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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.
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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 “T” test 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.