic variant of small lymphocytic lymphoma/leukemia. Am J Surg Pathol, 1988; 12: 907-917.
  17. Pombejara FN, Tulvatana W and Pungpapong K. Malignant tumors of the eye and ocular adnexa in Thailand: A six-year review at King Chulalongkorn Memorial Hospital. Asian Biomed, 2009; 3: 551-5.
  18. Toshida H, Mamada N, Fujimaki T, Funaki T, Ebihara N, Murakami A andOkisaka S. Incidence of Benign and Malignant Eyelid Tumors in Japan, Int J Ophthalmic Patho, 2012; l1(2): 112-14.
  19. Kerstern R, Ewing-Chow D, Kulwin DR. and Gallon M. Accuracy of clinical diagnosis of cutaneous eyelid lesions. Ophthalmology, 1997; 104: 479-484.
  20. Margo CE and Waltz K. Basal cell carcinoma of the eyelid and periocular skin, Survey of ophthalmol, 1999; 38(2):169-192.

Correlation of Ki-67 Proliferating Index with Histological Stage and Grade in Colorectal Carcinoma

Correlation of Ki-67 Proliferating Index with Histological Stage and Grade  in Colorectal  Carcinoma

 *Sultana S,1 Islam N,2 Kabir E,3 Akhter S,4 Paul R,5 Shirin A,6  Khan AA,7 Jahan N8

Abstract
Colorectal carcinoma is the most  common cancer of gastrointestinal tract. It is the 3rd most  commonly  diagnosed  cancer  and  the  3rd  leading  cause  of  cancer  death. The  growth  of  tumor  in  colorectal   carcinoma  is  highly  variable  and  its  histological grading and  staging  has  important  role  in  diagnosis, treatment  and  overall  prognosis. To observe the Ki-67 expression in colorectal carcinoma and find out the possible correlation of Ki-67 proliferating index with histological grading and Duke’s staging. This  cross sectional  study  was  conducted  at  Sir Salimullah Medical College  from  July 2014  to June 2016. 98  patients  with colorectal carcinoma  enrolled  in  this  study  by  purposive  sampling. The H&E staining was done on paraffin embedded tissue sample. Ki-67 expression by IHC method. Ki-67 is a proliferation associated  nuclear  antigen  which  can be recognize by  MIB-1 monoclonal antibody, correlate with histological staging and grading in  colorectal carcinoma. Then tumours were graded according to WHO grading criteria and pathological staging was done according to Duke’s staging system and immunohistochemical staining for Ki-67 antigen expression. Results were subjected to statistical analysis. The results were considered to be significant when the P< 0.05. Ki-67 proliferative index was high in well and moderately differentiated adenocarcinoma but low in poorly differentiated which is statistically significant (p <0.05).  Ki-67 expression was high in early Duke’s stage A and B but low expression  in advanced Duke’s stage C (p>0.05). The result of  this  study will enlighten the clinician regarding  the need for  doing Ki-67 in  colorectal carcinoma which   would  contribute to better understanding  of the  treatment as well as prognosis.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):125-133]

Key words: Colorectal carcinoma, Immunohistochemistry, Ki-67

  1. *Dr. Sahela Sultana, Assistant Professor, Department of Pathology, Dr. Sirajul Islam Medical College, Dhaka. sultana.sahela83@gmail.com
  2. Professor Dr. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  3. Professor Dr. Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Salma Akhter, Assistant Professor, Department of Pathology, Universal Medical College, Dhaka.
  5. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka.
  6. Afroz Shirin, Assistant Professor, Department of Pathology, Enam Medical College Savar, Dhaka.
  7. Abu Anis Khan, Assistant Professor, Department of Pathology, International Medical College, Dhaka.
  8. Nusrat Jahan, Lecturer, Department of Pathology, Sir Salimullah Medical College, Dhaka.

 

*For correspondence

Introduction

Colorectal carcinoma is the most common malignancy of gastrointesinal (GI) tract and is a major cause of morbidity and mortality worldwide.1 Colorectal cancer accounts for 10% of all cancers and it is the 2nd leading cause of death from malignancy in the industrialized world.2 There are nearly one million new cases of colorectal cancer diagnosed worldwide each year and half a million death.3 In 2013, there were an estimated 1,177,556 people living with colon and rectal cancer in the United States and the number of new cases of colon and rectal cancer was 41.0 per 100,000 men and women per year.4 Regarding age  incidence of colorectal carcinoma, recent reports show that in the USA it was the most frequent form of cancer among the person aged between 60-70 years and fewer than 20% of cases occurs before the age 50.5 The incidence of  colorectal cancer in Bangladesh is exactly not known but estimated population are approximately 15,10.1%.6 The distribution of colorectal carcinoma worldwide seems to be related to industrialization and socioeconomic standard and the incidence rate is higher in industrialized countries including Western Europe, Scandinavia and North America, whereas in the developing countries (sub-Saharan, Africa and Asia) the incidences appear to be lower.7

There are several staging system for colorectal carcinoma among these TNM and Duke’s staging systems are the most common way of staging and grading of colorectal carcinoma.8 The American Joint Committee (AJC) and the Union  for International  Cancer control (UICC) joined to produce the TNM system, which attempt to record clinical and pathological data, guide therapy and forecast prognosis, all in one.9 Whereas  the  classification   into  Duke’s stage A,B,C  cases   is  the  measurement  of  the  boundaries  reached and both   methods   permit the grouping  of  cases into favorable and unfavorable outcome.10 Histologically  the tumor is graded according to WHO grading criteria as well differentiated, moderately differentiated and poorly differentiated and the histological appearance of colorectal carcinoma may vary considerably with its major importance being related to prognosis.10

The use of monoclonal antibodies raised against specific antigens associated with the cell proliferation.11  Ki-67 is a proliferation associated nuclear antigen expressed in all cycling cell except resting cell in the G0 phase and it reflects cell in the S/G2+M phases in particularly.12  MIB-1 is a monoclonal antibody and it recognizes the Ki67 nuclear antigen in the formalin fixed paraffin embedded tissue section.13 Ki-67 expression is estimated as the percentage of tumors cells positively stained by the antibody with nuclear staining.12 The importance of Ki-67 as an indicator of tumor behavior and in colorectal cancer this index may be used as a marker of prognosis.12

The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grade and stage.2 In 2008 Uzma Nabi, Nagi A H and Waqas Sami, Department of Pathology, University of Health Sciences, Lahore, Pakistan conducted a study on Ki-67 proliferating index and histological stage and grade  of colorectal carcinoma and observed that proliferative index is high in well and moderately differentiated  adenocarcinoma and in an early Duke’s stage (A or B).2 But Ki-67 proliferating activity is low in poorly differentiated tumour and  in an advanced Duke’s stage C.13,8 But there are some studies of Lanza, Cavazzinil  in 1990 and  Yokoyama N, Okomoto H in Japan in 2005 contradicting the above mentioned  association of Ki-67 versus grading and staging of colorectal carcinoma, they concluded that proliferating index of Ki-67 was increasing with increasing grade, stage.14, 15

Methods

This  cross sectional study was conducted among the 98 histopathologically diagnosed patients having colorectal carcinoma  over a period of two years in the department of surgery, Sir Salimullah Medical College. Study population were the patients having colorectal cancer underwent surgical  treatment in the department of surgery of Sir Salimullah Medical College. The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grading  and staging of colorectal carcinoma. The representative sections were submitted for Immunohistochemical staining. The Ki-67 immunostaining were performed according to manufacture’s recommendation, using the MIB-1 clone (DAKO, Carpenteria, CA & Ventena Medical System, Tucson, AZ). Ki-67 immunostainined slides were examined via light microsccopy. Positive Ki-67 staining was observed brown granular nuclear staning. For Ki-67 scoring the most positive area of the tumor was selected avoiding foci of inflammation. The number of positive nuclei were counted in 500 tumor cells in a high power field. The average of the counts over the same slides was taken and expressed as the percentage of Ki-67 positive cells  in the tumor.

Statistical analysis were performed in SPSS statistical software program, Version 17.0. To correlate histological grading and staging of colorectal carcinoma with Ki-67 proliferating index were performed with  Mann- Whitney U test. The result were consider to be  significant when P<0.05. One way ANOVA followed by Bonferroni test was performed to compare between groups.

Results

98 cases were included in the present study.  Age incidence ranged from 28-78 years and their mean ± SD 47.38 ± 10.37. Maximum patients (30.6 %) were found in 41-50 years age group where M: F was 1.72: 1 (Table I). Regarding site of tumor more than 50% of patient had tumors in the left side of colon and in the rest of the cases tumors were present in caecum (26.5%; Table-II).  98 cases having different sizes of tumors and in most of the cases fifty nine cases (60.2%) tumors size were 3-4 cm and  their mean ± SD 4.8 ± 1.8 (Table-III). Different morphological types of tumor were observed in present study. In maximum forty cases (40.8%) the morphological types of tumors were ulcerative and only five cases (5.3%) tumors were infiltrative type. Rests were annular (33.5%) and polypoid (20.3%; Table IV).

In the present study, tumors were graded according to WHO grading system into well differentiated, moderately differentiated, poorly differentiated and grouped into A, B, C accordingly. The maximum cases 69 (70.4%) of colorectal carcinoma were well differentiated and their mean Ki-67 proliferating index was 47.83 ± 15.23. There were significant differences among the groups (A vs B vs C), when mean proliferating index of Ki-67 were compared among the three groups. The result was found statistically significant (P<0.05). However when compared in between groups only A vs C (between well differentiate & poorly differentiate) groups were found also statistically significant (P<0.05). In the present study, it was also observed that with increasing grade, Ki-67 proliferating index decreases (Table V) .

Regarding staging of the tumor (Duke’s staging) where  maximum  cases (44.8%) were in stage B1 and their mean Ki-67 proliferating index was 46.25±14.06. There was no significant differences among the stages and the result was not statistically significant (P>0.05). In the present study it was also observed that with increasing stage of the tumors, there was decreasing Ki-67 proliferating index (Table VI).

Table I: Distribution of patients according to age group with male female ratio (n=98)

Age groups         Frequency            M: F      Percentage
Total  Male Female
≤30

 

10    7    3           2.3:1       10.2
31-40 23   13   10           1.3: 1       23.5
 

41-50

 

30

 

19

 

11

 

1.72: 1

 

30.6

 

51-60

 

26

 

17

 

9

 

1.88: 1

 

26.5

 

≥60

 

9

 

9

 

0

 

9.00: 0

 

9.2

 

Total

 

98

 

Mean ± SD

 

 

47.01± 10.99

Range (Min-Max)                                              28 – 78

 

 

Table II: Distribution of tumors according to site (n=98)

Site of tumor Frequency (%)
Left side of colon
Sigmoid colon 34 (34.7)
Transverse colon 24 (24.6)
Rectum 14 (14.7)
Right side of colon
Caecum 26 (26.5)
Total 98 (100)

Table III: Distribution of patients according to tumor size (n=98)

Tumor size (cm) Frequency (%)
1 – 2 14 (14.3)
3 – 4 59 (60.2)
5 – 6 19 (19.4)
Mean ± SD 4.8 ± 1.8
Total 98 (100)

 

Table IV: Distribution of patients according to morphological types of tumor (n=98)

Morphology Frequency (%)
Ulcerative 40 (40.8)
Annular 33 (33.5)
Polypoid 20 (20.3)
Infiltrating 5 (5.3)
Total 98 (100)

 

Table V: Relation of Ki-67 proliferating index with histological grading (n=98)

Grading Frequency

n(%)

Ki-67 expression

(Mean ± SD)

P
Well differentiated (A) 69 (70.4) 47.83± 15.23
Moderate differentiate
(B)
15(15.3) 46.33 ± 18.07
Poor differentiated (C)  14(14.3) 35.35 ± 11.17
Statistical analysis
A vs B vs C 0.023*
A vs B 1.000ns
A vs C 0.019*
B vs C 0.162 ns

ANOVA followed by Bonferroni test was performed to compare between groups

Table VI: Relation of Ki-67 proliferating index with histological stage (n=98)

 

Duke’s staging

 

Frequency

n (%)

Ki-67 expression

(Mean ± SD)

p
Stage A 3 (3.3%) 53.33 ± 20.81 0.727ns
Stage B1 44 (44.8) 46.25 ± 14.06
Stage B2 20(20.5) 43.25 ± 14.06
Stage C1 14(14.8) 44.64 ± 21.07
Stage C2 17(17.5) 42.05 ± 13.69

 

ANOVA test was done to measure the level of significance.

 

 

 

 

 

Figure 1. Photomicrograph of histopathological section of well differentiated adenocarcinoma of colon (H&E method x100)

 

 

 

 

 

Figure 2. Photomicrograph of   well differentiated adenocarcinoma stained with Ki-67 immunostain showing high proliferative Index(x100).

 

 

 

 

Figure 3. Photomicrograph of histopathological section of poorly differentiated adenocarcinoma of colon ( H& E method x400).

 

 

 

 

 

Figure 4. Photomicrograph of   poorly differentiated adenocarcinoma stained with Ki-67 immunostain showing low Proliferative Index (x400).

Discussion

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide.16 The incidence of cancer colon and rectum was 41.0 per 100,000 men and women per year and the number of deaths was 15.1 per 100,000 men and women per year in Bangladesh.17 Management of  colorectal carcinoma depends on a number of  morphological and biological factors which include the pathological tumor stage (including involvement of lymph nodes, breach of serosa, distant spread etc.), primary tumor characteristics (including depth of tumor penetration in the bowel wall, histological subtype, histological grade and differentiation, venous and lymphatic invasion, perineural invasion and lymphocytic infiltration), status of surgical resection margins (free or involved).18 With assessment of tumor cell proliferation may predict tumor behavior.19 The aim of this study was to evaluate the proliferating index (PI) in formalin fixed, paraffin embedded tissue section of colorectal carcinoma, using monoclonal MIB-1 antibody (Ki-67) and to assess the relationship between proliferative index (PI) and various pathological findings in colorectal carcinoma including histological grade, and stage.

In the present study, the mean age of the patients was 47.01± 10.99 years and the highest number of malignant cases were seen in the 4th and 5th decades. Male female ratio was 1.72:1 and 1.88:1 in 4th and 5th decades accordingly. So, in present study, male were predominant than female. These findings were similar to other studies.20, 21

In 34.7% patients the tumors were located in sigmoid colon followed by caecum (26.5%), transverse colon (24.5%) and rectum (14.3%).  As per gross morphological type of cancer, maximum 40.8% were ulcerative and 33.5% were annular type and rest were polypoid (20.3%) and infiltrating type (5.3%). In one study, colon was more commonly affected site compared to rectum and most of the lesions were ulcerative.22 In another study rectum was the most common affected site and predominant lesions were annular.23

In present study, 60.2% of malignant cases had tumors size between 3-4 cm and 19.4% malignant cases had tumors size 5-6 cm. In a study in 2011 by Kornprat showed that maximum size of the tumor in colon cancer were in between 4.5 to 6.5 cm in majority of cases.24 In 2013 ASCO annual meeting showed a report where a study conducted on tumor size in colorectal cancer found that in majority of the cases the tumors sizes were in between 4-6 cm in colorectal carcinoma.25

In this present study it was observed that  the Ki-67 labelling index was high in grade I and grade II  compared to grade III. These results showed that proliferating index was low in poorly differentiated tumor compared to well and moderately differentiated tumor. When Ki-67 proliferating index was compared among three groups a statistically significant correlation was found in present study (p<0.05). These findings are similar to studies in Japan2 Pakistan12 and Finland.26 On the other hand, some of the studies showed contradictory result that Ki-67 proliferating index increased with increasing histological grade.26,27

In the present study, it was observed that Ki 67 labeling index was high in Duke’s stage A and B  and tumor in advanced stage (Duke’s C) have a low proliferating index  compared to tumors in  an early invasive stage (Duke’s A and B). Regarding the correlation of Ki-67 LI with staging, no statistically significant correlation was found. This result was similar with other studies in which they concluded that Ki-67 proliferating index was significantly lower in carcinoma in subserosa or deeper invasion compared to carcinoma with submucosa or muscularis mucosa invasion.2,8,26 In another study  contradictory results have been reported on associations of Ki-67 with prognosis and survival of colorectal tumors with a low proliferation index in Duke’s A and B tumors to be associated with survival impairment compared to those with high values.28 Tumors with high proliferative activity are known to be most responsive to radiotherapy and Willett and collaborators showed that radiation eradicates preferentially rapidly dividing cells in rectal cancer, whereas populations with slow proliferation show greater radioresistance.29

Conclusion

It is concluded that Ki-67 labeling index is high in well to moderately differentiated adenocarcinomas in an early Duke’s stage A or B compared to poorly differentiated adenocarcinomas, and  in an advanced Duke’s stage C. Thus Ki-67 proliferating index can be useful in a patient with colorectal carcinoma as a ancillary diagnostic support. Moreover, it may help in the prognostic evaluation of patient, survival as well as in considering them for post surgical treatment.

References

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  14. Lanza G Jr, Cavazzini L, Borghi L, Ferretti S, Buccoliero F and Rubbini M. Immunohistochemical assessment of growth fraction in colorectal adenocarcinomas with monoclonal antibody Ki-67. 1990;186 (1):608-18.
  15. Yokoyama N, Okamoto,Valentina V, Walter B,  Suda HT, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal  carcinoma Version of   Record British Journal of Surgery Society  2005; 10:4858-15.
  16. World Health Organization Cancer Incidence in Five Continents. Lyon: The World Health Organization and The International Agency for Research on Cancer; WHO, Geneva, 2002. http://www.iarc.fr/en/publications/pdfs-online/epi/index.php.
  17. National Institutes of Health and Human service. What You Need To Know About Cancer of the Colon and Rectum. Bethesda, MD: U.S. Department of Health and Human Services & National Institutes of Health.2010. http://www.cancer.gov/cancertopics/types/colon-and-rectal.
  18. Ola Marzouk and John Schofield. Review of Histopathological and Molecular Prognostic Features in Colorectal Cancer Cancers (Basel). 2011;3(2):2767–2810.
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  20. Bhagyalakshmi, Sreelekha A. Kasi Babu SV, Kumar P. Muralikrishnm Ki-67 proliferation index and clinicopathological patterns in colorectal carcinomas. Departments of   Pathology, Biochemistry, Surgery, Gastroenterology, Andhra Medical College, Visakhapatnam. 2015;4:119-28.
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  22. Missaouia N, Jaidaine L, Abdelkader AB, Beizig N, Anjorin A, Yaacoubi MT. . Clinicopathological patterns of colorectal cancer in Tunisia. Asian Pacific J Cancer Prev 2010; 11:1719-1722.
  23. Laishram RS, Kaiho N, Shimray R, Sorokhaibam BD, Pukhrambam P, Durlav CS. Histopathological evaluation of colorectal carcinomas status in Manipur, India. 2010; 8(1):5-8.
  24. Kornprat P, Pollheimer MJ, Lindtner RA, Schlemmer A, Rehak P, Langner C. Value of tumor size as a prognostic variable in colorectal cancer: a critical reappraisal. Am J Clin Oncol 2011; 34(1):43-9.
  25. ASCO 2013 Annual Meeting, Gastrointestinal Colorectal Cancer,ASCO university. https://meetinglibrary.asco.org/results/SessionTitle:/22Gastrointestinal/20/28Colorectal /Meeting:/222013/20ASCO.
  26. Eeva S, Salla P, Tero V, Peter J. Roberts, Karl-Owe Söderström. Increased proliferation activity measured by immunoreactive Ki-67 is associated with survival improvement in rectal/recto sigmoid cancer. Department of Oncology and Radiotherapy, Turku University Hospital, Finland 2005;11(21):3245–3249.
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  28. Palmqvist R, Sellberg P, Oberg A, Tavelin B, Rutegård JN. Low tumour cell proliferation at the invasive margin is associated with a poor prognosis in Dukes’ stage B colorectal cancers. Br J Cancer 1999;79:577-581.
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Cytogenetic Analysis and Clinical Findings in Patients with Disorders of Sex Development

Cytogenetic Analysis and Clinical Findings in Patients with Disorders of Sex Development

*Asaduzzaman,1 Khandkar T,2 Islam SS,3 Habib S4

Abstract

Disorder of sex development (DSD) is not so uncommon in Bangladesh. Frequency, types, clinical presentation and associated karyotypes in patients with sex differentiation errors is mostly unknown in our country. Genetic methods for the diagnosis of DSDs still include determination of the karyotype. It is impossible to manage a case without knowing the exact karyotype. This cross sectional study was conducted at the department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU) from January 2015 to December 2016 to determine the frequency, types, clinical presentation and associated karyotypes of disorders of sex development. A total of 93 cases of disorders of sex development were included in this study. All the cases attended the genetics laboratory, Department of Pathology, BSMMU. Suspected cases of DSD patients of any age group were included in the study and detailed clinical information were obtained. All suspected cases with clinical features, hormonal abnormality or radiological abnormality of disorders of sex development were confirmed by karyotyping analysis by using standard cytogenetic techniques. The commonest age of presentation was in age group between 11 to 20 years. History of parental consanguinity or endogamy was found in 31.18% patients. All the cases were classified according to Chicago nomenclature. Sex chromosome DSD (Turner syndrome and Klinefelter syndrome) was the commonest (69.89%), followed by 46,XY DSD (20.43%) and 46,XX DSD (9.68%). In the 55 studied cases with Turner syndrome phenotype, 39 patients (41.93%) had 45,X and 12 patients (12.90%) had mosaic [45,X/46,XX; 45,X/46,X,i(Xq); 45,X /46,XX /46, X,i(Xq) and 45,X/47,XXX ] and 4 patients (4.30%) had long arm isochromosome of X chromosome [46,Xi(Xq)]. In the 8 studied cases with Klinefelter syndrome phenotype, 7 (87.5%) had 47,XXY, and 1 (12.5%)  case was mosaic (46XY/47XXY). In our study, out of 93 patients 20.43% had 46, XY karyotype and 9.68% had 46,XX karyotype. In Turner syndrome most common presentation was primary amenorrhoea followed by short stature. In cases of Klinefelter syndrome, common clinical presentations were small atrophic testes, infertility  and gynaecomastia. Most common presentations of 46,XY DSD case were primary amenorrhoea, ambigious genitalia and delayed/ absent secondary sex characters. Most of the 46,XX DSD presented with ambigious genitalia, clitoromegaly and with hyperpigmentation of genitalia. This study showed that diagnosis and management of DSD in Bangladesh is possible in many cases despite the limitations of delayed presentation, incomplete investigations and unavailability of gene sequencing and molecular study. This study will guide the future planning and management of the patients with disorder of sex development.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):114-124]

Keywords: Disorder of sex development, clinical presentation, karyotype

  1. *Dr. Asaduzzaman, Lecturer, Department of Pathology, Sheikh SayeraKhatun Medical College, Gopalganj. dr.asad37@gmail.com
  2. Tahmina Khandkar, Resident, Department of Paediatric Nephrology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  3. SM Shahedul Islam, Scientific Officer, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.
  4. Saequa Habib, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka.

*For correspondence

Introduction

Phenotypic sex results from the differentiation of internal ducts and external genitalia under the influence of sex-determining genes and hormones.1-2 In one of every 4500 births, the genital appearance is abnormal and it is not always possible to decide the sex of the infant at first glance. The European Society for Paediatric Endocrinology and the Lawson Wilkins Pediatric Endocrine Society jointly organized a meeting of endocrinologists, surgeons, geneticists, psychologists, and patient advocacy group members, all representing a world community involved with the management of intersex disorders. A consensus document was subsequently published3 and it has become known as the Chicago Consensus by virtue of its generation in the ‘windy city’. There are numerous modes of classification to bewilder the reader with exhaustive lists of all the possible causes of DSD. Instead of using the confusing and/or controversial terms such as “intersex,” “hermaphroditism” and “sex reversal”, the Chicago consensus statement recommended a new taxonomy based on the umbrella term, “DSD”.4 The term disorders of sex development (DSD) embraces all the medical conditions characterized by an atypical chromosomal, gonadal, or phenotypical sex.4

The diagnostic terms that came out of the 2006 Chicago consensus meeting were designed to eliminate the more confusing and stigmatizing elements of the previous classification lexicon. They were confusing because a number of different terms and definitions could be used to describe a particular diagnosis. The terms such as ‘pseudo-hermaphrodite’ and ‘intersex’ were considered pejorative. The new classification system has made a significant improvement, in that it creates structure and definitions that are suitable for universal use, and also eliminates odious terminology.

Frequency of different types of DSD is mostly unknown in Bangladesh and many other countries of the world. Only limited data is available regarding these disorders. In our country DSD diagnosis is based on hormonal evaluation, imaging studies and most importantly, cytogenetic analysis which is done at cellular level. Cytogenetic analysis is a reliable procedure, can be done from peripheral blood and relatively inexpensive. It is done only in few centers in Dhaka.

Methods

This cross sectional study was conducted at the department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU) from January 2015 to December 2016. A total of 93 cases of disorders of sex development were included in this study. Most of the patients were included from outpatient department of Paediatrics, BSMMU and Department of Endocrinology and metabolic disorder, Dhaka Shishu (Children) Hospital. Other cases were included from various outpatient departments of BSMMU. All the cases attended the genetics laboratory, Department of Pathology, BSMMU.

Suspected cases of DSD patients based on clinical, biochemical and imaging studies of any age group were included in the study. Patient with autosomal disorder, severe psychiatric comorbidity and mental disabilities and patients not willing to take part in this study were excluded from the study.

A detailed history was taken including presence of similar conditions in the family. A thorough clinical examination was done including body hair distribution (hirsutism) and external genitalia examination with giving importance on body stature, genital ambiguity, apparent female genitalia with clitoromegaly, posterior labial fusion or inguinal/labial mass, and apparent male genitalia with non-palpable testis, micropenis, isolated perineal hypospadias or mild hypospadias with undescended testes. In ambiguous genitalia cases virilization was assessed by Prader score. Assessment of primary and secondary sex characteristics (pubic hair and breasts in females and testis, penis and pubic hair in males) were done according to Tanner sexual maturity ratings. Abdomino-pelvic ultrasound was done to evaluate ambiguous genitalia, anomalies of the pelvic organs and to see internal genital organ.

All suspected cases with clinical features, hormonal abnormality or radiological abnormality of disorders of sex development were confirmed by chromosomal analysis. Standard cytogenetic technique was used. With all aseptic precaution 2 – 3 ml of venous blood was taken in a heparinized syringe for cytogenetic analysis. After completion of all procedures two slides were made for each case. Each of the slides was scanned under low magnification (10X) first to locate good quality spread. Then in oil immersion (100 X) 15-20 well spread metaphase were counted and analyzed for aneuploidy and other structural abnormalities.

Classification of cases according to Chicago nomenclature

DSD cases were categorized on the basis of karyotype, hormonal level and results of imaging studies and sub classified according to Chicago nomenclature (Table I).

Table I: DSD Classification according to Chicago consensus nomenclature3

Sex Chromosome DSD 46,XY DSD 46,XX DSD
45,X (Turner syndrome and variants) Disorders of gonadal (testicular) development: (a) complete gonadal dysgenesis (Swyer syndrome); (b) partial gonadal dysgenesis; (c) gonadal regression; and (d) ovotesticular DSD Disorders of gonadal (ovarian) development: (a) ovotesticular DSD; (b) testicular DSD (eg, SRY+, duplicate SOX9); and (c) gonadal dysgenesis
47,XXY (Klinefelter syndrome and variants) Disorders in androgen synthesis or action: (a) androgen biosynthesis defect (eg, 17-hydroxysteroid dehydrogenase deficiency, 5αRD2 deficiency, StAR mutations); (b) defect in androgen action (eg, CAIS, PAIS); (c) luteinizing hormone receptor defects (eg, Leydig cell hypoplasia, aplasia); and (d) disorders of anti-Müllerian hormone and anti-Müllerian hormone receptor (persistent Müllerian duct syndrome) Androgen excess: (a) fetal (eg, 21-hydroxylase deficiency, 11-hydroxylase deficiency); (b) fetoplacental (aromatase deficiency, POR [P450 oxidoreductase]); and (c) maternal (luteoma, exogenous, etc)
45,X/46,XY (MGD, ovotesticular DSD) Other (eg, cloacalexstrophy, vaginal atresia, MURCS [Müllerian, renal, cervicothoracic somite abnormalities], other syndromes)
46,XX/46,XY (chimeric, ovotesticular DSD)

Statistical analysis and result

Statistical analyses have been carried out by using the Microsoft office 2013 packages software. The mean values were calculated for continuous variables. The quantitative observations were indicated by frequencies and percentages.

Ethical implication

Every ethical issue was discussed with the patients regarding the study and informed written consent was obtained. The research protocol was approved by the Institutional review board (I.R.B.) of BSMMU, Dhaka.

Results

A total of 160 suspected DSD cases were evaluated clinically and according to their hormonal and imaging status. Out of these 93 cases were cytogenetically and clinically proved as DSD. Remaining cases were diagnosed as normal or other disorders and excluded from the study.

Age of the patient at diagnosis

The commonest age of presentation was in between 11 to 20 years. Majority of DSD patients (60.22%) present at this age group. Only 20.43 % patients presented below 10 years. Above 20 years this rate was 18.28%. In this study no case of 46,XX DSD was presented after 20 years of age (Figure 1).

Figure 1. Age distribution of 93 DSD cases

Epidemiological Profile

Most of the DSD patients (82.80%) were found to be raised as female in this study. Among these 19.35% patients who raised as female proved to be male according to karyotyping. History of parental consanguinity or endogamy was seen in 31.18% patients. Small number of cases had family history of such disorders (5.38%).

Categorization of DSD cases according to Chicago nomenclature

Disorders of sex chromosomes (Turner syndrome and Klinefelter syndrome) were the commonest (69.89%), followed by 46,XY DSD (20.43%) and 46,XX DSD (9.68%). In many cases exact sub-classification was not possible as gene analysis was not included in this study. Classic Turner syndrome (41.93%) (Figure 2) and mosaic Turner syndrome (17.20%) (Figure 2) were subdivided according to cytogenetic analysis pattern. Klinefelter syndrome (7.53%) and mosaic Klinefelter syndrome (1.08%) were also classified accordingly. 46,XY DSD cases were found to be 20.43% and 46, XX DSD cases were 9.68%. Sub-classification and exact frequency found in this study are shown in Table II.

Table II: Frequency & types of disorders of sex development (DSDs)

Type of Disorder No. %
Sex chromosome DSD 65 69.89
     Turner syndrome and variants
          Classic Turner syndrome 39 41.93
          Mosaic Turner syndrome 16 17.20
Klinefelter syndrome
          Classic Klinefelter syndrome 7 7.53
           Mosaic Klinfelter syndrome 1 1.08
     Mixed (Gonadal dysgenesis/ Chimeric) 2 2.15
46, XY DSD 19 20.43
     Androgen insensitivity syndrome 5 5.38
     Defect in androgen synthesis/ action 4 4.30
     Gonadal dysgenesis 5 5.38
     Persistent mullerian duct syndrome 1 1.08
     Others 4 4.30
46,XX DSD 9 9.68
     Congenital adrenal hyperplasia 2 2.15
     Others 7 7.52
Total 93 100

 

 

Figure 2. Karyotypes of Sex Chromosome DSD; 45, X classic Turner Syndrome (Case no. 58) and 45,X/46,Xi(Xq) mosaic Turner syndrome (Case no. 7).

Chromosomal variations defined among the most common DSD

In the 55 studied cases with Turner syndrome phenotype, 39 (41.93%)  patients had 45,X and 12 (12.90%) patients had mosaic [45,X/46,XX; 45,X/46,X,i(Xq); 45,X /46,XX /46, X,i(Xq) and 45,X/47,XXX] and 4 (4.30%) patients had long arm isochromosome of X chromosome [46,Xi(Xq)]. In the 8 studied cases with Klinefelter syndrome phenotype, 7 (87.5%) had 47,XXY (Figure 3)  and one case (12.5%) was mosaic (46XY/47XXY). Among the rest, 19 (20.43%) had 46,XY DSD and 9(9.68%) had 46,XX DSD (Table II, Figure 3).

Table III: Cytogenetic findings of various DSD

Cytogenetic findings No of cases. %
Sex Chromosome DSD
Turner syndrome
     45,X 39 41.93
45,X/46,X,i(Xq) 6 6.45
45,X/46,XX 3 3.23
     45,X /46,XX /46, X,i(Xq) 1 1.08
     45,X/47,XXX 2 2.15
      46,X,i(Xq) 4 4.30
Klinefelter syndrome
     47,XXY 7 87.5
     47,XXY/46,XY 1 12.5
Others (Gonadal dysgenesis/ Chimeric)
     45,X/46XY 1 1.53
     47,XXX 1 1.53
46, XY DSD and 46, XX DSD
     46,XY 19 20.43
     46,XX 9 9.68

 

 

 

Figure 3. Karyotypes of Sex Chromosome DSD; 47,XXY, Klinefelter syndrome (Case no. 19) and 47,XXX syndrome (Case no.43).

Clinical presentations of DSD

The variability in the manifestation of DSD covers a spectrum ranging from normal external female and male phenotypes to ambiguous genitalia. In this study, major clinical manifestations of DSD cases were evaluated separately.

In Turner syndrome most common presentation was primary amenorrhoea (69.23% in classic and 56.25% in mosaic Turner syndrome) followed by short stature (51.28% in classic Turner syndrome). Twenty one (53.84%) patients of classic Turner syndrome presented with delayed/absent secondary sex characters, whereas 5 (31.25%) patient in mosaic Turner group. Webbing of neck was present in 9 (23.07%) patients and most of them were under 10 years of age. Two (5.13%) patient in classic TS group and 1 (6.25%) patient in mosaic TS group presented with infertility (Table IV).

Table IV: Clinical features in Turner syndrome

Clinical features Classic Turner syndrome (N=39) Frequency (%) Mosaic Turner syndrome (N=16) Frequency (%)
Primary amenorrhoea 27 69.23 9 56.25
Secondary amenorrhoea 2 5.13 1 6.25
Menstrual irregularity 2 5.13 0 0
Short stature 29 74.36 7 43.75
Delayed/Absent secondary sex character 21 53.84 5 31.25
Sparse axillary, pubic hair 9 23.01 2 12.25
Shield chest 6 15.38 1 6.25
Webbing of neck 9 23.07 0 0
Infertility 2 5.13 1 6.25

In cases of Klinefelter syndrome, common clinical presentations were small atrophic testes (62.5%), infertility (50%) and gynaecomastia (25%). One patient also presented with ambiguous genitalia and one with lack of secondary sex characters (Table V). Two other sex chromosome DSD (1.53% of each) includes XXX syndrome and mixed gonadal dysgenesis. The Triple X syndrome presented with normal female phenotype, with infertility and menstrual irregularity. The 45,X/46XY patient presented with female phenotype with virilization of external genitalia, absent secondary sex characters and primary amenorrhoea (Table V).

Table V: Clinical features in Klinfelter syndrome

Clinical features Klinefelter syndrome (N=8) Frequency (%)
Small atrophic testes 5 62.5
Infertility 4 50
Sparse axillary and pubic hair 1 12.5
Gynaecomastia 2 25
Ambiguous genitalia 1 12.5

Common presentations of 46, XY DSD cases were primary amenorrhoea (42.11%), ambigious genitalia (31.58%) and delayed/ absent sex characters (27.78%). Eleven (57.89%) patient was adolescence or adult in this category. Cases of 46,XX DSD presented with ambigious genitalia (55.56%), clitoromegaly (44.45%) and hyperpigmentation of genitalia (22.2%). Two (22.22%) patients of this category were in adolescent age. These results are shown in Table VI.

Table VI: Clinical features of 46,XX and 46,XY DSD

Clinical features 46, XY DSD

 

46,XX DSD
(N=19) Frequency (%) (N=9) Frequency (%)
Primary amenorrhoea 8 42.11 0 0.00
Delayed/absent secondary sex character 5 27.78 0 0.00
Ambiguous genitalia 6 31.58 5 55.56
Clitoromegaly 0 0.00 4 44.45
Small penis 5 26.32 0 0.00
Hypospadias 1 5.26 0 0.00
Hyperpigmentation of  genitalia 0 0.00 2 22.22

 

 

Figure 4. A child with 46, XX DSD presented with ambiguous genitalia

Discussion

The present study data demonstrated that sex chromosome DSD (Turner syndrome and Klinefelter syndrome) were the commonest disorders as it represented 69.89% of our patients. Turner syndrome constituted a significant proportion of DSD cases (59.14%) and Klinefelter syndrome represented 8.61%.  In present study 46,XY DSD was found 20.43% , followed by 46, XX DSD (9.68%). This finding is consistent with other studies by Erdogan et al. ( 2011) and Shawky et al.( 2012).5,6 A cross-sectional study was done at the department of Pediatric Surgery, Chittagong Medical College & Hospital (CMCH), Chittagong, Bangladesh, from January 2006 to December 2012 and they found that among 50 DSD patients, 22% had 46, XX DSD with congenital adrenal hyperplasia (CAH), 64% with 46, XY DSD, 8% with mixed gonadal dysgenesis (MGD), and 6% with ovotesticular DSD.7 Other studies like Mazen et al., have also reported a relatively higher incidence of 46,XY DSD excluding sex chromosome DSD.8 In present series 46, XY DSD was found to be 67.85% and 46,XX DSD 32.14%.

According to studies done by White and Speiser (2000) and Kovács et al. (2001), most (>80-90%) of the DSD patients of developed world presented in the neonatal period, with <10% presenting in adolescence.9,10 In contrast, in our study the commonest age of presentation was in between 11 to 20 years. Majority of DSD patients (60.22%) presented at this age group. Only 20.43 % patients were below 10 years. In developing countries like Bangladesh, this delayed presentation may be due to lack of awareness associated with other social factors.

Data on the actual prevalence of DSD in developing countries associated with high rates of consanguinity or endogamy is largely unavailable.11 In present study 31.18% patients have history of parental consanguinity or endogamy. A study done by Shawky et al.(2012) in Egyptian population comprised of 908 patients with sex differentiation errors showed that, consanguineous marriage was reported among parents of 504 patients (55.50%).6 This study result is consistent with the present study. Therefore, in case of disorders of sex development, consanguinity may have a role. Although most of the published data from western countries have showed low rates of consanguinity which may not be a true reflection of the worldwide prevalence.

Cytogenetic variants of our studied patients with Turner syndrome are consistent with other studies.Huang et al., (2002) reported the karotypes of Turner syndrome as, 45,X (53%); mosaicism 45X/46XX (15%); X isochromosome, 46,Xi(Xq) (10%); mosaicism 46,Xi(Xq)/46XX (8%); deletions 46,Xdel(Xp) or 46,X del (Xq) (6%); other mosaicism (8%).12 Another study on Turner syndrome patients in northeastern Malaysia (2008) showed that, the incidence of the most frequent karyotypes of the Turner syndrome were found to be 45,X (57.1%), followed by 46, Xi(Xq) (21.4%), 45,X/45,X,+mar (7.1%), 45, X/46,Xi(Xq) (7.1%) and 45, X/46,XY (7.1%)13. These results are similar with this present study.

In present study, The common causes of 46, XY DSD cases were androgen insensitivity syndrome(26.31%), gonadal dysgenesis (26.31%) anddefect in androgen synthesis/ action (21.05%). The causes of 46,XY DSD were numerous and heterogeneous, as described in other studies byLadjouze et al., (2016).14 In present study further sub categorization was not possible as gene analysis was not done and all the hormonal evaluation was not possible due to lack of resources.

In other studies, the 46,XX DSD group, the most common condition was CAH due to 21-hydroxylase deficiency, a finding compatible with its worldwide incidence of 1:14 000 live births.15 Present study results are not in agreement with these study results. In present study 22.22% cases were diagnosed as congenital adrenal hyperplasia. However a study done in Bangladesh by Chowdhury et al., in 2014 supports the present study findings.According to that study, the percentage of CAH patients, which usually accounts for more than half of the patients with DSD in a developed country, made up only 22%. This suggested many babies may have died of a salt-losing crisis in the second or third week of life, and hence are no longer represented. This was probably also true for boys with CAH, who have no genital anomaly but probably succumb to an adrenal crisis shortly after birth. This was more likely to be the case in lower socioeconomic classes, which is supported by the fact that they were under-represented the study.7 This explains the causes of reduced number of CAH in present study. Further sub categorization was not possible in 46, XX DSD cases, as it requires genetic analysis.

In present study, most common presentation in Turner syndrome was primary amenorrhoea, short stature, delayed/absent secondary sex characters and  webbing of neck. A small number of patients presented with infertility. In cases of Klinefelter syndrome, common clinical presentations were small atrophic testes, infertility andgynaecomastia. One patient also presents with ambiguous genitalia and one with lack of secondary sex characters. Most common presentation of 46, XY DSD case were primary amenorrhoea, ambigious genitalia and delayed/ absent sex characters. Most of the 46,XX DSD presented with ambigious genitalia, clitoromegalyand with hyperpigmentation of genitalia. These study results are consistent with study done by Shawky et al., in 20126. In that study presentation of DSD cases were primary infertility, primary amenorrhea, male infertility, ambiguous genitalia at birth, short stature and delayed secondary sexual characters, males with microtestes, and hirsutism. However frequency of different presentations slightly varies.

Conclusion

Although a number of diagnostic algorithms exist for DSD classification, no single evaluation protocol is suitable for all circumstances and some basic tests, such as hormone assay, ultrasonography and cytogenetic analysis are very important for classification and management of DSD. Further studies using molecular genetic analyses are needed to give a more precise diagnosis. This study will strengthen the proper management of DSDs andwill facilitate the sharing of experiences, thereby reducing the stress and isolation felt by patients and their families. Despite all the odds a number of individuals with DSD are highly resilient, true to the words of Helen Keller ‘Although the world is full of suffering, it is also full of overcoming it’.

References

  1. Allen L. Disorders of sexual development. Obstetrics and gynecology clinics of North America. 2009; 36:25-45.
  2. Dreger AD, Chase C, Sousa A, Gruppuso PA, Frader J. Changing the nomenclature/taxonomy for intersex: a scientific and clinical rationale. Journal of Pediatric Endocrinology and Metabolism. 2005;18:729-34.
  3. Hughes IA. Disorders of sex development: a new definition and classification. Best practice & research Clinical endocrinology & metabolism. 2008; 22:119-34.
  4. Hughes IA, Deeb A. Androgen resistance. Best practice & research Clinical endocrinology & metabolism. 2006; 20:577-98.
  5. Erdoğan S, Kara C, Uçaktürk A, Aydın M. Etiological classification and clinical assessment of children and adolescents with disorders of sex development. Journal of clinical research in pediatric endocrinology. 2011; 3:77.
  6. Shawky RM, Elsayed NS, Ibrahim DS, Seifeldin NS. Profile of genetic disorders prevalent in northeast region of Cairo, Egypt. Egyptian Journal of Medical Human Genetics. 2012;13:45-62.
  7. Chowdhury TK, Kabir M, Chowdhury MZ, Hutson JM, Banu T. The challenges in diagnosis and gender assignment in disorders of sex development presenting to a pediatric surgical unit in a developing country: the role of laparoscopy and simple tests for gender identity. Journal of pediatric urology. 2014;10:1255-60.
  8. Mazen I, Hiort O, Bassiouny R, El Gammal M. Differential diagnosis of disorders of sex development in Egypt. Hormone Research in Paediatrics. 2008;70:118-23.
  9. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocrine reviews. 2000; 21:245-91.
  10. Kovács J, Votava F, Heinze G, Sólyom J, Lebl J, Pribilincová Z, Frisch H, Battelino T, Waldhauser F, Middle European Workshop on Paediatric Endocrinology–Congenital Adrenal Hyperplasia Study Group. Lessons from 30 years of clinical diagnosis and treatment of congenital adrenal hyperplasia in five middle European countries. The Journal of Clinical Endocrinology & Metabolism. 2001; 86:2958-64.
  11. Bashamboo A, McElreavey K. Consanguinity and disorders of sex development. Human heredity. 2014;77:108-17.
  12. Huang B, Thangavelu M, Bhatt S, J. Sandlin C, Wang S. Prenatal diagnosis of 45, X and 45, X mosaicism: the need for thorough cytogenetic and clinical evaluations. Prenatal Diagnosis: Published in Affiliation With the International Society for Prenatal Diagnosis. 2002;22:105-10.
  13. Kannan TP, Azman BZ, Ahmad Tarmizi AB, Suhaida MA, Siti Mariam I, Ravindran A, Zilfalil BA. Turner syndrome diagnosed in northeastern Malaysia. Singapore medical journal. 2008;49:400.
  14. Ladjouze A, Philibert P, Taleb O, Kedji L, Maoudj A, Berkouk K, Bouhafs N, Dahmane N, Melzi S, Anane T, Sultan C. Aetiology of 46, XY DSD in Algeria; Putative Modifier Role of pV89L Polymorphism in the SRD5A2 Gene in Androgen Receptor Mutation-Negative Subjects. In55th Annual ESPE 2016 Aug 19 (Vol. 86). European Society for Paediatric Endocrinology.
  15. Pang S, Wallace MA, Hofman L, Thuline HC, Dorche C, Lyon IC, Dobbins RH, Kling S, Fujieda K, Suwa S. Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pediatrics. 1988;81:866-74.

CT Guided FNAC of Lung Mass – A Retrospective Study of Disease Spectrum

CT Guided FNAC of Lung Mass – A Retrospective Study of Disease Spectrum

 *Ahmed Z,1 Israt T,2 Raza AM,3 Hossain SA,4 Shahidullah M5

 

Abstract

Lung cancer is the major cause of cancer related deaths all over the world. CT guided FNAC of lung mass is an effective modality to diagnose lung cancer. The study was carried out in a specialized diagnostic center at the district of Feni, Bangladesh. A total of 100 cases were studied for a period of 2 years from July 2015 to July 2017. Aim of our study was to evaluate the pathological spectrum of diseases in the lesions of the lung  through CT guided FNAC. Total 100 cases were evaluated retrospectively for a period of 2 years. Out of 100 cases 66% were male and 34% patients were female. Mean age was 54. 34 years. In 56 cases lesions were at the right lung and in 44 cases were in left lung. 68%  cases had malignant lesion and 32% cases were have inflammatory conditions. Squamous cell carcinoma was the predominant malignant tumour. Among the complications, 2 cases developed pneumothorax which were managed conservatively, 3 had chest pain, 3 had mild haemorrhage from the lesion area and 1 had breathlessness. All were managed conservatively. CT guided FNAC can diagnose pulmonary lesion fairly accurately leading to early diagnosis which causes less morbidity and mortality as treatment can be started early.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):109-113]

Keywords: Computed tomography (CT), FNAC, Lung mass.

 

  1. *Dr. Zaman Ahmed, Assistant Professor of Pathology, Abdul Malek Ukil Medical College, Noakhali. drzahmed74@gmail.com
  2. Tasnim Israt, Assistant Professor of Pathology(CC), Cumilla Medical College, Cumilla.
  3. AKM Maruf Raza, Associate Professor of Pathology, Jahurul Islam Medical College, Kishoregonj .
  4. Shaikh Alamgir Hossain, Senior Consultant (Pathology), Bangladesh Secretariat Clinic, Dhaka.
  5. Mohammad Shahidullah,  Associate Professor (CC) of Medicine,  Abdul Malek Ukil Medical College, Noakhali.

 *For correspondece

Introduction

Computed tomography (CT) guided fine needle aspiration cytology (FNAC) is a well known modality for characterization of lung masses. It has been used to differentiate lung masses into benign, malignant and inflammatory types. Furthermore its use has been extended in differentiating lung malignancy into different cytopathological types which aids in proper management of the malignant lesion. CT guided FNAC is widely recognized technique in evaluating lung mass. It is a simple less invasive diagnostic method of relatively low cost, with negligible mortality and limited morbidity.1 In 1976 Haaga and Alfidi reported CT guided biopsy and since then this procedure has be shown to be both effective and accurate. The diagnostic accuracy is reported to be more than 80% in benign disease and more than 90% in malignant disease.2 Several post procedural complications have been reported for CT guided FNAC such as pulmonary hemorrhage, hemoptysis and pneumothorax. Pneumothorax has been observed to be 22% – 45% due to high sensitivity of CT in detecting pneumothorax.3 Relative contraindications to image guided FNAC are severe chronic obstructive airway disease, bleeding diathesis, contralateral pneumonectomy and pulmonary arterial hypertension.4

In Bangladesh CT guided FNAC of lung lesion is relatively a newer diagnostic technique and done mostly in the major cities. The purpose of this study was to evaluate the disease spectrum of lung lesion by CT guided FNAC in a district level diagnostic center.

Methods

The study was carried out in a specialized diagnostic center in the district of Feni. A total of 100 cases were studied for a period of 2 years from July 2015 to July 2017. CT guided FNAC was performed by pathologist in co-ordination with radiologist. Risk and benefit were explained and informed consent taken from each patients or his/her relatives. Skin was cleaned by betadine and 22G spinal needle was introduced through percutaneous transthoracic approach. The exact position of lesion was established by CT scan with site, angle, depth and route of needle introduction was determined. After the needle placement, CT scan done to ascertain that the tip of the needle was within the mass. The aspirate was obtained by to and fro movement of needle within the mass. All slides were fixed in 95% ethyl alcohol and were stained with papaniculaou stain.  All the slides were evaluated by an experienced pathologist. Patients were kept under observation for 2 hours to see any immediate complication.

 Statistical analysis

Microsoft Excel 2016 was used to generate tables. Only descriptive statistics were used to infer results.

 Results

Out of 100 cases 66 were male and 34 were female. Age group was from 25 years to 90 years with mean age of 54.34 years. In 56 cases lung lesion was in right lung and 44 cases were in left lung. Among 56 right lung lesion 45 were male and 11 were female. In 44 left lung lesion 31 were male and 13 were female (Table I).

Among 100 cases, 32 cases were inflammatory or benign lesion. Among the benign lesion, tubercular inflammation were the most common, accounting for 15 cases followed by chronic non specific inflammation 14 cases, suppurative inflammation 2 cases and Benign cystic lesion 1 case. 68 cases were malignant with squamous cell carcinoma exceeding adenocarcinoma, 35 and 25 cases respectively. Small cell carcinoma were 6 cases and poorly differentiated carcinoma were 3 cases (Table II).

Among the complications, 2 cases developed pneumothorax who were managed conservatively, 3 had chest pain, 3 had mild haemorrhage from the lesion area and 01 had breathlessness. All were managed conservatively (Table III).

Figure I show a radiological picture showing needle tip at the lesional site. Figure Ii and figure III show  picture of cytopathological slides of tubercular granuloma and adenocarcinoma respectively.

 

Table I: Lung lesion by site and sex (n=100)

 

Sex Site
Right lung Left lung Total (%)
Male 45 31 66 (66%)
Female 11 13 34 (24%)
Total 56 (56%) 44 (44%) 100 (100%)

Table II: Spectrum of disease in lung lesion on CT guided FNAC (n=100)

Disease Number of cases %
Squamous cell carcinoma 35 35
Adenocarcinoma 25 25
Small cell carcinoma 06 6
Undifferentiated carcinoma 02 2
Tubercular granuloma 15 15
Chronic nonspecific inflammation 14 14
Suppurative inflammation 02 2
Benign cystic lesion 01 1
Total 100 100

 

Table III: Complication of CT guided FNAC in this study (n=9)

Complication Number of cases %
Pneumothorax 02 22.2%
Chest pain 03 33.3%
Mild hemorrhage from overlying skin 03 33.3%
Breathlessness 01 11.1%
Total 09 100%

 

 

 

 

 

 

 

 

Figure 1. Showing needle inside the lung lesion

 

 

 

 

 

 

Figure 2. Showing tubercular granuloma in lung (Paps stain, 40X)

 

 

 

 

 

 

Figure 3. Showing adenocarcinoma of lung (Paps stain, 40X)

Discussion

CT guided transthoracic needle aspiration cytology is safe and accurate method for diagnosis and categorization of malignant and benign lesion. Accuracy of procedure varies in range from 64% to 97%.3 In this present study, 100 cases were studied over a period of 2 years time period. Conclusive cytodiagnosis were made in all the 100 cases. Most Patients tolerated the procedure well. Most common complaint was pain at the procedure site and mild bleeding at the skin puncture site which subsided without medicine in 2 hours. Two cases had pneumothorax which was mild and resolved conservatively. No chest tube insertion was needed. All the cases were adult. The mean age was 54.34 years similar to other studies. Mondal et al and Singh et al in their study found mean age 56.6 years and 56.4 years respectively, which is similar to our study.5,6 This indicates lung mass lesion especially malignant lung tumour come to clinical attention at middle to old age. There was male preponderance (66%) among the patients undergone FNAC for lung lesion. In this study, out of 100 patients male patient were 66% and female patient were 34%. Percentage of male patients in the studies by Saha et al7 78.9% and Tan et al8 71.1%. Bandyopadhyay et al9 found male patient 80.6%  which is high to other study and also high comparing to this study.

Out of the 100 cases, 32% were inflammatory or benign condition and 68% cases were malignant tumour. Mondal et al had benign lesion in 8.07% and malignant lesion in 91.93%.cases.5 This high percentage of malignant patient in this study and study done by Mondal et al probably due to as most of the inflammatory conditions are now a days effectively treated by antibiotics. The tuberculosis cases and malignant cases are non responsive to antibiotics and they suffer chronically and come to diagnostic CT guided FNAC.

The incidence of squamous cell carcinoma (35% cases) was higher than adenocarcinoma (25% cases) in our study similar to the study by Shah S.10 In their study, most common tumour was squamous cell carcinoma (45%) followed by adenocarcinoma (22%), small cell carcinoma (16%) and large cell carcinoma (8%).11 In his study adenocarcinoma was the most common malignant tumour. In that study, adenocarcinoma cases were 30%, squamous cell carcinoma 22.5% and undifferentiated carcinomas was 7.5%. The proportion of adenocarcinoma has risen in the last fifteen years. Adenocarcinoma is the most common histological type in women and the rising proportion of women in the lung cancer population is undoubtedly a factor in the relative increase in the incidence of adenocarcinoma.12

 Conclusion

CT guided FNAC is a well accepted, simple, accurate, safe and cost effective method for diagnosing a lung lesion with low morbidity rates. Combined with CT the aspiration needle can be guided safely into the lesion to improve the diagnosis of the cytological material. CT guided FNAC provides early diagnosis and sub classification of the lung masses hence directing the clinicians in proper management. Complication due to this procedure is not high and can be managed conservatively.

 References

  1. Martin HE, Ellis EB. Biopsy by needle puncture and aspiration. Ann Surg. 1930 Aug;92(2):169-81.
  2. Geraghty PR, Kee ST, Mc Farlane G, Razavi MK, Sze DY, Dake MD. CT-guided transthoracic needle aspiration biopsy of pulmonary nodules: needle size and pneumothorax rate.Radiology.2003 Nov;229(2):475-81.
  3. Herman PG, Hessel SJ. The diagnostic accuracy and complications of closed lung biopsies. Radiology. 1977 Oct;125(1):11-4.
  4. Mohammad GM. CT guided fine needle aspiration cytology in the diagnosis of thoracic lesions. JIMA 2001:99(10):1-5.
  5. Mondal SK, Nag D, Das R, Mandal PK, Biswas PK, Osta M. Computed tomogram guided fine-needle aspiration cytology of lung mass with histological correlation : A study in Eastern India. South Asian J Cancer. 2013 Jan;2(1):14-8. doi: 10.4103/2278-330X.105881.
  6. Singh JP, Garg L, Setia V. Computed tomography guided fine needle aspiration cytology in difficult thoracic mass lesions-not approchable by USG. Indian J radiology. Imaging 2004 May:14:395-400.
  7. Saha A, Kumar K, Choudhuri MK. Computed tomography-guided fine needle aspiration cytology of thoracic mass lesions: A study of 57 cases. J Cytol. 2009 Apr;26(2):55-9. doi: 10.4103/0970-9371.55222.
  8. Tan KB, Thamboo TP, Wang SC, Nilsson B, Rajwanshi A, Salto-Tellez M. Audit of transthoracic fine needle aspiration of the lung : cytological subclassification of bronchogenic carcinomas and diagnosis of tuberculosis. Singapore Med J. 2002 Nov; 43(11):570-5.
  9. Bandyopadhyay A, Laha R, Das TK et al. CT guided fine needle aspiration cytology of thoracic mass lesions: A prospective study of immediate cytological evaluation : Indian J pathological Microbiology. 2007 Jan; 50(1) : 51-5.
  10. Shah S, Shukla K, Patel P. Role of fine needle aspiration cytology in diagnosis of lung tumours-a study of 100 cases. Indian J Pathol Microbiol. 2007 Jan; 50(1):56-8.
  11. Madan M and Bannur H. Evaluation of FNAC in lung disease. Turk J pathology. 2010 Nov; 26(1): 1-6.
  12. Arslan S, Yilmaz A, Bayramgurler B, Uzman O, Unver E, Akkaya E: CT-guided transthoracic fine needle aspiration of pulmonary lesions: accuracy and complications in 294 patients. Med Sci Monit 2002, 8: 493-497.

Histomorphological Study of Urinary Bladder Tumor and Status of HER2/Neu and Ki67 Expression in Urothelial Carcinoma

Histomorphological Study of Urinary Bladder Tumor and Status of HER2/Neu and Ki67 Expression in Urothelial Carcinoma

*Haque S,1 Dewan RK,2 Saleh S,3 Jennah SA,4  Jahan F,5 Akter F,6 Sultana T,7 Ferdaus NJ8

 

Abstract:

Worldwide proliferation marker Kinase inhibitor Ki67 and Human epidermal growth factor receptor2(HER2/neu) both are focused as more reliable biomarker for the risk of prognosis and also useful for targeted therapies for urinary bladder tumor. The present study has used the 2004 WHO grading system of urothelial carcinoma and the AJCC/UICC T staging system of the urothelial carcinoma of the bladder. To observed the status of Ki67 and HER2/neu in uroepithelium as compared with different stages and grades of urothelial carcinoma with special emphasis on low grade and high grade lesions to reveal their help as an ancillary technique in the diagnosis. A cross sectional study was conducted in the Department of Pathology, Dhaka Medical College, Dhaka from January 2016 to December 2017 with 50 patients with urothelial carcinoma attending in Department of Urology, Dhaka Medical College Hospital, Dhaka. HER2/neu and Ki 67 IHC were assessed and compared by chi-square (x2) tests, unpaired student,s “Ttest or ANVOA test with p value <0.05 at 95% CI considered as significant. The mean age was 60.9±13.1 years old and the male to female ratio were 4:1. Among the histological variety, 100% of our patient showed urothelial carcinoma with significant male preponderance. A total of 72% of the patients had high grade and 28% had low grade urothelial carcinoma. A total of 100% of the patients presented with painless hematuria. Among 50 patients 68.0% had tumor level of extension up to PT1(sub epithelial connective tissue) and 32% up to PT2(muscularis propria) in their biopsy specimen. The incidence of smoking was much higher (72%) among patients with high grade urothelial carcinoma. Immunohistochemical expression of Her2/neu and Ki 67 revealed that there was no significant correlation between the expression of these markers with the age and gender (P value >0.05). There was significant association between the expression of Her2/neu (p value <0.030) and Ki67(P value <0.03) with the 2004 WHO grading system of urothelial carcinoma. Ki 67 and HER2/neu expression association with tumor grading can help in predicting the appropriate clinical outcome and selecting patients who may benefit by targeted therapy and avoid over treatment.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):99-108]

Key words: Bladder cancer; gender; smoking; transitional cell carcinoma; HER2/neu; Ki67

  1. *Dr. Sharmin Haque, Lecturer, Department of Pathology, Bangladesh Medical College, Dhaka. drsharminhaque@gmail.com
  2. Rezaul Karim Dewan, Professor, Department of Pathology, Dhaka Medical College, Dhaka.
  3. Suporna Saleh, Lecturer, Department of Pathology, National Medical College, Dhaka
  4. Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College, Dhaka.
  5. Fauzia Jahan, Associate Professor, Department of Pathology, Bangladesh Medical College.
  6. Fahmida Akter, Lecturer, Department of Pathology, Dhaka Medical College, Dhaka.
  7. Tahmina Sultana, Clinical Pathologist, Department of Pathology, Dhaka Medical College, Dhaka.
  8. Nur e Jannatul Ferdaus, Assistant Professor, Department of Pathology, North East Medical College, Sylhet.

*For correspondence

Intruduction

Urinary bladder carcinoma is one of the most common cancer of genitourinary system. It is the 4th commonest cancer in man   and 8th in women in the world with M: F ratio of 3:1.1,2 About 95% of the bladder tumors are of epithelial origin. Though the prevalence is more in developed countries, but now incidence is gradually increasing more in developing countries like Bangladesh, India ect due to industrialization and smoking habit. In our country bladder cancer also seems to be increasing due to increase number of aging people, expose to carcinogen and improved facilities of investigations. In Bangladesh a few populations based studies are available regarding the prevalence.3

The urothelial carcinoma represents about 90-95% of all urinary bladder tumor. Histological tumor grading and staging are known prognostic factors for bladder cancer. The accurate prognosis with any single factor is difficult to predict. There are literature data of numerous studies demonstrating the therapeutic and prognostic value of biomarkers involved in the biomolicular mechanism of urothelial carcinoma.4 Considerable attentions has been given to the identification of prognostic biomarkers of urinary bladder carcinoma.5 Therefore, the prime interest is being currently focused on protein and genetic markers as they may become therapeutic target. The therapeutic weapons are limited in UC and they permit only a limited improvement. 6,7

HER2/neu and Ki67 both are currently focused as more reliable prognostic factors to assess accurate prognosis and useful therapies. HER2/neu is a glycoprotein similar to EGFR family that has tyrosine kinase activity. It acts as an oncogene.8,9,10 Most of the studies on HER2/neu have been carried out in breast cancer. It has now been recognized in other forms of cancers such as colon, bladder, ovarian, uterine endometrial carcinoma, stomach and esophagus carcinoma.  It’s over expression seems to be correlated with recurrence, higher grade and worse prognosis.11 Ki67 is a non-histone nuclear protein, known to be strictly associated with cell proliferation.12,13 It established as an independent predictor of recurrence, progression and response to immunotherapy. Different studies observed that Ki67 proliferation index has increased in high grade carcinoma with or without invasion.14

Methods

A cross sectional study was conducted in the Department of Pathology, Dhaka Medical College, Dhaka from January 2016 to December 2017 with 50 patients with urothelial carcinoma attending in Department of Urology, Dhaka Medical College Hospital (DMCH), Dhaka. Clinically suspected patient (both male and female) were admitted as new cases of bladder tumor at Urology Department, DMCH with clinical symptoms like macroscopic hematuria, dysuria etc. Patients with these complain were advised for radiological examination. About 73 patients were reported as bladder tumor by the radiologists and were subjected to do cystoscopy and transurethral resection (TUR) or biopsies of the suspicious mass.

A total of 50 histologically diagnosed urinary bladder tumor cases were selected from 73 radiologically and clinically suspected bladder tumor cases. Twenty three samples were excluded for tissue necrosis, inadequacy and cautery effect. During the collection of specimen, all relevant information were recorded systematically in a prepared proforma. All the cases were numbered chronologically and the same number was given to histological as well as in immunohistochemical slides. Bladder tumors were sampled or removed with biopsy instrument. All obtained specimens were immersed in 10% buffered formalin. These samples were fixed for 6 hours to 48 hours which was required for proper H&E and immunostaining. Under fixation may cause false IHC result. HER2/neu and Ki 67 IHC were assessed and compared by chi-square (x2) tests, unpaired student,s ‘’T’’ test or ANVOA test with p value <0.05 at 95% CI considered as significant.

In this present study, the evaluation of HER2 was generally carry out using the American Society of Clinical Oncology/College of American Pathologists guideline for breast cancer.15,16 This guideline has been updated in 2013.

Assessment of Ki67 Immunohistochemical Staining done according to Jawad, Ali and Kamal (2016)17 and performed qualitatively by counting the percentage of positive cells (labeling index, LI) out of the total number tumor cells was calculated. Only distinct immune reactive tumor cell nuclei were counted.

Result

Table I: Demographic profile of the patients (n=50)

Frequency Percentage
Age (years)
≤60 29 58.0
>60 21 42.0
Mean ± SD (Min-Max) 60.9 ± 13.1 (20 – 88)
Gender
Male 40 80
Female 10 20
Socioeconomic status
Middle 19 38.0
Low 31 62.0
Personal history
Smoking 36 72.0
Betel nut chewing 36 72.0

Table I shows demographic profile of the patients.

Table II: Distribution of patients according to smoking habit (n=50)

Smoker Low grade

(n=14)

High grade

(n=36)

Total p
Yes 11 (78.6) 25 (69.4) 36 (72.0) 0.517
No 3 (21.4) 11 (30.6) 14 (28.0)
Total 14 (100.0) 36 (100.0) 50 (100.0)

Fisher’s Exact test was done to measure the level of significance.

Figures in the parenthesis denote corresponding %.

Table II shows there was no significant difference in smoking habit between low and high grade tumor.

Figure 1. Pie chart showing the tumor grading of study patients

Table III: Distribution of patients according to HER2/neu score in low and high grade tumor (n=50)

Grading Her2 expression p
Negative Equivocal Positive
Low grade 13 (92.9) 1 (7.1) 0 (0.00) 0.030s
High grade 15 (41.7) 11 (30.6) 10 (27.8)

Fisher’s Exact test was done to measure the level of significance, s= significant

Figures in the parenthesis denote corresponding %.

Table III shows that positive value of HER2/neu expression is significantly higher in high grade tumor (P<0.05).

Table IV: Distribution of patients according to Ki-67 expression in low and high grade tumor (n=50)

Grading Ki 67 p
Positive Negative
Low grade 7 (50.0) 7 (50.0) 0.031s
High grade 29 (80.6) 7 (19.4)

Chi-square test was done to measure the level of significance. s=significant

Figures in the parenthesis denote corresponding %.

Table IV shows Ki-67 expression was found more in high grade carcinoma then low grade carcinoma. The difference is statistically significant (p=0.031)

Table V: Distribution of the study patients according to grading with Her2/neu and Ki67 (n=50)

Grade HER2 (+ve) HER2 (-ve) HER2 (Equivocal)
KI67(+ve) KI67(-ve) KI67(+ve) KI67(-ve) KI67(+ve) KI67(-ve)
 Low grade 0 (0.0) 0 (0.0) 6 (37.5) 7 (58.3) 1 (10.0) 0 (0.0)
 High grade 10 (100.0) 0 (0.0) 10 (62.5) 5 (41.7) 9 (90.0) 2 (100.0)

Figures in the parenthesis denote corresponding %.

Table V shows HER2/neu and Ki67 expression according to histologic tumor grading

 

 

 

 

 

Figure 2. Photomicrography showing high grade urothelial carcinoma. (Case No: 45, H&E x400)

 

 

 

 

 

 

Figure 3. Photomicrography showing positive (score 3+) membrane reactivity of  HER2 protein in high grade urothelial carcinoma. (Case No:45,  IHC for HER2 x 400)

 

 

 

 

 

Figure 4. Photomicrography showing positive of Ki67 in high grade urothelial carcinoma (Case No:45, IHC for Ki67 x 400)

 

 

 

 

 

Figure 5. Photomicrography showing low grade urothelial carcinoma. (Case No: 40, H&E x400)

 

 

 

 

 

Figure 6.  Photomicrography showing no membrane reactivity of HER2 protein in low grade urothelial carcinoma. (Case No: 40, IHC for HER2x400)

 

 

 

 

 

Figure 7. Photomicrography showing negative Ki67 in low grade urothelial carcinoma. (Case No:40, IHC for Ki67 x400)

 

 

 

 

Figure 8. Photomicrography showing high grade urothelial carcinoma. (Case No: 12, H&E x400)

 

 

 

 

Figure 9.  Photomicrography showing no membrane reactivity of HER2 protein in high grade urothelial carcinoma (Case No:12, IHC for HER2x400)

 

 

 

 

Figure 10. Photomicrography showing positive Ki67 in high grade urothelial carcinoma. (Case No: 12, IHC for Ki67x400)

Discussion

In this study, maximum (58.0%) patients were below or equal to 60 years and 42.0% patients were more than 60 years old. mean age of the patients was 60.9 ± 13.1 and is similar to the study of Jawad, Ali and Kamal (2016) which was 58.72±1.6. In this study male to female ratio was found 4:1. Similar findings were also stated in the study of Jawad, Ali and Kamal, (2016).17 Male were predominant and This study shows the incidence of tumor is more in male (80.0%) than female (20.0%). Male to female ratio was found 4:1.

In our present study it was observed that majority (62.0%) of the patients came from low and 38.0% from middle socio economic condition and involved in different occupation. Two third of them were cultivator and only one patient worked in dye factory. Not only rapid industrialization and urbanization but also excess use of insecticide and fertilizer of the subcontinent particularly our country for the last few decades probably playing an important role for increasing incidence of UBC. Similar observation was also made by Kibria et al. (1997)18 in Bangladesh.

Maximum patients had habit of smoking and betel nut chewing. In this present study 72% patients had habit of smoking and betel nut chewing. Most of the male patients in this study had habit of both cigarette and betel nut and females had habit of betel leaf with betel nuts. Chinnasamy et al. (2016)19 revealed most of bladder cancer patients (71.2%) had smoking habit which was consistent with this study result. Chou et al. (2013)20 found 24.9% of urothelial cancer patients had smoking habit.

The histologic cell type of bladder cancer is geographically different. In our subcontinent urothelial carcinoma is the most common type. In our study, all the 50 (100%) cases were histologically transitional cell carcinoma. An Indian study in Kashmir by Jeelani et al. (2004)21 reported 98% TCC and 2% adenocarcinoma. A related study conducted by the Urology Department of BSMMU in 1088 patients of ten different hospital of Dhaka city, observed 96.7% TCC, 1.2% squamous cell carcinoma, 1.6% adenocarcinoma and 0.5% other type of urinary bladder cancer (Hossain, 2011).3 In another study in Egypt by Shawky (2013)10 reported 43.8% squamous cell carcinoma followed by 40.6% TCC. Jemal et al. (2008)22 found that the endemic infection with Schistosoma species in Africa and Egypt was responsible for squamous metaplasia and subsequently squamous cell carcinoma in urinary bladder.

The patients in this study were grouped according to WHO grading of urinary bladder carcinoma. It was observed that 36 (72.0%) patients had high grade urothelial carcinoma (HGUC) and 14 (28.0%) patients had low grade urothelial carcinoma (LGUC). Chou et al., (2013)20 in their study found 56.8% high grade and 43.2% low grade tumor.  Incidence of high grade UC patient was more in our study. In our country the probable cause may be poor economic condition, lack of knowledge, lack of urological facilities as well as social and religious restrictions especially for female patients which prevent them from utilizing hospital facilities.

The HER2/neu acts as an oncogene. HER2 expression was evaluated by immunohistochemistry in 50 cases of our study. Of the total 36 high grade Urothelial Carcinoma, HER2/neu expression was found (score3+) in 27.8% cases, equivocal (score 2+) in (30.6%) cases and rest (41.7%) were negative (score 0 & score 1+). No positive HER2/neu was observed in low grade. All the HER2/neu positive cases were found only in high grade cases but no positive expression was seen in low grade tumor.

Ki67 expression in high and low grade UC were 80.6% and 50% respectively. 7/36 (19.4%) showed negative expression of Ki67 in high grade UC. 7/14 (50%) low grade tumor showed positive expression of Ki67. Most of the positive Ki67 expression cases were found in high grade tumor.

10(100%) morphologically high grade tumor present score 3+ HER2/neu with positive Ki67 expression. 11(30.6%) high grade tumor showed equivocal expression of HER2/neu of which 90% showed Ki67 positivity.15 morphologically high grade tumor expressed Her2/neu negativity with 10(62.5%) positive Ki67 expression.

All Her2/neu positive cases were also Ki67 positive. Among the 12 HER2/neu equivocal cases, 09 show positive Ki67 expression. Ki67 also shows positive expression in both high and low grade UC that were HER2/neu negative. So to find out the accurate prognosis, Ki67 expression in low grade and HER2/neu in high grade must get proper attention.

Significant correlation was observed with different grading of UC according to 2014 grading system. Co expression of HER2/neu and Ki67 were observed in 10/36 high grade urothelial carcinoma which are aggressive in nature.

HER2/neu positive tumors can be benefited by Herceptin therapy. Low grade tumor with negative HER2/neu but high Ki67 may need more aggressive therapy. So to find out the accurate prognosis, Ki67 expression in low grade and HER2/neu in high grade must get proper attention. HER2/neu and Ki67 overexpression have a relationship with the grading of urothelial carcinoma and can be used to assess controversial cases. They can help us to estimate the accurate biological behavior of urothelial carcinoma to select the appropriate treatment protocol.

The expression profile of both biomarkers may be useful for the selecting high risk patients with bladder cancer for proper treatment. Hence patients who have a low risk of recurrence, need to identify in order to avoid over treatment as well as those who likely to progress in order to treat them more aggressively. In Bangladesh no study was conducted on both HER2/neu and Ki67 expression and association in urothelial carcinoma. This study could have been more effective if more number of urothelial carcinoma cases were included and follow up was done to see the progression of the disease and recurrence

Limitations

Reliability and reproducibility of IHC technique was a major limitation.

Fluorescent in situ hybridization (FISH) could not be done for the equivocal cases due to financial limitation.

Recommendation

Use of immunohistochemistry in urinary bladder carcinoma for the screening of high risk patients.

Second confirmatory test with FISH for equivocal cases in IHC.

Further study with more sample size with cystectomy specimen and with follow up.

References

  1. Seigel R, Ma J, Zou Z, Jemal A. Cancer statistics 2014.CA Cancer J Clin. 2014; 649-29.
  2. Parkin DM. The global health burden of urinary bladder cancer. Scand J Urol Nephrol Suppl, 2008; 218:12-20.
  3. Hossain MDA. 2011, Frequency of carcinoma of urinary bladder in bladder tissue samples collected from different tertiary level hospitals in Dhaka city- a retrospective study, MS (Urology) thesis, BSMMU, Dhaka, Bangladesh.
  4. Jamal A, Tiwari RC and Murray T. Cancer Statistics. CA Cancer J Clin 2004, 54:8-29.
  5. Zhao J, Xu W, Zhang Z, Song R, Zeng S, Sun Y et al. Prognostic role of HER2 expression in bladder cancer: a systematic review and meta-analysis. International urology and nephrology, 2015; 47(1):87-94.
  6. Kassouf W, Black PC, Tuziak T, Bondaruk J, Lee S, Brown GA et al. Distinctive expression pattern of ErbB family receptors signifies an aggressive variant of bladder cancer. The Journal of urology, 2008;179(1):353-358.
  7. Latif Z, Watters AD, Dunn I, Grigor KM, Underwood MA, Bartlett JM. 2003.HER2/neu overexpression in the development of muscle invasive transitional cell carcinoma of the bladder. Br J Cancer 2003; 89:1305-9.
  8. Burger M, van der Aa MN, van Oers JM, Brinkmann A, van der Kwast TH, Steyerberg EC et al.. Prediction of progression of non–muscle-invasive bladder cancer by WHO 1973 and 2004 grading and by FGFR3 mutation status: a prospective study. European urology, 2008; 54(4):835-844.
  9. Wang L, Feng C, Ding G, Zhou Z, Jiang H and Wu Z.. Relationship of TP53 and Ki67 expression in bladder cancer under WHO 2004 classification. J BUON, 2013;18(2):420-4.
  10. Shawky AEA, Elosaily G, Al-Matubsi H and Farahat A.. Her-2/Neu overexpression in invasive bladder carcinoma among a Cohort of Egyptian Patients. World Journal of Nephrology and Urology, 2013;2(2):70-75.
  11. Alexa A, Baderca F, Zahoi DE, Lighezan R, Izvernariu D, Raica M. Clinical significance of Her2/neu overexpression in urothelial carcinoma. Rom J Morphol Embryol. 2010; 51(2):277-282.
  12. Li R, Heydon K, Hammond ME, Grignon DJ, Roach M, Wolkov HB et al.. Ki-67 staining index predicts distant metastasis and survival in locally advanced prostate cancer treated with radiotherapy. Clinical Cancer Research, 2004;10(12):4118-4124.
  13. Margulis V, Shariat SF, Ashfaq R, Sagalowsky AI and Lotan Y. Ki-67 is an independent predictor of bladder cancer outcome in patients treated with radical cystectomy for organ-confined disease. Clinical cancer research, 2006;12(24):7369-7373.
  14. Quintero A, Alvarez-Kindelan J, Luque RJ, Gonzalez-Campora R, Requena MJ, Montironi R et al. Ki-67 MIB1 labelling index and the prognosis of primary TaT1 urothelial cell carcinoma of the bladder. Journal of clinical pathology, 2006;59(1):83-88.
  15. Hansel DE, Swain E, Dreicer R and Tubbs RR. HER2 overexpression and amplification in urothelial carcinoma of the bladder is associated with MYC coamplification in a subset of cases. American journal of clinical pathology, 2008; 130(2):274-281.
  16. Olsson H, Fyhr IM, Hultman P, Jahnson S. HER2 status in primary stage T1 urothelial cell carcinoma of the urinary bladder.Scand J Urol Nephrol 2012;46:102 -7.
  17. Jawad NA, Ali HH, Kamal MS. Her2/ Neu and Ki-67 Immunohistochemical Expression in Transitional Cell Carcinoma of the Urinary Bladder (A Clinicopathological Study). Journal of Dental and Medical Sciences, 2016; 15(2);6-12.
  18. Kibria SAMG, Islam MF, Hasan MS, Wahab ANM. Management of carcinoma of urinary bladder. Eight-year experience in a teaching hospital. J Dhaka Medical College, 1997; 6(1): 12-14.
  19. Chinnasamy R, Krishnamoorthy S, Joseph L, Kumaresan N and Ramanan V. Clinico-pathological Study of Bladder Cancer in a Tertiary Care Center of South India and Impact of Age, Gender, and Tobacco in Causing Bladder Cancer: A Single Center Experience. International Journal of Scientific Study, 2016;3(10):72-77.
  20. Chou YH, Chang WC, Wu W, Li CC, Yeh HC, Hou MF et al. The association between gender and outcome of patients with upper tract urothelial cancer. The Kaohsiung Journal of Medical Sciences, 2013; 29(1):37-42.
  21. Jeelani G, Waseem Qureshi, MD, Khan, NA, Mohammad Shafi, MS, Mumtaz-ud-Din, MS, Shabnam Khan, MBBS et al. 2004. Pathology of Bladder Tumors in Kashmir.
  22. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA: a cancer journal for clinicians, 2008;58(2):71-96.

Histomorphological Pattern of Childhood CNS Tumor: An Experience at National Institute of Neurosciences & Hospital, Bangladesh


Histomorphological Pattern of Childhood CNS Tumor: An Experience at National Institute of Neurosciences & Hospital, Bangladesh

 *Huq N,1 Haque ME,2 Baqui MN,3 Yusuf A,4 Islam N5

Abstract

CNS tumors in childhood differ considerably from adult in term of geographic distribution, histological patterns, clinical and therapeutic aspects, prognosis and outcome. There is a paucity of study about the distribution pattern of CNS tumors in pediatric age group in Bangladesh. National Institute of Neurosciences & Hospital (NINS&H) is a tertiary health care hospital in Bangladesh dealing with the neurological diseases having a well developed Pediatric Neurosurgery department. The purpose of the present study was to see the histomorphological pattern of childhood CNS tumors and the frequency distribution in Bangladesh. We analyzed the data compiled from CNS lesions biopsied in Pediatric Neurosurgical department and reported from the Department of Neuropathology of NINS&H during the time period of June 2013 to April 2016. A total of 239 cases of pediatric CNS lesions were included in this study. 180 cases were CNS tumors and 59 were tumor like lesions which was not further analyzed. The mean age was 10.30±5.48 years. Most of the patients were in 10 to 15 years age group. A slight male predominance was seen. Out of 239 cases 140 were intracranial and 50 were spinal. There were (103, 43.5%) supratentorial lesions and (37, 15.6%) infratentorial lesions. Grade I tumor (39.3%) was most frequent followed by grade IV (14.6%). Within the grade I tumor, Pilocytic astrocytoma was the most common variant (33, 36.3%) followed by Craniopharyngioma. Medulloblastoma was the common tumor (13, 38.2%) in grade IV group followed by Central Primitive Neuroectodermal tumor (PNET). Supratentorial tumors were more common   than infratentorial. Pilocytic astrocytoma was the commonest pediatric CNS tumor. Craniopharyngioma, Ependymoma, Medulloblastoma and PNET were next in frequency.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):92-98]

 Keywords: Childhood CNS tumor, pilocytic astrocytoma, medulloblastoma

 

  1. *Dr. Naila Huq, Associate Professor (Pathology), Department of Neuropathology, National Institute of Neurosciences & Hospital, Dhaka. nailahuqpopy@gmail.com
  2. Mohammed Enamul Haque, Junior Consultant, General Hospital, Munshigonj.
  3. Muhammad Nazmul Baqui, Senior lecturer, Unit of Pathology, Faculty of Medicine, AIMST University, Kedah, Malaysia
  4. Abdullah Yusuf, Assistant Professor, Microbiology, National Institute of Neurosciences & Hospital, Dhaka.
  5. Professor Nowfel Islam, Head of the Department, Department of Neuropathology, National Institute of Neurosciences & Hospital, Dhaka.

 

*For correspondence

 

Introduction

Primary CNS lesions are a varied group of lesions occurring in brain and spinal region. It includes a wide variety of diseases ranging from neoplastic lesions to infectious diseases as well as some cystic lesions common in CNS.1 Brain tumor in childhood differs considerably from adult in term of distribution, histological patterns, clinical and therapeutic aspects, prognosis and outcome.2,3,4 The incidence of childhood brain tumors varies greatly throughout the world depending on its type. National Institute of Neurosciences & Hospital is the one of the referral centre in Bangladesh dealing with the neurological diseases both for pediatric and adult patients. Reports on the pattern of childhood CNS tumors in Bangladesh are rare.  We analyzed the data compiled from CNS lesions biopsied in Neurosurgical Department and reported from the Department of Neuropathology of NINS&H during the time period of June 2013 to April 2016. Although exact incidence cannot be provided by a hospital based study, but the information derived from this study will be useful in showing pattern of childhood CNS tumors in our regions. It will have implications for future research, treatment and prognostic factors.  It will provide information about the extent of diseases, address the weight of the problem and finally it will help to design the human resources and fund needed to face the problem. The etiology of CNS tumors is mostly unknown. Some CNS tumors have proven associations with some genetic and environmental factors.3,4,5 So the study will be also helpful to decline the disease prevalence in our country by prenatal diagnosis and molecular treatment.  The objectives of this study are to determine the histomorphological pattern of CNS lesions and the frequency distribution.

Methods

The present study is based on the data collected from the Neuropathology department of NINS &H. The study is a retrospective analytical study. The study included all the patients up to eighteen years of age having CNS lesions and has biopsy proven diagnosis during the period of June 2013 to April, 2016. Skin, soft tissue, bony tumors and tumor like lesions were excluded from the study. All the samples were stained by routine Hematoxyline & Eosin stain. Beside H& E stain, immunohistochemistry was performed in only selective cases where there was diagnostic dilemma. Histological diagnosis and grading of the tumor was based on the, “WHO classification of the tumor of the CNS” and other reference books of histopathology.6,7,8,9

 Result

A total of 239 cases of pediatric CNS lesions biopsied in the Neurosurgical department of NINS&H were included in the study. The mean age at diagnosis was 10.30±5.48   years with age range of 3 months to 18 years. Children were stratified into four age groups: Group I (0-5); group 2 (5-10); group III (10-15); group IV (15-18) years. Most of the patients were in 10 to 15 years age group and least number of patients in 5 to 10 years.

Table I: Age distribution among the study population

 

Age Group Frequency Percent
Less Than 5 Years 60 25.1
5 to 10 Years 48 20.1
10 to 15 Years 82 34.3
More Than 15 Years 49 20.5
Total 239 100.0
Mean±SD (Range) 10.30±5.488 (1-18)

 

The study shows a slight male predominance with a male to female ratio of 1.4:1.

 Anatomical location of the lesions

In this study out of 239 cases 140 were intracranial and 50 were spinal.  Exact site was not mentioned in 49 cases.  There were (103, 43.5%) supratentorial lesions and (37, 15.6%) infratentorial lesions. Among the supratentorial location sellar lesions (28, 28%) were most common and in case of infratentorial site posterior fossa (24, 63.2%) lesions were commonest.

 Histological types of the lesions

The tumors were sub grouped into grade I to IV according to WHO grading system. The most commonly encountered group was grade I tumor (39.3%) followed by grade IV (14.6%) and grade II(16.7%) There was only 4.4% of grade III tumor. In 7.2% cases tumor grade cannot be determined due to inadequate biopsy material, technical error, type of tumor and some other causes.

Pilocytic astrocytoma was the most common variant (33, 36.3%) followed by Craniopharyngioma(19,7.9%) and  Ependymoma (17,7.1%). Medulloblastoma (13, 38.2%) and PNET (11,4.6%) was next in frequency which was grade IV tumor . Fibrillary astrocytoma(8,3.3%), Gemistocytic astrocytoma, Nerve sheath tumor, Meningioma, Hemangioma and other tumors were also found in pediatric age groups in different frequency. Even Glioblastoma(4,1.7%) was not uncommon in this age .

Table II: Distribution of Different Grades of Tumor

 

Grade of Tumour Frequency Percent
Grade I 94 52.2
Grade II 30 16.7
Grade III 8 4.4
Grade IV 35 19.4
Undetermined Grade 13 7.2
Total 180 100.0

 

Table III: Distribution of Different Tumors according to frequency

 

TYPES Frequency Percentage
Pilocytic astrocytoma 33 13.8%
Craniopharyngioma 19 7.9%
Ependymoma 17 7.1%
Medulloblastoma 13 5.4%
PNET 11 4.6%

 

A B

 

Fig 1A. Photomicrograph showing pilocytes and Rosenthal fibres in a case of Pilocytic astrocytoma.
1B. MRI showing hyperintense mural nodule with a large cyst

 

 
A B

 

Fig 2A. Photomicrograph showing nests of squamous epithelial cells with peripheral palisading by columnar cells  in a case of Craniopharyngioma.

2B. MRI showing cyst with hyperintense contrast enhancement in solid area.

 

 

A B

Fig 3A. Photomicrograph showing nodules of undifferentiated ells with zones of  reduced cellularity in a case of Medulloblastoma.

3B. MRI showing homogenous enhancement in posterior fossa

 

A B

 

Fig 4A. Photomicrograph showing sheets of poorly differentiated cells.

4B. Immunohistochemistry showing positive reaction for Synaptophysin

 Discussion

The present study was designed to determine the spectrum of childhood CNS tumors, their  site, grade and morphological pattern. In many of the cases tumor like lesions cannot be differentiated preoperatively by clinical and radiological evaluation. We excluded these cases from our study. There have a paucity of publications regarding CNS lesions in Bangladesh.

Slight male predominance is found in this study (1.4:1). Other study of Asian region also found a high male to female ratio.2,4, 10

Present study revealed most cases in 10 to 15 years age group with a mean age of   10.30±5.48 years which is higher than other studies. The variation may be due to difference in sample size and selection of cases. This study defines pediatric age group up to 18 years of age whereas most of the study includes 14 or 15 years of age.2,3,4 In this study most of the tumors were supratentorial (43.5%) and only (15.6%) were infratentorial. But other study showed that infratentorial tumors are most common in childhood.11 As it is a hospital based study and NINS & H is a referral and research oriented hospital there are some selection bias. Some national and international workshop occurs in NINS&H regarding newer technological approach addressing tumor of some special site. This may be the cause of high sellar lesion in this study.  However Pollack, 1999 found higher supratentorial tumor then infratentorial. Among the sellar lesion Craniopharyngioma was commonest.

Pattern of primary brain tumor in children differs significantly from adult. In this study tumors are further subdivided according to WHO grading system into 4 grades. However there are some tumors in which cases grade cannot be determined as Pituitary adenoma. We found that grade I tumor is more common in this age group (52.2%) followed by grade IV (19.4%). Among the grade I tumor Pilocytic astrocytoma (33, 13.8%) is the commonest tumor having better prognosis followed by Craniopharyngioma (19, 7.9%). In case of grade-II tumors, Ependymoma is (17, 7.1%) common in childhood. Medulloblastoma have a high frequency (13, 5.4%) followed by Central PNET (11, 4.6%) among the grade IV tumors. Jahan et al.4 also found Medulloblastoma as the commonest childhood CNS tumor followed by Ependymoma in Bangladesh. That study was done with a small sample size in comparison to the present study. On the other hand other publications noted Astrocytoma  as the commonest childhood CNS tumor.10,12,13

 Conclusion

The study reflects supratentorial region as the commonest site and Pilocytic astrocytoma as the commonest pediatric CNS tumor. Craniophayngioma, Ependymoma, Medulloblastoma, PNET are next in frequency. The current study is a single institutional study. A population based study including a larger sample size and long study period is required to determine the tumor burden and histopathological pattern of childhood CNS tumor in Bangladesh.

References

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  7. Rosenblum MK.Central nervous system. In: Rosai J. Ed. Rosai and Ackermanss Surgical Pathology 10th St Louis, Mo. Elsevier. 2010.Vol-2, pp23
  8. Frosch MP, Anthony DC, Girolami UD. The Central Nervous System in: Robbins and Cotran pathologic Basis of Disease. 8th Elsevier.2012, pp 1279-1342.
  9. Burger PC and Scheithauer BW, Tumours of the central nervous system.In: Rosai J, ed.Atlas of Tumor Pathology,3rd series, Fascicle 10.Armed Forces Institute of Pathology, Washington DC. 1994; 25-952.
  10. Deopujan CE, Kumar A, Karmakar VS et al. Paediatric suprasellar lesions. J Pediatr Neurosci. 2011; 6(Suppl1): S46–S55.
  11. Katchy KC, Alexander S, Al-Nashmi N M et al. Epidemiology of primary brain tumors in childhood and adolescence in Kuwait. Pp 1-8.
  12. Siqueira EB, Rahm B, Kanaan I, and Jallu A. Brain tumors in pediatric patients at king faisal specialist hospital and research centre. Surgical neurology 1993; 39(6): 443-450.
  13. Mckinney P. A, Parslow R.C, Lane. S. A et al. Epidemiology of childhood brain tumors in     Yorkshire, UK, 1974-95: geographical distribution and changing patterns of occurrence.  Br J Cancer. 1998; 78(7): 974-979.

Study of ER, PR and Ki-67 Expression in Different Histopathological Pattern of Endometrial Hyperplasia



Study of ER, PR and Ki-67 Expression in Different Histopathological Pattern of Endometrial Hyperplasia

*Fardousi F,1Sultana SS,2 Kaizer N,3 Dewan RK,4 Jinnah MS,5 Jeba R,6 Haque WS,7 Alam SM.8 Hussain M9

Abstract
Endometrial hyperplasia is one of the major gynaecological problem in peri and postmenopausal women worldwide. It deserves special attention because of its relationship with endometrial carcinoma. To study the histopathological pattern of endometrial hyperplasia in peri and postmenopausal women and their association with ER, PR and Ki-67 expression by immunohistochemistry is essential for early diagnosis with effective treatment. Out of 70 endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. ER expression in majority (35) of the cases of endometrial hyperplasia was between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia express ER less than 10%. The PR expression in majority of the cases (37) of endometrial hyperplasia was between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia expressed PR less than 40%. Ki-67 expression in majority of the cases of all types of endometrial hyperplasia 32 (78%) was less than 35%. All the cases (24) of simple endometrial hyperplasia without atypia had less than 35% Ki-67 expression. Only 6 cases had Ki-67 expression of more than 35%. In this study, 5 cases had negative (0%) Ki-67 expression. It can be inferred that after evaluating the ER, PR and Ki-67 expression, conservative treatment with progestogen and GnRH-agonists can be effective in selected cases of simple endometrial hyperplasia without or with atypia and complex endometrial hyperplasia without atypia.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):74-84]

 Key words: Endometrial hyperplasia, Gynecological problem, Peri and postmenopausal women, Histopathological pattern, Immunohistochemistry, ER, PR and Ki-67 expression

 

  1. *Dr. Farzana Fardousi, Lecturer, Department of Cytopathology, National Institute of Cancer Research and Hospital (NICRH), Mohakhali, Dhaka. farzanafardousi@yahoo.com.
  2. Sk Salowa Sultana, Assistant Professor, Department of Pathology, Ad Din Women’s Medical College. Dhaka.
  3. Nahid Kaizer, Associate Professor (CC), Department of Pathology, Shahabuddin Medical College.
  4. Professor Dr. Rezaul Karim Dewan, Head of the Department of Pathology, Dhaka Medical College.
  5. Mohammed Shahed Ali Jinnah, Associate Professor, Department of Pathology, Dhaka Medical College.
  6. Ruksana Jeba, Associate Professor, Department of Pathology, Dhaka Medical College.
  7. Lt Col (Dr) Wasim Selimul Haque. Classified spl in Pathology, Armed Forces Institute of Pathology.
  8. Prof Brig Gen Dr. SM. Mahbubul Alam, Ex Deputy Commandment, Armed Forces Institute of Pathology, Senior consultant- Histopathology, Apollo Hospitals Dhaka.
  9. Professor Dr. Maleeha Hussain, Ex Head of the Department of Pathology, Dhaka Medical College.

 

*For corespondence

 Introduction
Endometrial hyperplasia has a high risk for malignant transformation into endometrial carcinoma in peri and postmenopausal women.1,2 Eendometrial hyperplasia usually develops due to the continuous estrogen stimulation unopposed by progesterone. In the years before menopause, women may have numerous cycles without ovulation (anovulatory) during which there is sustained and unopposed estrogen activity. It is also likely that hormone replacement therapy (HRT) consisting of estrogen without progesterone may lead to endometrial hyperplasia.3 The postmenopausal endometrium which  despite being atrophic, retain a weak proliferative pattern for many years probably as a response to continuous low level of estrogenic stimulation. These are at a higher risk of progression to endometrial hyperplasia and subsequently to endometrial malignancy. Although the diagnosis of endometrial hyperplasia can be made by histopathological examination; the immunohistochemistry aids for the prognosis, in order to establish the best treatment.4

The receptor status for estrogen and progesterone in endometrial hyperplasia can be a prognostic indicator in the treatment. The expression of estrogen and progesterone receptors varies in different types of endometrial hyperplasia. Several studies have found that, all types of hyperplasia present a smaller number of receptors compared to the endometrium in proliferative phase, but higher compared with secretory endometrium and endometrial carcinoma.5 The proliferative activity of endometrial hyperplasia can be examined by using an antibody to Ki-67 antigen, a non-histone protein, a well established marker of proliferative activity. The expression of the human Ki-67 protein is strictly associated with cell proliferation.

The endometrium of reproductive-aged women undergoes cyclic developmental changes in response to the steroids – estrogen and progesterone. The highest score of estrogen and progesterone receptors are observed in the epithelial and stromal cells of the normal uterine endometrium at the early proliferative phase; then, throughout the secretory phase, the ER and PR scores decline. Again, the highest score of estrogen receptors and progesterone receptors are observed in non-atypical hyperplasia and lowest score of estrogen receptors and progesterone receptors are observed in atypical hyperplasia.6

Ferrandina et al. (2005)7 found that, ER and PR positive cases of endometrial hyperplasias had a statistically significant association with the clinicopathological parameters, which correlates with a more favorable prognosis. In addition, the hormone receptor status appears to correlate with the treatment response to the progesterone therapy. This finding may be of particular clinical importance, since almost all endometrial hyperplasia cases contain estrogen/ progesterone receptors and progesterone therapy could be beneficial in this cases.8

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells G(0), makes it an excellent marker for determining the so-called growth fraction of a given cell population. The highest score of Ki-67 expression is observed in atypical hyperplasia and lowest score of Ki-67 expression is observed in non-atypical hyperplasia. Scholzen and Gerdes, (2009)9 in their study showed that, cell proliferation is highly correlated with Ki67 expression.

Perez-Medina et al have observed that, ER, PR and Ki-67 expression rate, along with the histopathological features of endometrial hyperplasia are important prognostic factors.10 After evaluating the ER, PR and Ki-67 expression rate, they have shown that, for all cases of endometrial hyperplasia, the general treatment protocol i.e. hysterectomy is not rationally justified. Patients who want to complete their family or who have heart disease or surgical or anaesthetic risk history, if they have positive ER, PR expression and low Ki-67 expression; they can be selected for conservative treatment with progestogens and GnRH-agonists. These patients should be followed for 5 years by biopsies every 6 months.

Hysterectomy is usually recommended for cases with endometrial hyperplasia.2 Several studies have revealed that less than 5 % of simple and complex non-atypical hyperplasia cases undergo malignant transformation in the long term (20 years), whereas this percentage increases to 30 % when there is an atypical hyperplasia.11 So, the rationality of hysterectomy in the treatment of endometrial hyperplasia in general needs to be evaluated.

The evaluation of receptor status by markers for hormone receptors (estrogen and progesterone receptors) and proliferative activity by proliferative marker (Ki-67) in patients with endometrial hyperplasia can predict the treatment option in selected cases.12 -16

 Methods
This is a descriptive cross sectional study which was carried out at the Department of Pathology, Dhaka Medical College, and Dhaka during the period of January 2013 to December 2014. A total of 110 peri and postmenopausal women with dysfunctional uterine bleeding or postmenopausal bleeding who underwent D&C or hysterectomy were screened.  A total of seventy histopathologically diagnosed cases of endometrial hyperplasia who met the enrollment criteria (inclusion & exclusion criteria) were included in this study. Among 70 cases, endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Routine Hematoxylin and Eosin staining was done on all 70 samples. Out of these 70 cases, ER, PR and Ki-67 immunostaining was done on 41 cases. Ethical clearance was taken for this study from institutional ethical committee of Dhaka Medical College. Each patient was interviewed before collection of the specimen and relevant information was recorded in a prescribed clinical proforma. Detail history with particular attention to age, clinical features, age at menarche, parity, obesity, history of contraceptives, history of hormone replacement therapy, history of diabetes, history of hypertension, history of estrogen producing ovarian tumor, age at menopause were taken.

 Histopathological examination

The gross examination of specimens, routine tissue processing and Hematoxylin & Eosin staining were done at the Department of Pathology, Dhaka Medical College.

 Microscopic analysis

According to WHO classification endometrial hyperplasia is classified into simple endometrial hyperplasia without atypia, Simple endometrial hyperplasia with atypia, Complex endometrial hyperplasia without atypia, Complex endometrial hyperplasia with atypia.17

 Immunohistochemical examination

Immunostaining for ER, PR and Ki-67 was done at AFIP (Armed Forces Institute of Pathology, Dhaka). A total of 41 cases were selected for immunohistochemical examination for ER, PR and Ki-67 expression. For immunostain, 3cases of proliferative phase of endometrium, 3 cases of secretory phase of endometrium and 3 cases of well differentiated endometrial carcinoma were enrolled as control.

All the data were recorded in data sheet along with patient’s clinical findings. The main objectives of this study were to observe the histopathological pattern of endometrial hyperplasia in peri and postmenopausal women and their association with ER, PR and Ki-67 expression by immunohistochemical method.

1) Primary antibody –Mouse monoclonal Anti-Human Estrogen Receptor, clone 1D5, code M7047(1:60 dilution), Mouse monoclonal Anti-Human Progesterone Receptor, clone PgR636(1:50 dilution) and Mouse monoclonal antibody against ki67 antigen(1:100 dilution)  were used as primary antibody.

2) Secondary antibody – Envision (ready to use, Dako), was used as secondary antibody.

3) Positive control –

  1. High grade breast carcinoma was taken as positive control for ER and PR.
  2. Follicular hyperplasia of lymph node was taken as positive control for Ki-67.

 Immunohistochemical analysis

For ER and PR expression in endometrial tissue: 18

Score for proportion staining Score for staining intensity
1. 0-25 % nuclei 1. Absent or weak staining
2. 26-75 % nuclei 2. Strong staining
3. ≥ 76 % nuclei 3. Very strong staining

 

Cat – I Total score 2 Immuno negative
Cat – II Total score 3-4 Immuno reactive
Cat – III Total score 5-6 Immuno reactive

For Ki-67 expression in endometrial tissue:

The patients are divided into two groups:

  1. Low Ki-67 expression (≤ 35 % cells are Ki-67 positive)
  2. High Ki-67 expression (> 35 % cells are Ki-67 positive)

(Nuclear staining in endometrial glandular epithelial cells are evaluated)

 Statistical analysis

Statistical analyses of the results were obtained by using window based computer software devised with Statistical Packages for Social Sciences (SPSS-16). Percentages were calculated to find out the proportion of the findings. The results are presented in Tables and Figures.

 Results
A total of 70 histopathologically diagnosed endometrial hyperplasia cases were included in this study.  Among 70 cases endometrial curettage biopsy specimens were 45 and hysterectomy specimens were 25. Out of all endometrial hyperplasia cases, 53 cases were simple endometrial hyperplasia without atypia, 8 cases were simple endometrial hyperplasia with atypia, 6 cases were complex endometrial hyperplasia without atypia and 3 cases were complex endometrial hyperplasia with atypia. Out of these all the 8 cases of simple endometrial hyperplasia with atypia, 6 cases of complex endometrial hyperplasia without atypia, 3 cases of complex endometrial hyperplasia with atypia and 24 cases of simple endometrial hyperplasia without atypia.

Table I: Distribution of the study patients by immunohistochemistry findings (ER %) (n=50)

 

Immunohisto-chemistry
findings (ER %)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
≤10 0 1 0 0 0 3 1 5 10.0
11-20 0 0 0 0 0 0 1 1 2.0
21-30 0 0 0 0 0 0 1 1 2.0
31-40 0 1 0 0 0 0 0 1 2.0
41-50 0 0 3 0 0 0 0 3 6.0
51-60 0 0 2 2 0 0 0 4 8.0
61-70 0 1 2 0 1 0 0 4 8.0
71-80 0 0 10 0 0 0 0 10 20.0
81-90 2 0 5 5 4 0 0 16 32.0
91-100 1 0 2 1 1 0 0 5 10.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

 Distributions of the patients with PR expression (%). PR expression in majority of the cases (37) of endometrial hyperplasia is between 51-100%. All the cases (3) of complex endometrial hyperplasia with atypia express PR less than 40% (Table II).

 Table II: Distribution of the study patients by immunohistochemistry findings (PR %) (n=50)

 

Immunohist-ochemistry
findings (PR %)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
≤10 1 0 0 0 0 0 0 1 2.0
11-20 0 0 0 0 0 2 1 3 6.0
21-30 0 0 0 0 0 0 0 0 0.0
31-40 0 0 1 0 0 1 0 2 4.0
41-50 0 0 0 0 0 0 0 0 0.0
51-60 0 0 3 0 0 0 0 3 6.0
61-70 0 0 2 0 0 0 1 3 6.0
71-80 0 0 6 0 0 0 0 6 12.0
81-90 2.0 1 6 5 1 0 1 16 32.0
91-100 0 2 6 3 5 0 0 16 32.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

Table-III shows distributions of the patients with Ki-67 expression (%). Ki-67 expression in majority of the cases of all types of endometrial hyperplasia 32(78%) is less than 35%.

 Table III: Distribution of the study patients by immunohistochemistry findings (Ki-67%) (n=50)

 

Immunohisto
-chemistry
findings (KI-67%)
PP

(n=3)

SP

(n=3)

SEH without atypia

(n=24)

SEH with atypia

(n=8)

CEH without atypia

(n=6)

CEH with atypia

(n=3)

End. Ca. grade 1

(n=3)

Total

(n=50)

n %
0 0 0 4 0 1 0 0 5 10.0
1-10 0 2 10 1 1 0 0 14 28.0
11-20 0 1 1 2 2 0 0 6 12.0
21-30 0 0 3 0 1 0 0 4 8.0
31-35 0 0 6 3 0 0 1 12 24.0
36-50 0 0 0 0 1 2 0 1 2.0
51-60 1 0 0 2 0 1 1 5 10.0
61-70 0 0 0 0 0 0 0 0 0.0
71-80 1 0 0 0 0 0 0 1 2.0
81-90 1 0 0 0 0 0 0 1 2.0
91-100 0 0 0 0 0 0 1 1 2.0
Total 3 3 24 8 6 3 3 50 100

(PP- Proliferative phase of endometrium, SP- Secretory phase of endometrium, SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia and End. Ca. grade 1- Endometrial carcinoma Grade-I)

(SEH without atypia- Simple endometrial hyperplasia without atypia, SEH with atypia- Simple endometrial hyperplasia with atypia, CEH without atypia – Complex endometrial hyperplasia without atypia, CEH with atypia- Complex endometrial hyperplasia with atypia I)

 

Figure 1.  Pie chart showing distribution of the patients by diagnosis (n=70)

Pie chart showing the commonest diagnosis in 70 patients was SEH without atypia (75.7%) followed by SEH with atypia (11.4%) (Fig 1).

Discussion
Endometrial hyperplasia has a significant place in gynecological morbidity in women of reproductive age (10% to 18%).19 Endometrial hyperplasia is associated with menstrual irregularities and anaemia in women and poses a high risk for malignant transformation into endometrial cancer.20 World wide endometrial cancer is the most common gynecological cancer in peri and postmenopausal women.21,22 The incidence of endometrial adenocarcinoma not only has remained high but in recent years has tended to significantly increase in many countries, including Bangladesh.19,23-32

In this study, the commonest diagnosed lesion was simple endometrial hyperplasia without atypia which was 53(75.7%) followed by simple endometrial hyperplasia with atypia 8(11.4%), complex endometrial hyperplasia without atypia 6(8.6%) and then complex endometrial hyperplasia with atypia 3(4.3%).

From the present study it was observed that, in all types of endometrial hyperplasia except complex endometrial hyperplasia with atypia, the ER expression was lower than proliferative phase but higher than secretory phase of endometrium. However, the ER expression in complex endometrial hyperplasia with atypia was lower than any other type of endometrial hyperplasia or proliferative phase or secretory phase of endometrium. It was even lower than endometrial carcinoma. Similar study done by Ilie et al. (2011)33 also found that the ER expression in different types of endometrial hyperplasia and endometrial cancer is lower than proliferative phase but higher than secretory phase of endometrium.33 Found that, the expression of ER in different types of endometrial hyperplasia was much lower (41.5%) than the present study (75%) except that of complex endometrial hyperplasia with atypia. This variation may be due to inclusion of menopausal patients in their study.

In this study it was observed that, the mean ER expression was 75±15.1% in simple endometrial hyperplasia without atypia. Similar observations was found by Goncharenko et al. (2013)20 they have found 75.6% ER expression in simple endometrial hyperplasia without atypia. This may be due to inclusion of perimenopausal women in their study like present study. It was also observed that the mean ER expression was 10±0% in complex endometrial hyperplasia with atypia in the current study. These findings differed from Goncharenko et al. (2013)20 they found 65.2% ER expression in complex endometrial hyperplasia with atypia. This variation may be due to inclusion of many patients in their study at their reproductive age.

From the present study it was observed that, the  PR expression  in all types of endometrial hyperplasia except complex endometrial hyperplasia with atypia, was higher than proliferative phase of normal endometrium and endometrial carcinoma ,but they were lower than secretory phase of normal endometrium. However, the PR expression was lowest in complex endometrial hyperplasia with atypia.  In the study done by Ilie et al. (2011)33 found that, the PR expression in different types of endometrial hyperplasia was lower than proliferative phase but higher than secretory phase of endometrium and endometrial carcinoma. This variation may be due to inclusion of only menopausal women in their study. In this current study it was observed that, the mean PR expression was 80.8±16.4% in simple endometrial hyperplasia without atypia. This finding was nearly similar to the observations found by Goncharenko et al. (2013).20 They have found 69.3% PR expression in simple endometrial hyperplasia without atypia as they also included perimenopausal patients in their study.

It was observed in our study that the mean PR expression was 25±8.7% in complex endometrial hyperplasia with atypia. These findings differed from Goncharenko et al. (2013)20 they found 44.3% PR expression in complex endometrial hyperplasia with atypia. This may be due to inclusion of many patients in their study at their reproductive age. This result is in concordance with that of, Uchikawa  et al. (2003)5 in which it was found that, the expression of ER and PR decreased in endometrial hyperplasia compared with normal proliferative endometrium.

In present study, the expression of Ki-67 was compared with proliferative phase (73.3±12.6%) and secretory phase (12.3±5.8 %) of normal endometrium and with endometrial carcinoma, grade I (66.7±30.5%). We found that, the presence of mitotic activity in normal endometrium was observed more in the proliferative phase than the secretory phase. Mitotic activity in neoplastic and hyperplasic endometrium was low compared with proliferative phase of normal endometrium, but more than the endometrium in secretory phase.This findings are similar to the study done by Ilie et al. (2011).33

In the present study, the mean Ki-67 expression was 36.3±18.5% in simple endometrial hyperplasia with atypia. This finding are nearly similar to the study done by Goncharenko et al. (2013)20 who found upto 50% Ki-67 expression in simple endometrial hyperplasia with atypia. This may be due to inclusion of perimenopausal patients like our study. The present study result is in concordance with that of Uchikawa et al. (2003)5 in which they found that, ki-67 expression was more in atypical hyperplasia (50%) than non-atypical hyperplasia (10%).

In patients with simple endometrial hyperplasia with atypia and complex endometrial hyperplasia without atypia, Ki-67 expression can predict the treatment protocol. The patients with low Ki-67 expression can be selected for conservative treatment with hormone therapy and with high Ki-67 expression should undergo hysterectomy.  In complex endometrial hyperplasia with atypia, they should preferably be treated by hysterectomy operation. If, patients who want to complete their family and have low Ki-67 expression, they can be temporarily treated by conservative treatment. After delivery, they must undergo surgical intervention.16

The treatment response with hormone therapy depends on expression of ER & PR. So, before hormonal intervention, ER & PR should be evaluated, because in few cases, where expressions are low, have the chance of poor response to treatment. 10

 Limitation of the study

The study population was selected from the department of Pathology of Dhaka Medical College Hospital. But there are many patients with endometrial hyperplasia who are attending other hospitals than DMCH. Therefore, the sample lacks representation of the population. Thus, the study place was selected purposively and the respondents, those are interviewed, were attended a particular department of a specific hospital.

 Conclusion

The study revealed that highest ER & PR expression were observed in complex endometrial hyperplasia without atypia and lowest ER & PR expression were observed in complex endometrial hyperplasia with atypia. Again, highest Ki-67 expression was observed in complex endometrial hyperplasia with atypia and lowest Ki-67 expression was observed in simple endometrial hyperplasia without atypia. In complex endometrial hyperplasia with atypia, if patient desires to complete the family, after evaluation of ER, PR and Ki-67 expression, conservative treatment can be given and allowed to conceive. After delivery, if Ki-67 expression remains high, then hysterectomy must be done.

 Recommendations

A large follow–up study is recommended for patients of endometrial hyperplasia selected for conservative treatment with progestogen and GnRH-agonists. Monitoring should be done by observing the Ki-67 expression in these patients. If the Ki-67 expression increases, they should be treated by surgical intervention.

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