Relationship of Gleason's Score with Age, Cellularity of Tumor and PSA Immunohistochemical Stain in Prostatic Carcinoma

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1 Proceeding S.Z.P.G.M.J. vol: 21(1): pp , 27. Relationship of Gleason's Score with Age, Cellularity of Tumor and PSA mmunohistochemical Stain in Prostatic Carcinoma Afshan Kamran Hussain, Sabiha Riaz and Saeed ur Rahman Fatima Memorial Hospital Medical & Dental College, Shadman, Lahore ABSTRACT Purpose of the study: To study the relationship between Gleason's Score with age, cellularity of Tumor and PSA immunohistochemical staining in prostatic carcinoma. Basic procedures: An exploratory study carried out at the Department of Histopathology of a Tertiary Care Hospital Lahore, from January 1999 to July 22. Sixty-two (62) cases of prostatic adenocarcinoma were graded with Gleason's score and degree of cellularity of the prostatic cancer were determined on each slide, along with degree of positive staining with immunohistochemical stain marking tissue PSA. These were correlated and relationship was evaluated by ANOVA and simple regression. Main findings: Age of the 62 cases ranged between 5-9 years and no significant difference was found between age groups regarding tumor cell differentiation. Cellularity of the prostate cancer showed a weak positive relationship with Gleason's score. Relationship between staining positivity and Gleason's score showed an inverse quadratic relationship with an F statistic of 76.2 (p.1) and Beta of Conclusions: From this study it can be concluded that cellularity of tumor tissue in a specimen bears little or no relationship with Gleason's Score. There is an inverse relationship between Gleason's Score and percentage of tumor cells showing positive staining with PSA immunohistochemical stain. Key words: mmunohistochemical - Tissue PSA - Prostate Carcinoma - Gleason's Score Cellularity Staining Positivity percentage NTRODUCTON Prostate carcinoma is the most common malignancy among men 1 n the European Union, 1% of the malignancies in men are prostate carcinoma 2 Approximately 2% cases occur in men below 65 years of age n Egypt, it is the fifth most prevalent malignant disease 1 The magnitude of the problem is not known in Pakistan 4 The prevalence at autopsy is % where as clinical prevalence is 1.2%, implying that most of the prostate carcinomas do not progress to clinical disease 2 The incidence and prevalence of prostate carcinoma continue to increase with advancing age. Ninety-five percent of prostate carcinoma is diagnosed bet\veen the ages of 45-9 years with a median age of 72 years 5 TNM staging system is commonly used to determine the clinical stage 6 Histological grading is a valuable tool that should be used more systematically and extensively in patient care to determine severity of prostate cancer 7 The clinical course of prostate carcinoma is highly variable and cannot satisfactorily be predicted by histological criteria alone 1 8, although histological grading of the degree of malignancy has been shown to correlate with survival and other measures of biological malignancy9. t helps to predict the mortality rate 1 Gleason's method is a common reference system for histological grading 11 Because of histological variation in tumors, two sets of patterns are recorded for each case. The predominant pattern is designated the primary pattern and the lesser

2 A.K. Hussain et al. pattern is a secondary pattern JO. This grading system takes into account the heterogeneity by scoring both primary and secondary growth patterns The two numerical grades are then added to obtain a combined Gleason score from 2-1. Gleason's grading system has been found to be satisfactory and reproducible by different pathologists 12 The introduction of immunohistochemistry to the study of prostate cancer has provided an opportuni1( for further sub-classification of prostate carcinoma 1 Since the isolation of Prostate Specific Antigen (PSA), specific monoclonal antibodies have been raised 14 These have led to the development of immunohistochemical techniques for diagnostic identification of prostate specific antigen in tissue 14. Tissue prostate specific antigen is currently the most frequently used marker for the identification of normal and pathologically altered prostate tissue 8 A definite correlation exists between tissue prostate specific antigen (PSA) and histological grading for each pathological grade 15 Tissue PSA reactivity in tumor has been used as a predictor of severity of prostate carcinoma. Prostate carcinoma with areas of weak or negative reactivity for PSA, behave in more aggressive manner 16 Tumor associated antigen distribution pattern and tumor heterogeneity can be readily examined by PSA staining 17 Gleason's score is a well-established measure of severity of prostate cancer and tissue PSA staining is currently being tried to ascertain severity and predictive behavior of prostate cancer. Cellularity and percentage of cells exhibiting a positive reaction to the presence of tissue PSA, has been evaluated in this study for possible relationship with Gleason's score. MATERALS AND METHODS This study included a total of 62 cases of prostatic adenocarcinoma received from the Department of Urology and the Department of General Surgery of Shaikh Zayed Hospital, Lahore. The specimens were collected prospectively between the period of January 1999 to July 22, assessing tissue prostate specific antigen expression on prostatic adenocarcinoma and its relationship with the Gleason's score. Detailed history of each of the 62 cases, was recorded, which included age, clinical complaints, nature of biopsy and the histological diagnosis. Gleason's score was performed on routine Haematoxylin and Eosin stained slides. Percentage cellularity was estimated subjectively on the most representative H&E slides. n addition immunohistochemical stains of prostate specific antigen (PSA) was carried out on one of the representative sections from each of the 62 cases, and percentage of cells staining positively with the PSA antibody was estimated. Criteria of inclusion of cases were; biopsy or surgical specimens received in 1% formalin, with histological diagnosis of adenocarcinoma prostate and with well preserved morphology in haematoxylin and eosin sections. Specimens were excluded from the study if received without 1% formalin, were poorly preserved specimens or sections, benign prostate lesions and all other malignancies (local and metastatic) except adenocarcinoma prostate. Relevant clinical data was obtained from the patients and hospital records. The specimens included mostly prostate chippings from transuretheral resections and total prostatectomies. Hematoxylin & Eosin stained sections of slides established the diagnosis of adenocarcinoma. Histological grading and Gleason's scoring was done. Percentage cellularity of the tumour was ascertained by the pathologist's observation of an approximate percentage of tumour cells occupying the section of the tumor visible in the slide. PSA immunohistochemical staining was performed by peroxidase antiperoxidase technique, using commercially available antibodies (YHl) and reagents (DAKO Corporation, USA) according to manufacturers and recommendations. The antibody is reactive to epithelial cells of normal prostate, benign prostate hyperplasia and prostate carcinoma. When tested, no reaction with a panel of other normal tissue was demonstrated. Positive and negative controls were used with each batch of slides. On the immune-peroxidase stained sections observations were made under 4x magnification. Percentage positivity of the tumour cells was estimated by counting 2 tumors cells or acini were counted in at least 2 contiguous fields and 16

3 Relationship of Gleason's Score with Age, Cellularity of Tumor and PSA determining how many of those were positively stained. After staining following formula was applied: y Percentage positivity of tumor cells = ----X 1 2 Y = umber of positively stained tumours cells. 2 = Total number of tumour cells includino-. negative and positively staining cells. When acini were present, one whole acinus was counted as one cell. Therefore 2 acini would be equal to 2 cells in histological grade 1 to. n grade 4, rosette formation of at least 6-7 cells or a single cell was counted as 1. n grade 5 grostate cancer, individual cells were counted 2 Data was tabulated and analyzed with SPSS. For variables, age, Gleason's score, percentage cellularity and percentage positivity of cancer cases, descriptive analysis was carried out, with mean, mode, median and standard deviation. Data was further analyzed with ANOV A and Linear regression exploring the relationships between, age and Gleason's score, percentage cellularity and Gleason's score, and percentage positivity of PSA stain with Gleason's score. RESULTS The age range of the 62 cases of prostatic adenocarcinoma in this study was from 5-9 years, with a mean of 69 years (SD 8.45). Modal age was 7 years, with 15 cases (24.2%) (Table 1). Between the ages of 66-7 years, 29% of the prostate cancer cases were noted. Almost 2 percent cases were 6 years or less of age and 2 percent of the patients with adenocarcinoma were more than 75 years of age. A little less than half of the cases in this study had Gleason's score less than or equal to 6. Commonest Gleason score was 7 (24.2%). Scores less than 4 were noted in 14.5% of the specimens and score of more than 8 were noted in 17. 7% of the carcinoma cases. One fifth of the prostate cancer cases in the study had Gleason's score of 5. Mean Gleason's score of the 62 cases was 6.45 (SD 1.9). Tumours with Gleason's score of 5-7 (moderately differentiated adenocarcinoma) constituted the largest sub-group of 5 out of 62 cases, representing 56.4% of the cancer. Poorly differentiated carcinoma with Gleason's score of 8-1 was found in 29.1 % (18) cases (Table 1 ). ANOV A and Linear regression between age and Gleason's score revealed that no relationship existed between age and Gleason's score, suggesting that varying degree of severity in prostatic carcinoma may exist in all the age group categories in the study (Table & Fig. 1)... 1 VO <> 8 8 <> <> 7 a ~ ~ 9 <> s 8 <> c 6 <> <> ~ s a. 5- <> <> Observed.. Ol 4 < +---~--~ <> Unear Gleason's Score Fig. 1: Relationship between Gleason's score & age of patient Percentage cellularity of the tumours was assessed subjectively from the H&E stained sections. Most of the tumour specimens had high tumor cellularity i.e. 7% of the cases had cellularity greater than 6% in the tissue sections (Fig. 2). Forty percent of specimens had cellularity between 6-8%. As it can be observed that percentage cellularity has minimal positive relationship with Gleason's Score in this study, the histogram of the cellularity percentage distribution among the tissue specimens of prostate cancer revealed a negatively skewed curve with mode around 8%. Regression analysis revealed a low F statistic of with relatively flat slope. This suggests that degree of cellular differentiation (measured by Gleason's score) has little relationship to cellularity of the carcinoma. High and low cellularity could equally exist in cancers that are well differentiated or poorly differentiated, although the statistical estimates suggest a weak positive relationship (Fig. 2). ~ 17

4 A.K. Hussain et al. Table 1: Gleason's score and age groups Gleason's score Age Groups Total < > O Total ~ i 1 E 8 8 C/) Cl) "c: 4 Cl) "' Q)... Observed Rsq.2795 Unoar 1 2 Rsq Cellularity percentage R Square =.27952; F Statistic = ; significant at.1 Fig. 2: Relationship between Gleasons score and percentage cellularity of prostate cancer. On immunohistochemistry, the control slides (normal/benign prostatic hyperplasi~) showed _a strong uniform positive reaction for tissue prostat1c specific ant igen in secretary cells lining the acini, prostatic ducts including the chromaffin ~ells. Basal cells of prostate acini did not contain prostate specific antigen. Transitional epithelium of p~osta~ic ducts and the prostate urethra also did not stam with PSA. mmunohistochemical staining of prostatic adenocarcinoma by PSA was of found to be variable. Prostatic adenocarcinoma, which appeared to consist of a homogenous eosin stained sections, was found to be moderately heterogeneous with respect to the functional differentiation of the cells as stained by immunohistochemistry (Fig. ). The intensity of the immuno staining reaction varied from cell to cell and from area to area. The reaction in neoplastic cells was confined to the cytoplasm with greater concentration of brown granules in the paranuclear area on the luminal aspects of the cells. The tumors with Gleason's score of -4 were composed of acinar structures in well-defined or vague nodules, prostate specific antigen was demonstrated in most cells although intensity of staining varied. The tumours with Gleason's score ranging from 5_ 7 showed cribiform pattern, cord and fused glands of tumours cells, which revealed cytoplasmic PSA staining with a strong positive reaction in the apical cytoplasm of cells on the luminal side of present. Tumours with Gleason's score of 8-1 showed the most heterogeneity with PSA. Some tumours were strongly positive in most of the cells while others showed only clones of cells that were positive. On the whole, as the Gleason's score increased the ability to express prostate specific antigen by the tumor cells seemed to disappear. Degree of nuclear anaplasia seemed to correlate inversely with the degree of glandular acinar differentiation and prostate specific antigen expression of the tumor cells (Fig. 4) Tumors consisting predominantly of cells with higher degree of nuclear anaplasia contained less number of cells positive for prostate specific antigen than those with milder degree of nuclear anaplasia. Evaluating the percentage of positive tumor cells in 62 cases of prostatic adenocarcinoma revealed the mean percentage positivity to be 8.18%. The minimum positivity was 1%. t was seen in one cas~ _w_ith Gleason's score of 1 (Table 2). 1% pos1t1v1ty 18

5 Relationship of Gleason's Score with Age, Cellularity of Tumor and PSA positivity of the tumor. The regression curve of Gleason's score and percentage positivity of tumour cells revealed an inverse quadratic relationship between the two variables, with Beta of -.75 and R square value of.56. The F statistic (ANOVA) was 76.2, significant at.1 (Fig. 5). Percentage positivity varied little between Gleason's score of -7, whereas it varied considerably from Gleason's score 8-1, suggesting that the more differentiated the tumor, higher is the percentage of cells staining positively with PSA antibody reagents. Fig.. The cytoplasm of prostatic cells acquiring brown color with the immunohistochemical stain. o Observed Rsq.749 o linear Rsq 1. Percentage Positivity of tumor cells Fig. 5: Relationship between Gleason's score and percentage positivity of prostatic carcinoma DSCUSSON Fig. 4. Poorly differentiated sheets of tumor cells stained negatively by PSA immunohistochemical stain, whereas neoplastic glandular structure showed strong staining. was seen in 7 case_s with Gleason's score of & 4. The frequency of cancer specimens showing percentage of tumor cells staining positively with tissue PSA immunohistochemical techniqu~ is shown in Table 2. Percentage categories shown here have been made for the purpose of simplification. Majority (12.5%) of the adenocarcinoma specimens had positively staining cells with percentage positivity greater than 8%, negatively skewed on the histogram, suggesting that more cases had cells taking up the immunohistochemical stain. A pattern can be observed in the Table 2 that shows Gleason's score in inverse relationship with percentage Clinical staging is used to determine initial therapy while pathological staging is important in determining prognosis. There is general agreement regarding the prognostic value of histological grade, i.e. poorly differentiated carcinoma shows more aggressive behaviour than well differentiate tumours 1 Of the various grading systems proposed, Gleason's grading/scoring is the most widely accepted 18 Many parameters have been evaluated for their ability to predict outcome in patients with prostate carcinoma, such as clinical stage, pathologic stage, microscopic grading, surgical margins, tumor volume and age: men under 45 with prostate carcinoma have shown poor differentiation and aggressive behaviour of tumor 19 Generally no definite relationship between age and survival is seen 5 19

6 A.K. Hussain et al. Table 2: Gleason's score and percentage positivity of tumor. Gleason's score Percentage Positivi!l'. of Tumor Total Total n our country, autopsies are limited to medicolegal cases only. Histopathological studies are limited to pre-mortem surgical specimens, which include mostly those biopsies that are either from operable tumours or from surgical procedures performed to relieve prostatic symptoms and for diagnostic purposes. Therefore the data obtained does not coincide with the incidence of prostate carcinoma in the general population 8 For this reason the inclusion criteria in this series of 62 cases is based on histopathological confirmation only. Despite the limitations, this study shares several features with earlier published work. The age incidence of our study (5-9 years) is close to the ranges of other studies, such as reported by Aihara et al in , of 4 to 79 years for prostatic carcinoma. Pathological diagnosis of prostate carcinoma is based on nuclear anaplasia, invasion and architectural disturbances 8. Glands of adenocarcinoma of prostate tend to have discreet, crisp, sharp, luminal border without undulation or ruffling of cytoplasm 18 Nuclei are usually larger and hyperchromatic 8 Some studies have demonstrated an apparent correlation between tissue PSA staining variability in field to field /cell-to-cell and increasing tumor grades 19 2 Similar to normal and hyperplastic prostatic epithelium, the tissue PSA reaction in neoplastic cells was confined to the cytoplasm with greater concentration of brown granules in the paranuclear area on the luminal aspect of cells 14 Attempts have been made to correlate degree of tumour differentiation with immunohistochemical staining. The immuno-peroxidase method for PSA is highly sensitive and specific for staining of prostatic epithelium. Nadji et al. 14 noted 1% diagnostic specificity and 94% sensitivi7i of PSA, confirmed by Wright et al. 17, Aihara et al. 2 Prostate carcinoma demonstrates heterogeneity in cell-to-cell and field-to-field staining. These features were also highlighted in our study. Allsbrook and Simms 1 noted a correlation between staining variability and tumour grades. This study also revealed that staining with PSA decreased with increased Gleason's scoring i.e. the percentage of cells staining positively with PSA antibody decreased with decreasing differentiation 21 Cellularity and age of patient had little or no relationship to histological severity of prostate cancer, suggesting that prostate cancers may have high or low proportions of cells in histological specimens and that prostatic cancer of high or low Gleason's score may be found in men, irrespective of age. Partin et al. 15 and Grande et al. 22 observed lowering of tumor PSA expression with increasing Gleason's scores. However, Hammond et a!. 2 found no statistically significant correlation between scoring and extent of staining. Jung et al. 21 also did not find any relationships between tissue PSA content and the cell differentiation. Observations and calculations made in this study of 62 prostatic adenocarcinomas do not reveal results in agreement with the studies of Hammond et al and Jung et al. Grande et al. 22 observations of higher scoring 2

7 Relationship of Gleason's Score with Age, Cellularity of Tumor and PSA malignancy is correlated to lower tissue PSA expression and that represents loss of function by decrease production of secretary proteins, which corresponds to nuclear DNA abnormalities. Epstein and Eggleston 24 found statistically significant correlation between foci of poor immunohistochemical staining and progression of disease. Pousette et al. 25 concluded that a single analysis of tissue PSA at the time of diagnosis could predict the clinical out come in most cases. This study can be compared to that of Pousette's, in which single immuno-histological analysis of surgical specimen has been utilized for PSA staining to determine severity of histological grade. CONCLUSON As our study was limited to histological examinations, further studies will be required that include patients follow-up to measure and to conduct survival analysis. Cellularity of tumor tissue in a specimen bears little or no relationship with Gleason's score, however percentage positivity of staining with immunohistochemical tissue PSA inversely correlate with Gleason's score. Percentage positivity can be used to complement Gleason's score for more definitive diagnosis of the histological grade, particularly for scores less than 8 and percentage cellularity of more than 8%. REFERENCES 1. Ahmad M, Abd-elmotelib F, Farag RM, Zaida NA, Khalifa A. Evaluation of some tissue and serum biomarkers in prostatic carcinoma among Egyptian males. Clin Biochem: 1999; 2: Duffy MJ. PSA as a marker of prostate cancer. Ann Clin Biochem: 1996; : saac J. Molecular markers for prostate cancer metastasis. Am J Pathol: 1997; 15: Ghafoor F, Khan S, Suleman B, Khan AU. Evaluation of prostate specific antigen as a tumour marker in cancer prostate. J Pak Med Assoc: 1998; 48: Meikle AW, Smith JA. Epidemiology of prostate cancer. Urol Clin North Am: 199; 17: Speissal B, Beahrs OH, Hamanck P, Hutter RVP, Scheibe, Sobin LH. UCC TNM Atlas, illustrated guide to the JNM/pTNM classification of malignant tumors. Springier and Verb rd Ed: 1992; Oesterling JE, Rice DC, Olenski WJ, Bergstralh EJ. Effect of cystoscopy, prostate biopsy and transuretheral resection of prostate on serum prostate-specific antigen concentration. Adult Urology: 199; 42: Mostofi FK, Davis CJ, Sesterhenn A. Pathology of carcinoma of the prostate. Cancer: 1992; 7: Mohler JL, Partin AW, Epstein J, Becker RL, Mikel VV, Sesterhenn A. Prediction of prognosis in untreated stage A2 prostatic carcinoma. Cancer: 1992; 69: Gleason DF, Mellinger GT. Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J Urol: 1974; 111: Gleason DF. Histologic grading of prostate cancer: A perspective. Hum Pathol: 1992; 2: Aihara M, Lebovitz RM, Wheeler TM, Kinner BM, Ohorio M, Scardino PT. Prostate specific antigen and Gleason grade: an immunohistochemical study of prostate cancer. JUrol: 1994; 151: Allsbrook WC, Simms WW. Histochemistry of prostate. Hum Pathol: 1992; 2: Nadji M, Tabei SZ, Castro A, Cu TM, Murphy GP, Wang MC. Prostate acid phosphtase: an immunohistologic marker for prostatic neoplasm. Cancer: 1981; 48: Partin AW, Carter HB, Chan DW, Epstein J, Oesterling JE, Rock RC. Prostate-specific antigen in staging of localized prostate cancer influence of tumor differentiation. J Urol: 199; 14: Bemer A, Waare H, Nesland JM, Paus E, Danielsen HE, Fossa SD. DNA ploidy, serum prostate specific antigen histological grade and immunohistochemistry as predictive parameters of lymphnode metastasis in Tl -T 21

8 A.K. Hussain et al. MO prostate adenocarcinoma. Br J Urol: 1995; 75: Wright GL, Beckett ML, Starling JJ, Schellhammer PF, Seig SM, Ladaga LE. mmunohistochemical localization of prostate Ca antigens. Can Res: 198; 4: Vernon SE, William WD. Pre-treatment and post-treatment evaluation of prostatic adenocarcinoma for prostatic specific acid phosphatase and prostate specific antigen by immunohistochemistry. J Urol: 198; 1: Mikulajczyk SD, Millar LS, Wang TJ, Rettinboose HG, Wolfert RL, Mark SL. A specific molecular form of free prostate specific antigen in found hyperplasia. Adult Urol: 2; 55: Hussain A, Riaz S, Rahman S. Relationship between Gleason's Score and mmunohistochemical staining, intensity of tissue PSA in prostate cancer. Pak. J. of path: 26; 17: Jung K, Brox B, Lein M, Rudolph B, Kristiansen G, Hauptmann S, et al. Molecular forms of prostatic antigen in malignant and benign prostatic tissue: Biochemical and diagnostic implication. Clin Chem: 2; 46: Grande M, Calstorm K, Lundh RB, Eneroth P, Stege R, Pousette A. Tissue concentration of tissue polypeptide antigen and prostatic specific antigen in 42 patients with prostatic carcinoma. Prostate: 2; 45: Hammond NE, Sause WT, Martz KL, Plepich MV, Asbell SO, Rubin P, Myers RP, Farrow GM. Correlation of prostate specific acid phosphatase and prostate specific antigen immunocytochemistry with survival in prostate carcinoma. Cancer: 1989; 6: Epstein n, Eggleston JC. mmunohisto- chemical localization of prostate specific acid phosphatase and prostate specific antigen in stage A2 adenocarcinoma of the prostatic. Human Pathol: 1984; 15: Pousette A, Grande M, Codstrom K, Sege R. Tissue PSA is the best predicting variable for the outcome of endocrine treatment of prostatic carcinoma. Scand J Clin Lab nvest Suppl: 1999; 229: The Authors: Afshan Kamran Hussain Assistant Professor, Department of Pathology Fatima Memorial Hospital Medical & College, Shadman, Lahore Address: afskamhus@hotmail.com Sabiha Riaz Professor of Histopathology Fatima Memorial Hospital Medical & Dental College, Shadman, Lahore Address: sabihariaz@hotmail.com Dental Saeed ur Rahman Assistant Professor, Department of Community Medicine Lahore Medical & Dental College, Tulspura, Canal Bank North, Lahore sanjhi@hotmail.com Address for Correspondence: Afshan Kamran Hussain Assistant Professor, Department of Pathology Fatima Memorial Hospital Medical & College, Shadman, Lahore Address: afskamhus@hotmail.com Dental 22