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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

  1. Kumar V, Abul K Abbas, Nelson F, Jon C Aster. The Gastrointestinal Tract, Robbins and Cotran Pathologic Basis of Disease, 8th edition,Saunders, 2010;17: 822-826.
  2. Uzma N, Nagi A.H, Waqas S. Correlation of Ki 67 with histological grade, stage in colorectal carcinoma. Dept of pathology,University of health science, Lahore  Pakistan, J Ayub Med Coll, 2008;20(4):44-8.
  3. Bisgaard ML, Jager AC, Myrhoj T, Bernstein I, Nielsen FC. Hereditary non-polyposis colorectal cancer (HNPCC): phenotype-genotype correlation between patients with and without identified mutation. 2002; 20(1):20-7.
  4. Andrew RH, Hongmei N. Epidemiology of colorectal cancer. Int Journal of Molecular Epidemiology and  Genetics, 2016;7(3):105–114.
  5. Soreide K, Janssen MA, Soiland H, H, Baak APJ. Microsatellite  instability in colorectal cancer. Br J Surg, 2006; 93(4):395-406.
  6. Sayed A H, Richard S. Cancer control in Bangladesh. Jpn J Clin Oncol, 2013; 43(12):1159-1169.
  7. Alice G, Davies A, Liz G, Evropi T, Harry Cl. Estimating the incidence of colorectal cancer in  Sub-Saharan Africa: A systematic analysis. J Glob Health, 2012;2(2):020404.
  8. Ishida H, Sadahiro S, Suzuki T, Ishikawa K, Kamijo A, Tajima T. Proliferative, infiltrative, and metastatic activitie s in colorectal tumors assessed by MIB-1 anti-body. 2003;10(6):1741-5.
  9. Sobin LH, Godspodarowicz MK, Wittekind C. UICC TNM Classification of Malignant Tumours. 7th edition. Wiley-Blackwell, 2009;12(4):100-105.
  10. Gordon PH, Nivatvongs S. Missouri: Karen Berger, Principles and practice of surgery for the colon, rectum  and anus.1992;23(3):503–520.
  11. Mokrý J, Nĕmecek S. Immunohistochemical detection of proliferative cells’,Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove.1995;38(3):107-.130.
  12. Oshima FTC, Iriya K, Forones MN. Ki-67 as a prognostic marker in colorectal cancer but not in gastric cancer. Neoplasm, 2005;5(4):420-4.
  13. Parkin D, Bray F, Ferlay. Global cancer statistics. CA Cancer J Clinic, 2005;55(2):74-108.
  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.
  19. Valera V, Yokoyama N, Walter B, Okamoto H, Suda.T, Hatakeyama K. Clinical significance of Ki-67 proliferation index in disease progression and prognosis of patients with resected colorectal carcinoma’. Br J Surg 2005;92:1002-7.
  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.
  21. El-Bolkainy TN, Sakr MA, Nouh AA, El-Din NH. A comparative study of rectal and colonic carcinoma: demographic, pathologic and TNM staging analysis. J Egypt Natl Canc Inst, 2006;18: 258-263.
  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.
  27. Saleh HA, Jackson H, Khatib G, Banerjee M. Correlation of bcl2 oncoprotein immunohistochemical expression with proliferation index and histopathologic parameters in  colorectal neoplasia. Appl mmunohistochem Mol Morphol, 2002;8:175-182.
  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.
  29. Willett CG, Warland G, Hagan MP, Daly WJ, Coen J, Shellito PC, Compton CC. Tumor proliferation in rectal cancer following preoperative irradiation. J Clin Oncol 1999;13:1417-1424.
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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.
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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.