Histopathological Spectrum of Prostatic Lesions Evaluated in a Tertiary Hospital

 *Sultana SS,¹ Hossain S,² Rahman A³

 

1. *Dr. Sk Salowa Sultana, Assistant Professor, Department of Pathology, Ad-Din Women’s Medical College. salowasultana257@gmail.com
2. Shahadat Hossain, Associate Professor, Department of Pathology, Ad-Din Women’s Medical College.
3. Afiqur Rahman, Professor of Urology, Department of Urology, Ad-Din Women’s Medical College.

*For correspondence

 Abstract

Objective: To determine the age distribution of various prostatic lesions, to evaluate histopathological  pattern  of prostatic lesions  and  to analyze adenocarcinoma  of prostate according to Gleason  system.

Methods: This was a retrospective study comprising of 178 cases, carried out at the department of Pathology, Ad-din Women´s  Medical College Hospital during the  period of January 2017 to December 2019.

Results: Out of 178 cases, 159(89.32%) were diagnosed as benign lesion, nodular hyperplasia being the commonest one. Prostatic adenocarcinoma was found in 16(9%) cases, majority of which belonged to Gleasons score 7.

Conclusion: Histopathological examination of prostatic biopsy specimen is essential for diagnosis of benign and malignant lesions, to rule out the incidental carcinoma and HGPIN.

 [Journal of Histopathology and Cytopathology, 2020 Jan; 4 (1):33-37]

 Keywords: Nodular hyperplasia of prostate, prostatic carcinoma, HGPIN

Introduction

The  prostate that weights up to 20 gm in normal adult depends for its subsequent growth and differentiation on androgenic hormones.^1,2  Lesions of prostate are extremely common over the age of 50 years and important cause of morbidity and mortality in male in advance age. Pathological processes affecting this organ include inflammation, benign nodular enlargement and tumor.¹ The clinical incidence of the diseases is only 8% during the 4^th decade  but it reaches 50% in the 5^th decade and 75% in the 8^th decade.² Benign prostatic  hyperplasia(BPH) and carcinoma are increasingly frequent with advancing age.³  BPH results from hyperplasia of both stromal and epithelial cells, gradually being enlarged, compress and narrow the urethral canal and often leads to urinary obstruction. Inflammation of prostate is known as prostatitis and divided into acute, chronic and granulomatous prostatitis. Prostatic carcinoma is one of the most common malignancies affecting men, also it is the sixth leading cause of cancer death in male.⁴ Among the prostatic malignancies 95% is adenocarcinoma. premalignant lesion of prostatic adenocarcinoma  is known as high grade intraepithelial neoplasia (HGPIN). The objective of this study was to determine the age distribution of various prostatic lesions, to evaluate histopathological  pattern  of prostatic lesions  and  to analyze adenocarcinoma  of prostate according to Gleason  system.

Methods

The study was conducted in the Department  of Pathology, Ad-din Women´s Medical College Hospital – a  tertiary level hospital of Dhaka, Bangladesh. The study period was from January 2017 to December 2019. The data were collected retrospectively from histopathology register. A  total of 178 TURP (Transurethral resection of prostate) were evaluated. The received specimen were fixed in 10% formalin and routine paraffin processing followed by hematoxylin and eosin staining was done and the slides were examined under light microscope. The specimens were ananlyzed as type of specimen, age of the patient, histopathological  pattern and final diagnosis. The tumour were classified according to 2016 WHO classification and histological grading was done using modified Gleason´s system. Data  were analyzed using tables, figures and percentage.

 Results

A total of 178 cases were included in our study, all of which (100%) were TURP specimen.  In the present study, most of the prostatic lesions were benign(89.32%) followed by malignant (9.6%) and HPIN (1.12%) and ratio of benign and malignant lesions is 1:9, approximately.

In the present study, benign lesions were mostly found (45%) in the age group of 61-70 years  and 34% in the age group of 51-60 years,  whereas malignant lesions were common  41% in the age group of 71-80 years and 29% in the age group of 61-70 years of age. In our study, benign lesions(159) were nodular hyperplasia of prostate 130(73.03%) and nodular hyperplasia of prostate with co-existing prostatitis 29(16.3%). Out of 29 cases of prostatitis, 24 cases were chronic non specific prostatitis, 2 cases were granulomatous prostatitis and 3 cases were acute prostatitis.  In our study, 2(1.12%) cases were diagnosed as HGPIN and 17(9.6%) cases were diagnosed as malignant. Most of the malignant cases 16(9%)  were prostatic adenocarcinoma and 1(0.56%) was metastatic adenocarcinoma. In this study, 41% cases of malignant belong to 71-80 years age group and 29% cases belong to 61-70 years age group. According to Gleason score, most commonly (81.25%) found grade was grade 7, in this study.

Discussion

We enrolled a total of 178 cases in our study, all of which (100%) were TURP specimen. Bhatta S et al⁵ have found 88.54% specimen of TURP in their study. Screening procedure like trans-rectal ultrasound, prostate specific antigen (PSA) are still used, but biopsy remains the gold standard for final diagnosis.⁶ In the present study, most of the prostatic lesions were benign (89.32%) followed by malignant (9.6%) and HPIN (1.12%). These findings are similar to the studies done by Bhatta S et al,⁵ Bal et al,⁷ Jehoram  et al,⁸ and Bhat S et al.⁹ They found (89.58%), (87%), (93%) and (92.4%) cases of BPH in their study, respectively. In our study, ratio of benign and malignant lesions is 1:9, approximately.

In the present study, benign lesions were mostly found 45% in the age group of 61-70  years  and 34% in the age group of 51-60 years,  whereas malignant lesions were common  41% in the age group of 71-80 years and 29% in the age group of 61-70 years of age. These findings are in concordance with other studies.^10,11,12

In our study, benign lesions(159) were nodular hyperplasia of prostate 130(73.03%) and nodular hyperplasia of prostate with co-existing prostatitis 29(16.3%). Out of 29 cases of prostatitis, 24 cases were chronic non specific prostatitis, 2 cases were granulomatous prostatitis and 3 cases were acute prostatitis.  Bhatta S et al found 24(25%) cases of prostatitis associated with nodular hyperplasia of prostate, out of which 22 cases were chronic non specific prostatis and two cases were acuteprostatis.⁵ Prostatic carcinoma is one of the most common malignancies affecting men. In our study, 2(1.12%) cases were diagnosed as HGPIN and 17(9.6%) cases were diagnosed as malignant. Most of the malignant cases 16(9%)  were prostatic adenocarcinoma and 1(0.56%) was metastatic adenocarcinoma. Bhatta S et. al⁵found 2.08% of HGPIN and 8.34% of prostatic carcinoma. Also Deshmukh BD et. al¹¹ and Bhat S et. al⁹ found similar observation. In this study, 41% cases of malignant  belong to 71-80 years age group and 29% cases belong to 61-70 years age group. Bhat S et. al¹²  found 75% cases of malignancy diagnosed after 60 years. All the cases of prostatic adenocarcinoma were graded according to Gleason score which determine the tumour aggressiveness.¹  In this study, most commonly 13(81.25%) found grade was grade 7. Albasri et al.¹³ found Gleasons score 5-7 as the commonest among 71 cases of prostatic adenocarcinoma. Bhat S et. al⁹ found 56.16% of adenocarcinoma with Gleason score 8-9.

 Conclusion

Histopathological  examination of prostatic lesions is essential for diagnosis and management. Prostatic lesions are common in the age group of 60-70 years. All the specimen received were TURP. Benign prostatic hyperplasia (BPH) was predominant type among all the lesions. Majority of the malignant lesions were incidental diagnosis and most of the malignant lesions were prostatic adenocarcinoma. Emphasis should be  given to identify premalignant lesions.

References

1. Epstein JI. The lower urinary tract and male genital system. 8th rev ed. Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran. New Delhi: Elsevier Pathologic Basis of Disease, 2010. p993-1002.
2. Rosai J. Male reproductive system. In: Rosai J, editor. Rosai and Ackerman’s Surgical Pathology. 10thed. New Delhi: Elsevier, 2011:1287-1333.
3. A Josephin-2014, clinicopathological study of prostatic biopsy, ©2014journal of Clinical and Diagnostic 2014 Sep; 8(9): FC04-FC06.
4. Chandanwale S, Jadhav PS, Anwekar SC, Kumar H, Buch AC, Chaudhari US, et al. Clinico-pathological study of benign and malignant lesions of prostate. IJPBS. 2013;3:162-178.
5. Bhatta S, Hirachan S. Prostatic lesions: Histopathological Study in a Tertiary Care Hospital. JMMIHS. 2018;4(1):12-19.
6. Garg M, Kaur G, Malhotra V, Garg R. Histopathological spectrum of 364 prostatic specimens including immunohistochemistry with special reference to grey zone lesions. Prostate Intl. 2013;1:146-151.
7. Bal MS, Kanwal S, Goyal  AK, Singla N, Prostatic lesions in surgical biopsyspecimen. JK Pract 2011;16:33-4.
8. Jehoram TA, Sitara AS, Mohammed EB. Hyperplastic , premalignant and malignant lesions of the prostate gland. Hum Pathol. 2005;36:480-5.
9. Bhat S, Chaudhri S, Bhat P, Hatwal D. Histopathological study of prostatic Diseases in Garhwal Region. Int  J Sci stud. 2015: 3(8):136-140.
10. Shakya G, Malla S, Shakya KN. Salient and co-morbid features in benign prostatic hyperplasia: A histopathological study of the prostate. Kathmandu Univ Med J. 2003;2:104-109.
11. Deshmukh BD, Ramteerthakar NA, Sulhyan KR. Histopathological study of lesions of prostate- A five year study. Int J Health Sci Res. 2014;4:1-9.
12. Yadav M, Desai H, Goswami H. Study of Various Histopathological Patterns in Prostate Biopsy. IJCRR. 2017;9(21):59-63.
13. Albasri A, EL-Sidding A, Hussainy A, Mahrous M, Alhosaini AA, Alhujaily A. Histopathologic characterization of prostatic diseases in Madinah, Saudi Arabia. Asian Pac J Cancer prev. 2014;15:4175-9.

Histopathological Patterns of Ovarian Tumours and HER2 Expression in Epithelial Ovarian Carcinoma

^*Barua M,¹  Hossain MI,²  Bini UH,³ Nasreen S,⁴ Ahamad MU,⁵ Bhattecharjee P,⁶ RahmanM Z⁷

 

 

1. ^* Mitasree Barua, Lecturer, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh. mitakmc12@gmail.com
2. Mohammad Ismail Hossain, Lecturer, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh
3. Umme Habiba Bini, Assistant Professor, Department of Pathology, Pabna Medical College, Pabna, Bangladesh.
4. Sayeeda Nasreen, Assistant Professor, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh.
5. M. Sahab Uddin Ahamad, Associate Professor, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh.
6. Pradip Bhattacharjee, Associate Professor, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh.
7. Md. Zillur Rahman, Professor & Head, Department of Pathology, Chittagong Medical College, Chattogram, Bangladesh.

 ^*For correspondence

 Abstract

Background: Ovarian tumours are a heterogenous group of neoplasm of epithelial, stromal and germ cell origin. The management of which depends on the histological type of the tumour. Neoadjuvant chemotherapy is limited by toxicity and resistance. Hence targeted therapy is now being proposed to overcome these hurdle.

Objectives: To find out the histopathological patterns of ovarian tumours and to evaluate HER2 overexpression in epithelial ovarian carcinoma.

Methods: This study was carried out in the Department of Pathology, Chittagong Medical College, Chattogram, received from Department of Gynaecology and Obstetrics, Chittagong Medical College Hospital, Chattogram during the period from January 2016 to December 2016. A total of 92 cases of ovarian tumours were selected consecutively. The age range was 12-80 years. Histopathological sections were stained with Hematoxylin and Eosin stain and epithelial ovarian carcinoma were subjected to HER2/neu immunohistochemical stain. Histopathological patterns of ovarian neoplasm, histopathological subtyping and grading of epithelial ovarian tumours and the expression of HER2/neu among epithelial ovarian carcinomas on immunohistochemistry was analyzed.

Results: Out of total 92 cases, histopathological diagnosis showed 65 (70.7%) were benign tumours and 27 (29.3%) were malignant tumours. Histologically, surface epithelial tumours were the most common (74%) followed by germ cell tumours (21.7%) and sex cord stromal tumours (4.3%). Age incidence of benign tumour was age group of 21-40 yrs and malignant 41-60 years. One in 23 epithelial ovarian carcinoma cases (4.3%) exhibits HER2 overexpression. Only one HER2 positive case was mucinous cystadenocarcinoma grade II.

Conclusion: Surface epithelial tumours were most common followed by germ cell tumours. Serous cystadenocarcinoma were most common epithelial ovarian carcinoma.

 

[Journal of Histopathology and Cytopathology, 2020 Jan; 4 (1):23-32]

 Keywords: Ovarian tumour, Epithelial Carcinoma, HER2/neu.

 

Introduction

Ovarian tumours account for 3% of all cancers in females, being the second most common cancer of the female genital tract, next only to uterine cancer.¹ Ovarian cancer, traditionally known as the silent killer, is a dreaded disease because of its vague, nonspecific symptoms and late presentation. Worldwide, it is the seventh most common cause of cancer and eighth most common cause of cancer-related death among women.² It is be a disease of developed and industrialized countries where parity of women is low. As the standard of living increases and resources for a medical infrastructure improve in Bangladesh, there will be an increased need to address less common but highly lethal cancers such as ovarian cancer. Globocan predicts a change in the reported incidence of ovarian cancer from 2912 in year 2012 to 3132 in year 2015.³

Risk factors for ovarian cancer are not well defined. However, there is general agreement on two: nulliparity and family history. Women 40 to 59 years of age who have taken oral contraceptives or undergone tubal ligation have a reduced risk of developing ovarian cancer.⁴ Two treatment options, available for ovarian cancers in advanced stage, are either a primary surgical cytoreduction/debulking or chemotherapy in an attempt to downstage the tumor followed by surgery. Though platinum-based therapy has produced an impressive result, chemoresistance and toxicity are creating hurdles. To overcome this, monoclonal antibodies targeting HER2/neu, a transmembrane protein homologous to epidermal growth factor receptor, has been introduced recently that might bring a new era of management.⁵

HER2/neu protein expression is commonly measured using immunohistochemistry (IHC).⁶ Overexpression of HER2/neu is seen in 20–30% patients with ovarian cancer. Some of these differences are likely to be attributable to the diagnostic technique used to measure HER2 expression. HER2/neu overexpression in epithelial ovarian carcinomas has not been studied as extensively as it has been studied in case of breast carcinomas, especially in Bangladesh scenario.

The aim of my study was to determine histopathological patterns of ovarian tumor and to evaluate the frequency of the expression of HER2 among epithelial ovarian carcinomas.

 Methods

It was a cross-sectional descriptive study carried out in the Department of Pathology of Chittagong Medical College, Chattogram, Bangladesh. Study period was for one year from 1^st January, 2016 to 31^st December, 2016. All female patients clinically diagnosed as ovarian neoplasm received at the Department of Pathology of Chittagong Medical College, Chattogram referred from Department of Gynaecology and Obstetrics, Chittagong Medical College Hospital (CMCH) during the specified time duration. Finally, 92 consecutive patients were selected according to time frame.

 Inclusion criteria

Patient with clinically diagnosed as ovarian tumours who underwent intervention like diagnostic laparoscopy or laparotomy; at least for biopsy in Chittagong Medical College Hospital, Chattogram.  Patient with histopathologically diagnosed ovarian tumour in the Department of Pathology, Chittagong Medical College.

 Exclusion criteria

Previously diagnosed case of ovarian tumour who already has got treatment. Patient unwilling to give written consent.

Protocol was ethically reviewed and approved by the Ethical review Committee of Chittagong Medical College, Chattogram. All the patients included in the study were informed and explained about the nature of study. Informed written consent was taken from all the subjects after full explanation of nature, purpose and potential risks of all the procedures.

A brief clinical history was taken from each patient with particular reference to the age, occupation, marital history, parity, menstrual history, family history, history of breast feeding, use of contraceptives etc. Clinical history, questionnaire, thorough physical examination, and relevant investigations were recorded in details in all cases.Routine histopathology with H & E stain and Immunohistochemistry for ovarian carcinoma were done.

H&E stained slides were examined under light microscope to get a definitive diagnosis of the ovarian tumour and its type. Histological classification was based on the 2003 World Health Organization classification of ovarian tumours. The Shimizu-Silverberg three-grade histological grading system was used in epithelial ovarian carcinoma (EOC).

The most representative tumor tissue was chosen from each epithelial ovarian carcinoma case and 4 μm sections were taken to poly-L-lysine coated slides for immunohistochemical staining. For immunohistochemistry, epitope retrieval was done with a polymer-based detection system (Envision; Dako) using a Herceptin kit (HercepTest; Dako) according to the manufacturer’s instructions.Antigen retrieval for HER2 using Hercep Test was performed by immersing and incubating the slides in 10 mmol/L citrate buffer in calibrated water bath (95- 99°C) for 40 minutes. After decanting the epitope-retrieval solution, the sections was rinsed in the wash buffer and later, soaked in the buffer for 5 to 20 minutes before staining. Then the slides were rinsed, placed in 200 μL peroxidase-blocking reagent for 5 minutes, rinsed, placed in 200 μL primary anti-HER2 protein (or negative control reagent) for 30 minutes, rinsed twice, and finally immersed in 200 μL substrate chromogen solution (3,3´-diaminobenzidinetetrahydrochloride, DAB) for 10 minutes. The slides were counter stained with hematoxylin and finally were cover slipped.

 Results

In this study the age range of 92 ovarian tumours was 12-80 years, were divided into seven age groups and it was observed that maximum number of benign lesions 19 (20.6%) were in age groups 21-30 years. Maximum number of malignant tumours were found 9 (9.8%) in age group 41-50 years. Among 92 ovarian lesions, 68 (74%) were surface epithelial tumour, 20 (21.7%) were germ cell tumour, 4 (4.3%) were sex cord stromal tumours .

Discussion 

Current study was carried out in Departmant of pathology, Chittagong Medical College to see the histopathological patterns of ovarian tumours and HER2 expression in epithelial ovarian carcinoma (EOC).Total number of the patients in this study was 92. The age range was 12-80 years. The mean age was 34.64 years. Patients were divided into seven groups consisting of each decade as a single group and maximum number patients 23 (25%) were in 21 – 30 years group, followed by 21 (22.8%) were in 41-50 years age group. Study done in Nepal by Vaidya et al.(2014) showed highest number of patients (58%) were in 21-40 years age group which is similar to present study.⁷ In this study in 21-40 years age group total number of patients were 42 (45.65%). Study done in India by Bhagyalakshmi et al (2014)) patients age ranged from 11-70 years, majority of benign cases and malignant cases were between 21 to 40 years and 41 to 60 years respectively.⁸ Agrawal et al. (2015) also showed age ranged from 12 to 80 years with most common benign and malignant lesions cases were 21 to 40 years and 41 to 60 years respectively which was consistent with this study.⁹

In this study, among 92 cases, 72 (78.3%) were married and 20 (21.7%) cases were unmarried women. This is probably due to more married women presented to the outpatient department than unmarried ones. Among 72 married women 08 (11.1%) were nulliparous, 03(4.2%) were primiparous and 64 (84.7%) were multiparous.Study done in Dhaka by Dhar et al (2015) showed 8% were unmarried and 92% were married, of whom 12% were nulliparous and 80% was parous.¹⁰

Regarding socio-economic status 68 (73.9%) cases were from average socioeconomic condition, 23 (25%) were from poor and only 1 (1.1%) cases from high socioeconomic family. Among the malignant cases maximum 13 (48.1%) cases were from average socioeconomic condition. This is similar to the finding of  Dhar et al (2015).¹⁰This may be due to the fact that patients attended at a government hospital and most of the people of average and poor socioeconomic conditions usually come here to get treatment.

In this study, it was seen that, 19 (20.7%) had history of hormonal contraceptive use and 49 (53.3%) had no history. Among 27 malignant cases 3 (11.1%) had history of contraceptive use. Ness et al (2011) had reported that oral contraceptives reduce the risk of developing ovarian cancer.¹¹

In this study 75 (81.5%) patients presented with lower abdominal pain 74 (80.4%) with lower abdominal mass. Study done by Jaffar et al (2013) showed, abdominal pain was in 90% cases, abdominal mass was present in 24% of the patients with benign tumours and in 66% with malignant tumours.¹²

In the present study, among 65 benign lesions 20 (21.7%) cases had increased level of CA-125 and among 27 malignant lesions 3(3.3%) cases were found within normal level.Miralles et al. (2003) suggested that variety of malignancies and benign conditions courses with increased CA-125 level and it is observed that CA-125 is very important as tumour marker for malignancy.¹³

In the present study, out of 65 benign tumours 62(95.3%) were unilateral and 3(4.6%) were bilateral. Out of 27 malignant tumours 5(18.51%) were bilateral and 22(81.48%) were unilateral. Modepalli et al (2016) reported 93.8% of the neoplasms were unilateral and remaining 6.2% of the lesions were bilateral.¹⁴ Benign tumours were more unilateral than malignant tumours. In this present study, out of 92 cases, maximum number of lesions were 48 (52.2%) in the right side of abdomen which is similar to the study done by Modepalli et al (2016).¹⁴

Among 92 cases 43 (46.7%) were solid and cystic lesion, 41 (44.6%) were cystic lesions and 8 (8.7%) were solid. Similar study done by Agrawal et al (2017) showed 83.3% benign tumours were cystic whereas 65.3% malignant tumours were solid and cystic.⁹ Benign tumors were more often cystic in consistency in this study and malignant tumors were solid consistency which was comparable to the study of Kanthikar et al. (2014).¹⁵

In this study of the 92 cases of ovarian tumours, 67 (70.7%) were benign and 27 (29.3%) were malignant. Similar studies in India carried out by GG swamy et al. (2010) showed 86 (71.6%) benign tumours, 30 (25.1%) malignant and 4 (3.3%) borderline tumors.¹⁶ In Nepal R Jha et al reported 83.9% benign, 16.1% malignant and 2.8% borderilne ovarian tumour.¹⁷ In  Pakistan a similar study by Ahmed et al showed 59.18% benign and 40.81% malignant tumour. In this study there was no borderline malignancy which were similar to the study done by Yasmine et al. (2008).¹⁸

Among the histologic types, in the present study, surface epithelial tumours were most common 68 (74%), germ cell tumour was 20 (21.7%), and sex cord stromal tumour was 4 (4.3%).Similar observation made by Pilli et al.(2002) showed 70.9% surface epithelial tumours, 21.2% germ cell tumours and 6.7% sex cord stromal tumours.¹⁹

In this present study Among 92 cases the most common benign surface epithelial tumour was mucinous cystadenoma 26 (28.3%) followed by serous cyst adenoma 18 (19.6%) and benign brenner tumour 1(1.1%). The most common benign germ cell tumour was mature cystic teratoma 18 (19.6%).In this study benign sexcord stromal tumour was fibroma 2(2.2%). Studies done by Bhagyalakshmi et al (2014)⁸ and Mondol et al (2011)²⁰ showed serous cystadenoma was the most common benign tumour. Study done by Ahmed et al (2000) however found the most common benign tumour to be benign cystic teratoma.²¹

The commonest malignant tumour was serous cyst adenocarcinoma 13 (14.1%), followed by mucinous cystadenocarcinoma 8 (8.7%), malignant brenner tumour 1 (1.1%) and clear cell carcinoma 1 (1.1%). Similar results were shown in studies by Mondol et al (2011) and Yogambal et al (2014).²²

In this present study 23 cases of epithelial ovarian carcinoma (EOC) were evaluated for HER2/neu protein expression using immunohistochemistry.The positive expression rate of HER2/neu in this study was 1(4.3%).This is comparable to the rate of  HER2/neu positivity in EOC reported in the literature,which ranges from 7-50%Verri et al (2005),²³ Berchuck et al (1990),²⁴ Mayr et al (2006).²⁵ The variation in HER2/neu protein expression rate in different studies may be early stage and 17% of patients with advanced stage disease in the study by Kacinsky et al (1992). However, multiple factor may be involved in producing a wide range of positivity, such as sample size, the detection technique and type of monoclonal antibody used, staining intensity and the tissue where analysis is performed. Studies with populations smaller than 100 patients reported a frequency of 1.8-76%,(Serrano-Olvera et al., 2006).²⁶

In this study among 8 mucinous cystadenocarcinoma, HER2 expression was positive in 1 (12.5%) case and was in Grade-II. In the study done by Missaoui et al (2014),²⁷ HER2 expression was described in 14.3% of mucinous carcinoma. Study in Nijeria done by Ajani et al.(2015) ²⁸ observed that a higher proportion of serous carcinoma (as opposed to mucinous carcinoma) was HER2/neu positive. Sarkar et al (2015)² also showed that HER2 expression was more intense for high grade serous carcinoma.

In this study HER2 expression is relatively common in mucinous epithelial carcinoma of the ovary and trastuzumab therapy would be an option for patients with mucinous carcinoma when the tumour has HER2 overexpression.

The study also has some limitations. This study was severely constrained by the limited duration that led to have small population size and this study was done in patient who underwent surgery and samples were collected from Chittagong Medical College and Hospital only. In spite of these reason the present study showed more or less acceptable findings with consideration of the observations by others.

 Conclusion

Ovarian cancer presents a tremendous clinical challenge to gynecologists, medical oncologists and radiotherapists. It is a silent menace and is not associated with significant symptoms. Surface Epithelial Tumours were the commonest group followed by Germ Cell Tumours. Most common Germ cell tumours were mature cystic teratoma occured in much younger women. Malignant ovarian neoplasms were more common in patients above 40 years. Sex Cord Stromal Tumours were also common with Fibroma and Granulosa cell tumour. Unilateral involvement of ovarian neoplasms was more common than bilateral and involvement of right ovary was more than the left. Mucinous cystadenoma was the commonest benign ovarian neoplasm while the commonest malignant tumour was serous cystadenocarcinoma. Mucinous epithelial carcinoma have expressed HER2.

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26. Serrano-OlveraA,Dueñas-González A, Gallardo-Rincón D, Candelaria M, Garza J. et al . Prognostic, predictive and therapeutic implications of HER2 in invasive epithelial ovarian cancer. Cancer treatment reviews. 2006;32(3):180-90.
27. Missaoui N, Ben AS, Ayachi M, Hmissa S,  Yaacoubi M et al. (2014). HER2 Expression in Ovarian Mucinous Carcinomas in Tunisia. Asian Pacific journal of cancer prevention: APJCP. 2014; 15(19): 8121-5.
28. Ajania MA, Salamia A, Awoludeb OA,Oluwasolaa AO, and Akang EU, etal. The expression status of human epidermal growth factor receptor 2 in epithelial ovarian cancer in Ibadan, Nigeria South Afr J Gynaecol Oncol. 2016; 8(1):1–13.

Correlation of Lymphovascular Density with Histological Prognostic Parameters in Gastric Carcinoma

*Hassan MI,¹ Kabir AN,² Poly FH,³ Ray S,⁴ Munmun UK,⁵ Mehjabin M⁶

 

1. * Mohammed Imran Hassan, Assistant Professor (CC), Department of Pathology, National Institute of Diseases of Chest and Hospital, Mohakhali, Dhaka. imran.vua@gmail.com
2. AKM Nurul Kabir, Associate Professor, Department of Pathology, BangabandhuSheikh Mujib Medical University, Shahbag,Dhaka.
3. Ferdousi Hossain Poly, Medical Officer, OSD, DGHS, Attached- Department of Pediatric Neurology, National Institute of Neurosciences and Hospital, Dhaka.
4. Subrata Ray, Assistant Professor, Department of Pathology, Jashore Medical College.
5. Umme Kulsum Munmun, Lecturer, Department of Pathology, Dhaka Medical College, Dhaka.
6. Monika Mehjabin, Lecturer, Department of Pathology, Shahid Suhrawardy Medical College, Dhaka.

*For correspondence

 Abstract

Background: Carcinoma of the stomach is a major cause of cancer mortality worldwide. Gastric cancer is actually a curable disease if it is detected at an appropriate stage and treated adequately. It rarely disseminates widely before it has involved the lymph nodes and, therefore, there is an opportunity to cure the disease prior to dissemination. Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. However, the significance of lymph vessel density has been controversial in gastric cancer. Specific lymphatic markers are now available making possible analysis of lymphatic in cancer. The aim of this study was to investigate the relation of lymph vessel density with histological prognostic factors in gastric cancer as potential indicator.
Objectives: To estimate of the lymph vessel density using Podoplanin immunostain in gastric cancer and then to correlate lymph vessel density with histological prognostic factors.
Methods: Sections from Paraffin blocks of gastric adenocarcinoma with routine stain were assessed to detect prognostic factors and then assessed with immunostain Podoplanin to count lymph vessel density. The mean number of lymph vessels in five hot spots was calculated in both intratumoral and peritumoral areas. Finally correlation was seen between histological prognostic factors and lymph vessel density in gastric carcinoma.
Results: A positive significant correlation was found between lymph node metastasis with lymph vessel density in both intratumoral and peritumoral area. Similarly, a positive significant correlation was found between tumor stage with lymph vessel density in both intratumoral and peritumoral area.

 [Journal of Histopathology and Cytopathology, 2020 Jan; 4 (1):12-22]

 Keywords: Podoplanin, Lymphovascular density, Gastric carcinoma

 Introduction

Carcinoma of the stomach is a major cause of cancer mortality worldwide. The prognosis of gastric carcinoma is poor with cure rates little better than 5–10%, although better results are obtained in Japan where the disease is common. Gastric cancer is actually a curable disease provided that it is detected at an appropriate stage and treated adequately. It rarely disseminates widely before it has involved the lymph nodes and, therefore, there is an opportunity to cure the disease prior to dissemination.¹

Worldwide gastric cancer ranks fourth in frequency and second in cancer mortality rate; with a 5-year survival rate not exceeding 30% in Western countries.² Gastric cancer incidence varies markedly with geography. In Japan, Chile, Costa Rica, and Eastern Europe the incidence is up to 20-fold higher than in North America, northern Europe, Africa, and Southeast Asia.¹ The incidence of gastric carcinoma in Bangladesh is 5.2 per 100,000.³

 

Gastric cancer is a multi-factorial disease. Epidemiological studies point to a role for Helicobacter pylori, although its importance is disputed. Helicobacter pylori seem to be principally associated with carcinoma of the body of the stomach and the distal stomach rather than the proximal stomach. As Helicobacter is associated with gastritis, gastric atrophy and intestinal metaplasia, the association with malignancy is perhaps not surprising. Patients with pernicious anemia, gastric atrophy, duodenogastric reflux and reflux gastritis, intestinal metaplasia and cigarette smoking are at increased risk, as are those with gastric polyps. Diet appears to be important factor. The ingestion of substances such as spirits may induce gastritis and, in the long term, cancer. Excessive salt intake, deficiency of antioxidants and exposure to N-nitroso compounds are also implicated.¹

The most useful classification of gastric cancer is the Lauren classification.⁴ In this system, there are principally two forms of gastric cancer: intestinal gastric cancer and diffuse gastric cancer. In intestinal gastric cancer, the tumor resembles carcinomas found elsewhere in the tubular gastrointestinal tract and forms polypoid tumors or ulcers. It probably arises in areas of intestinal metaplasia. In contrast, diffuse gastric cancer infiltrates deeply into the stomach without forming obvious mass lesions but spreading widely in the gastric wall.¹

These tumors have several prognostic and predictive factors. The depth of invasion and the nodal and distant metastasis at the time of diagnosis remain the most important prognostic indicators for gastric carcinoma. Currently tumor size, nuclear grade and patient’s age are well known prognostic factors for patients with operable gastric cancer. These prognostic factors are widely used to determine whether to apply neo-adjuvant therapy in patient with gastric cancer.  In advanced cases, gastric carcinoma may first be detected as metastases to the supraclavicular sentinel lymph nodes, also called Virchow’s node. Gastric carcinoma can also metastasize to the periumbilical region to form a subcutaneous nodule, termed as Sister Mary Joseph nodule. Local invasion into the duodenum, pancreas, and retroperitoneum is also characteristic. In such cases efforts are usually focused on chemotherapy or radiation therapy and palliative care. However, surgical resection remains the preferred treatment for gastric carcinoma.⁵

Lymphangiogenesis is a critical process for tumor growth, invasion and metastasis.  That is why measurement of lymph vessel density may be clinically important in gastric cancer specimens. The induction of lymphangiogenesis by a tumor is controlled process, influenced by lymphangiogenic factors, which involve a complex interaction between tumor and endothelial cells.⁶ The pathologic approach to assess lymphangiogenesis involves microscopic estimation of lymph vessel density by using endothelial markers in immunohistochemistry.

On previous studies detection of lymphangiogenesis was limited due to lack of specific lymphatic endothelial markers. The recent discovery of anti-Podoplanin antibody has a major impact on lymphatic studies. Podoplanin has become a valuable marker for detecting lymph vessels and for identifying lymph vessel invasion in tumor.⁷

Podoplanin is a specific marker of the lymphatic endothelium and is not expressed in blood vessels. It is expressed by both developing and mature lymphatic endothelial cells and seems to be a more specific marker of lymphatic endothelial cells.  By electron microscopy and immunoelectron microscopy, it was demonstrated that Podoplanin is mainly expressed on the luminal surface of lymphatic endothelial cells.  Peritumoral (PT) lymphatic vessels are larger and more irregular than the intratumoral (IT) lymphatic’s, with a significantly lower density. Intratumoral lymphatic vessels are found in a large variety of tumors and are usually small, flattened and irregular. It was suggested that Podoplanin has potential role in invasion and metastasis of tumor. This hypothesis is mainly based on the observation that high expression of Podoplanin is consistently correlated with the presence of metastases. It was reported that Podoplanin expressing cells were found at the invasion.⁸ The use of an anti-Podoplanin-based therapeutic strategy could be suggested in the treatment of lymphatic metastases based on three considerations: (i) Podoplanin is a well-known marker of lymphatic endothelial cells; (ii) its expression seems to be associated with bad prognosis and high risk for lymph node metastases; (iii) it is involved in tumor invasion. In conclusion, Podoplanin is a sensitive marker of lymphatic endothelial cells and is very useful in evaluating lymphatic micro vessel density. Immunohistochemical detection of Podoplanin is helpful in the diagnosis of lymphovascular invasion.⁸ Some studies have suggested that lymph vessel density is associated with an increased risk of lymph node metastasis;⁹ however, this conclusion is not supported by all of the published studies.¹⁰

 

 

 

 

 

 

 

 

 

 

 

The study was conducted a) to investigate morphological prognostic factors of gastric adenocarcinoma, b) to estimate lymph vessel density by using immunostain Podoplanin and c) to correlate lymph vessel density with prognostic factors.

 Methods

It was a cross sectional observational study carried out from March, 2016 to July, 2018at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh. The study population was paraffin blocks of gastric adenocarcinoma from pathological laboratory in the same Institute with consecutive convenience sampling. Inclusion Criteria was paraffin blocks of gastric samples diagnosed as gastric adenocarcinoma in pathology laboratory and Exclusion criteria were with history of treated gastric carcinoma cases like neo adjuvant therapy, blocks containing autolyzed tissue, extensive necrosis, hemorrhage and desmoplasia, blocks having no tissue or damaged blocks and blocks containing other cancers like lymphoma, carcinoid tumor, malignant GIST etc. The sample size was 53. Monoclonal Anti-Human Podoplanin antibody, clone D2-40, Ready to use (code- IR072) was used for detecting lymph vessel. In this study, sections of normal vermiform appendix were taken as positive control (as recommended by Dako). Podoplanin antigen expression was detected by immunohistochemistry in the submitted blocks (paraffin blocks with maximum tumor bulk were chosen) and were performed using DAKO Cytomation at immunohistochemistry laboratory, department Pathology, BSMMU.

Histopathological categorization of tumor and grading of all the cases were done. Lymphovascular invasion was recorded. Each lymph node was histologically examined to determine metastasis. Numbers of lymph vessels were counted by Weidner’s method in peritumoral (PT) and intratumoral (IT) areas.¹¹

 Micro vessel Quantification

Determination of lymph vessel density (LVD) were performed according to Weidner et al.¹¹Brown staining of cytoplasmic membrane of endothelial cells of lymph vessels were considered as positive reaction. Lymph vessel density was calculated according to Weidner’s method by Olympus microscope model BH51. The immunostained sections were scanned by light microscopy at low magnification (10x) and the areas of tissue with the greatest numbers of distinctly highlighted lymph vessels (“hot spots”) were selected. So, “hot spot” is an area with the most dense lymph vessels growth. Five hot spots were selected in intratumoral (IT) and peritumoral (PT) areas. When the hot spots were defined, lymph vessel count were performed by counting the individual stained lymph vessel (at power 20x) representing a field size of 0.74mm2 (i.e. – 20x objective lens, 10x ocular lens; equivalent to 0.7386 mm² per 200x field).¹¹ The mean number of lymph vessels in each case were determined and considered as the lymph vessel density.⁷So, at first five hot spots were chosen in intratumoral (IT) and peritumoral (PT) area. In each hot spot, lymph vessel count was performed at power x20. Finally lymph vessel density was calculated as the mean of the total number of lymph vessels in these five hot spots. Lymph vessel density (LVD) assessed in this manner may be not equivalent to LVD seen in multiple areas of multiple samples of the stomach; however, it is well accepted that these hot spots, not arbitrary areas, are representative of the entire tumor. Although there is some variability in the distribution of lymph vessels within the layers of the gastric wall, the five ‘hot spot’ areas were chosen to obtain an objective assessment and to avoid observer variation.⁷

Results

A total number of fifty three diagnosed cases of gastric adenocarcinoma were taken for this study. Forty six samples were partial/subtotal gastrectomy specimen and seven samples were total gastrectomy specimen. All fifty three samples contain epigastric lymph nodes ranging one to twenty five. Thirty seven cases had lymph node metastasis and the rest sixteen were free of tumor metastasis. Thirty seven cases were LVI positive in H&E routine stain and forty two cases were LVI positive with Podoplaninimmunostain.

In the group of 53 patients with gastric carcinoma, mean patients’ age was 49.87±11.38 years (range 20-70).33 patients ware male and 20 patients ware female with a male/female ratio of 1.6:1. According to Lauren’s classification, 30 cases ware diffuse type and 23 cases were intestinal type. Two third (60.4%) samples belonged to tumor size ≤ 5 cm. The mean tumor size was 5.61±1.92 cm, ranged from (3.0 – 11.0) cm. Lymph vessel density (LVD) was more frequent in cases having tumor size > 5 cm in diameter.

The histological grade was assessed according to WHO grading system. 33(62.3%) samples belonged to grade-III (poorly differentiation), followed by grade-II (moderate differentiation) (24.5%) and grade-I (well differentiation)(13.2%). Lymph vessel density (LVD) was most frequent in grade- II group.

Cases were grouped according to depth of invasion (tumor stage) as T1, T2, T3 and T4. 41.5% samples belonged to subserosal invasion (T3). T3 was followed by T2(32.1%), T4 (20.8%) and T1(5.7%). Lymph vessel density(LVD) was most frequent in T3 group.

Numbers of lymph node ranged from 1-25. Cases were grouped according to the numbers of lymph node metastasis as N0, N1, N2 and N3. 30.2% samples had no lymph node metastasis (nodal stage N0). 28.3% samples belonged to nodal stage N1, followed by nodal stage N2 (20.8%) and N3 (20.8%). Nodal stages were increasing with increase of Lymph vessel density (LVD).

Lymph vessels in intratumoral (IT) area ranged from 1-12 and peritumoral (PT) area ranged 2-11. Mean LVD in IT area was 5.93 ±2.79 cm and mean LVD in PT area was 5.50±2.38 cm.

A positive correlation was found between Lymph vessel density(LVD) and tumor stage in intratumoral (IT) area (Figure 7).

The value of Pearson’s correlation coefficient was 0.377 and it was significant (p=0.005). Therefore, there was linear association between tumor stage and lymph vessel density (LDV) in intratumoral (IT) area with Podoplanin stain.

A positive correlation was found between Lymph vessel density(LVD) and tumor stage in peritumoral (PT) area (Figure 8).  The value of Pearson’s correlation coefficient was 0.334 and it was significant (p=0.014). Therefore, there was linear association between tumor stage and lymph vessel density (LVD) in peritumoral (PT) area with Podoplanin stain.

A positive correlation was found between Lymph vessel density (LVD) and Lymph node metastases inintratumoral (IT) area (Figure 9).

The value of Pearson’s correlation coefficient was 0.855 and it was significant (p=0.000). Therefore, there was linear association between lymph node metastasis and lymph vessel density (LVD) in intratumoral (IT) area with Podoplanin stain.

A positive correlation was found between Lymph vessel density (LVD) and Lymph node metastases in peritumoral (PT) area (Figure 10).

The value of Pearson’s correlation coefficient was 0.791 and it was significant (p=0.000). Therefore, there was linear association between lymph node metastasis and lymph vessel density (LVD) in peritumoral (PT) area with Podoplanin stain.

A positive correlation was found between LVD in IT and PT.

The value of Pearson’s correlation coefficient was 0.937 and it was significant (p=0.000). Therefore, there was linear association between LVD in intratumoral (IT) and peritumoral (PT) area with Podoplanin stain.

Discussion

Gastric carcinoma is the most common leading tumor of the world. Although great efforts have been made in the field of early diagnosis and adjuvant therapy, the incidence and overall mortality of gastric carcinoma continues to increase. Since gastric tumor cells commonly infiltrate into the lymphatic system, lymph node status is routinely used to identify a patient’s prognosis, tumor stage, and treatment modality. Inhibition of tumor cells to lymph node metastasis (LNM) is a promising way to prevent distant metastasis. However, the relationship between lymphangiogenesis, lymphovascular invasion and lymph node metastasis (LNM) remains ambiguous.

This cross sectional study was carried out with an aim to estimate the density of expression of Podoplanin in lymphangiogenic vessel in gastric adenocarcinoma and to see the correlation between the percentages of area covered by lymphangiogenic vessels with prognostic factors. The present study findings were discussed and compared with previously published relevant studies.

In this study, 62.3% sample had grade-III (poorly differentiation) followed by 24.5% grade-II (moderately differentiation) and 13.2 % grade-I (well differentiation). Significant relation was found between lymph vessel density-intratumoral (LVD-IT) area (p=0.013) and lymph vessel density-peritumoral (LVD-PT) area (p= 0.006) with different histological differentiation. In their study, Pak et al., 2015¹² found that 53.3 % sample had grade-III and significant relation (p= 0.021) between LVD-IT area with histological differentiation, which is comparable with the current study.

 

It was observed that 41.5 % of samples belonged to T3, followed by 32.1% T2, 20.8% T4  and 5.7% T1 and significant relation found between LVD-IT area (p=0.019) and LVD-PT area (p=0.041) with different tumor stage. Gresta, Rodrigues Jr and Cabral, 2014¹³ found 63.5% cases were T3, followed by 19.2% T2, 13.4% T1 and 3.8% T4. Pak et al., 2015 found 39.3% samples were T3 followed by 16.6 % T2 and T4. They also found significant association between LVD-IT area (p=0.024) with different tumor stage. Raica et al., 2008⁸ found significant correlation between LVD with tumor stage (p<0.002) and Nakamura et al., 2006⁷ found significant positive correlation between LVD with tumor stage (p=0.0008). So, present study result is consistent with previous study.

According to number of lymph node metastasis (LNM), it was observed that 30.2% sample belonged to nodal stage 0 (N0), followed by 28.3% stage-I (N1), 20.8% stage II (N2) and stage III (N3). There was significant relation was found between LVD-IT area (p<0.001) and LVD-PT area (p<0.001) with nodal status. Yan et al., 2008¹⁴ reported that LVD was obviously higher in the colorectal cancer sample with metastasis (12.08 ± 4.96) than in those without (8.26 ± 4.08) (p<0.001). Schoppmann et al., 2001¹⁵ demonstrated that lymph vascular invasion (LVI), assessed by anti-Podoplanin immunostain, has been strongly associated with presence of lymph node metastasis. Pak et al. 2015¹² found LVD-IT area was a higher than LVD-PT area (12.29± 4.36 vs. 11.01±3.62), Nodal stage N0 cases were more frequent and significant relation between LVD-PT in area (p=0.040) with nodal status. Nakamura et al., 2006⁷ found significant positive correlation between LVD with nodal metastasis (p= 0.0094). Raica et al., 2008⁸ found significant correlation between LVD with nodal metastasis (p<0.031) which is comparable with current study.

In this study, there was a positive significant Pearson’s correlation (r= 0.855, p= 0.000) was found between lymph node (LN) metastasis with LVD in IT area. Similarly, there was a positive significant Pearson’s correlation (r= 0.791 p= 0.000) was found between LN metastasis with LVD in PT area. Pak et al., 2015¹² found in their study that LVD in PT area was significantly associated with LN metastasis (p=0.040). Nakamura et al., 2006⁷ found significant correlation between LN metastasis with LVD (p= 0.0094). Raica et al., 2008⁸ reported that there was significant positive correlation between both LVD in IT and PT areas with LN metastasis (P<0.031). So current study result is consistent with previous study.

In this current study, there was a positive significant Pearson’s correlation (r= 0.377, p= 0.005) was found between tumor stages with LVD in IT area. Similarly, there was a positive significant Pearson’s correlation (r= 0.334, p= 0.014) was found between tumor stage with LVD in PT area. Nakamura et al., 2006⁷ found significant positive correlation between tumor stage with LVD (p= 0.0008). Raica et al., 2008⁸ reported that there was significant positive correlation between both LVD-IT area and LVD-PT area with tumor stage (P<0.02).

In this current study, there was a positive significant Pearson’s correlation (r= 0.937, p= 0.000) was found between LVD-IT areas with LVD-PT areas. Pak et al., 2015¹² found significant correlation (p=0.028) between LVD-IT area with LVD-PT area.

Conclusion

Total fifty three cases of gastric adenocarcinoma were examined for lymphangiogenic vessels in peritumoral (PT) and intratumoral (IT) areas. Thirty seven cases had lymph node metastasis and the rest sixteen were free of tumor metastasis. Both peritumoral and intratumoral lymphangiogenic vessel count (density) stained by Podoplanin antibody correlated with lymph node metastasis and tumor stage. Lymphatic vessel count is more in the intratumoral area.  The specific lymphatic endothelial marker Podoplanin proved to be a valuable tool in highlighting lymph vessel density (LVD) and lymphovascular invasion, and therefore a predictor of lymph node metastasis.

Limitation 

The study population was selected from one institute in Dhaka city, so that the results may not be reflect the exact picture of the country.  The study period was short. Sample size was small. Podoplanin was only marker used to detect lymph vessels. Sometimes differentiation between blood vessels and lymph vessels were difficult in tissue sections. Differentiation between blood vessels and lymph vessels was also a limitation of this study.  Use of blood vessel immunostain like factor VIIIa, CD31, and CD 34 etc. could have overcome the limitation and overall no control was used.

 

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