Μελέτη του καρκίνου του προστάτου με την εφαρμογή μεθόδων ανοσοκυτταροχημείας, in situ υβριδισμού και ανάλυσης εικόνας: συσχέτιση με προγνωστικούς παράγοντες και την κλινική πορεία των ασθενών

Abstract

The aim of this study was to investigate by in situ hybridization procedure the Telomerase expression, by immunostaining methods the expression of cell proliferation markers (p120, Ki-67, PCNA), apoptotic markers (p53 and bcl-2) and DNA ploidy by the image analysis, and to correlate these results with several prognostic factors concerning prostate cancer.Samples were obtained, from 70 patients who underwent radical prostatectomy for prostatic adenocarcinoma immediately after prostate removal at the operating theatre. Patients age ranged from 59 to 75 years (mean 67.11 years). Imprint smears were taken from different areas of macroscopically estimated prostatic cancer.The histopathological diagnoses were performed using sections from the same samples that were used for the imprints. The TNM system (based on the staging system of the American Joint Committee on Cancer) was used for pathologic staging and grading and the primary cancer was evaluated according to the Gleason score system.According to the pathological examination the tumour stages of the patients were as follows: stage T2a,T2b in 61(87,2%), stages T2cand T3a in 9 (12,8%). The Gleason score was:2-6 in 49 (70%) and >7 in 21 (30) of the cases. The tumour differentiation was high and moderate in 60 (85,7%) and low in 10 (14,3%) of the patients. The PSA value of our patients was 0-9,9 ng/ml in 49 (70%) and >10 ng/ml in 21 (30%).In situ hybridization method was used for the detection of Telomerase activity . Telomerase positivity was scored as follows: staining of <5% of celis(-), 5-25%(+), 26-74%(++), 75-100%(+++). Immunocytochemical staining was performed by the Avidin Biotin method (ABC complex technique) for the proliferate markers and apoptotic markers. PCNA, Ki-67 p120, p53 and bcl-2 positivity was scored as follows: staining of <10% of cells(-), 10-25%(+), 26- 74%(++), 75-100%(+++). The DNA content (DNA index) was studied (Feulgen stain, SAMBA 2005 and Compaq P.C) by the image analysis technique.Telomerase positive expression was detected in 62 (88.6%) prostate cancer studied smears, 8 (11.4%) prostate cancer smears were entirely negative. Telomerase expression was shown to correlate significantly with the degree of Gleason score (p<0.001 ), with tumour differentiation (p<0.001 ), with increase pretreatment PSA serum levels (>10ng/ml) (p<0.002). in contrast there was no correlation between Telomerase expression and pathological stage(p<0.56). The multiple linear regression model showed that the most significant variables associated with Telomerase expression were the Gleason score (p=0,0001) and PSA serum levels (p=0.0125)The hormone levels were found to be in normal levels according to the age of the patients. There was a statistically significant increase in serum testosterone (p<0,001), FSH (p=0,0001) and LH (p=0,0004), and decrease in serum DFIT (p<0,0001) one year after the surgical procedure.According to the results of our study during the first postoperative month was observed a decrease of PSA levels (0,0 ng/ml) a part of two who had PSA value 0,02 and 0,4 ng/ml respectively. During the first postoperative year the majority of PSA values remained 0,0 ng/ml, thought in two new patients an increase in PSA value was observed, who were characterized as biochemical recurrence (0,09-2,15 ng/ml). In the second yearly control we observed a limited increase in PSA levels and a new recurrence (3,00-4,00ng/ml) in the end of third there was no other increase of PSA value in new patient, negative patients 65(92,3%) and positive patients 5 (7,7%).The DNA ploidy patterns (DNA index) of the entire group were as follows: 67,1 % were euploid, 32,9 % were aneuploid. The DNA index was statistically correlated with Gleason score (p<0.001), with tumour differentiation (pO.001), pretreatment PSA serum levels (>10ng/ml ) (p<0.001). There was not statistically significant correlation between DNA index and pathological stage (p:0,33). The multiple linear regression model(Pearson test) showed that the most significant variables associated with DNA index were the Gleason score (p<0,0001) , PSA serum levels (p<0,0001 ) and tumour differentiation (p<0,0001 ).Positive expression of p120, Ki-67 and PCNA was detected in 64 (91.4%), 63 (90%) and 60 (85.7%) of the prostate adenocarcinoma smears, respectively. The expression of proliferative markers was statistically correlated with Gleason score (p<0.001 ), tumour differentiation (p<0.001 ) and pretreatment PSA serum levels (>10ng/ml ) (p<0.001). There was not correlation between P 120, ki-67 and pathological stage (p:0,22 and p=0,69 for p120 and ki67 respectively). In contrast PCNA immunostaining values were statistically significant (p<0.001).Positive expression of p53 and bcl-2 was detected in 50 (71,4%), 16 (22,85%) prostate adenocarcinoma smears, respectively. The expression of markers p 53 and bcl-2, was correlated with Gleason score (p<0.001 for p53 and p=0,005 for bcl-2)), with tumour differentiation (p<0.001 for p53 and bcl-2) and pretreatment PSA serum levels (>10ng/ml ) (p<0.001 for p53 and bcl-2). The distribution of p 53 and bcl-2 expression in prostate carcinomas according to histopathological staging has no statistical difference for T2a (p:0,61 for p53) and T2b (p:0,24 for bcl-2). In contrast p53 and bcl-2 immunostaining values were statistically significant (p<0.001) for T2c and T3a stages.Long rank test and Cox's Model demonstrated that Telomerase (p=0,009), PCNA (p=0,001), Ki-67 (p=0,003), p120 (p=0,009), p53 (p=0,005) and bcl-2 (p=0,007) expression and DNA index, had significant prognostic values for disease free recurrence. Recurrence function was calculated according to the Kaplan-Meier method with Telomerase expression, proliferate and apoptotic markers and DNA index.P120, Ki-67, PCNA, p53 and bcl-2 expression had significant prognostic value for the disease free recurrence. However, a higher recurrence was noted for those patients (5) who had high (++ or +++) expression in compared with those (65) who had low (+) expression for a period of 6-36 months (mean 21 months)Conclusions: Our results suggest that Telomerase, p120, ki67,PCNA, p53 and bcl-2 expression and DNA ploidy appear to be additional The aim of this study was to investigate by in situ hybridization procedure the Telomerase expression, by immunostaining methods the expression of cell proliferation markers (p120, Ki-67, PCNA), apoptotic markers (p53 and bcl-2) and DNA ploidy by the image analysis, and to correlate these results with several prognostic factors concerning prostate cancer.Samples were obtained, from 70 patients who underwent radical prostatectomy for prostatic adenocarcinoma immediately after prostate removal at the operating theatre. Patients age ranged from 59 to 75 years (mean 67.11 years). Imprint smears were taken from different areas of macroscopically estimated prostatic cancer.The histopathological diagnoses were performed using sections from the same samples that were used for the imprints. The TNM system (based on the staging system of the American Joint Committee on Cancer) was used for pathologic staging and grading and the primary cancer was evaluated according to the Gleason score system.According to the pathological examination the tumour stages of the patients were as follows: stage T2a,T2b in 61(87,2%), stages T2cand T3a in 9 (12,8%). The Gleason score was:2-6 in 49 (70%) and >7 in 21 (30) of the cases. The tumour differentiation was high and moderate in 60 (85,7%) and low in 10 (14,3%) of the patients. The PSA value of our patients was 0-9,9 ng/ml in 49 (70%) and >10 ng/ml in 21 (30%).In situ hybridization method was used for the detection of Telomerase activity . Telomerase positivity was scored as follows: staining of <5% of celis(-), 5-25%(+), 26-74%(++), 75-100%(+++). Immunocytochemical staining was performed by the Avidin Biotin method (ABC complex technique) for the proliferate markers and apoptotic markers. PCNA, Ki-67 p120, p53 and bcl-2 positivity was scored as follows: staining of <10% of cells(-), 10-25%(+), 26- 74%(++), 75-100%(+++). The DNA content (DNA index) was studied (Feulgen stain, SAMBA 2005 and Compaq P.C) by the image analysis technique.Telomerase positive expression was detected in 62 (88.6%) prostate cancer studied smears, 8 (11.4%) prostate cancer smears were entirely negative. Telomerase expression was shown to correlate significantly with the degree of Gleason score (p<0.001 ), with tumour differentiation (p<0.001 ), with increase pretreatment PSA serum levels (>10ng/ml) (p<0.002). in contrast there was no correlation between Telomerase expression and pathological stage(p<0.56). The multiple linear regression model showed that the most significant variables associated with Telomerase expression were the Gleason score (p=0,0001) and PSA serum levels (p=0.0125)The hormone levels were found to be in normal levels according to the age of the patients. There was a statistically significant increase in serum testosterone (p<0,001), FSH (p=0,0001) and LH (p=0,0004), and decrease in serum DFIT (p<0,0001) one year after the surgical procedure.According to the results of our study during the first postoperative month was observed a decrease of PSA levels (0,0 ng/ml) a part of two who had PSA value 0,02 and 0,4 ng/ml respectively. During the first postoperative year the majority of PSA values remained 0,0 ng/ml, thought in two new patients an increase in PSA value was observed, who were characterized as biochemical recurrence (0,09-2,15 ng/ml). In the second yearly control we observed a limited increase in PSA levels and a new recurrence (3,00-4,00ng/ml) in the end of third there was no other increase of PSA value in new patient, negative patients 65(92,3%) and positive patients 5 (7,7%).The DNA ploidy patterns (DNA index) of the entire group were as follows: 67,1 % were euploid, 32,9 % were aneuploid. The DNA index was statistically correlated with Gleason score (p<0.001), with tumour differentiation (pO.001), pretreatment PSA serum levels (>10ng/ml ) (p<0.001). There was not statistically significant correlation between DNA index and pathological stage (p:0,33). The multiple linear regression model(Pearson test) showed that the most significant variables associated with DNA index were the Gleason score (p<0,0001) , PSA serum levels (p<0,0001 ) and tumour differentiation (p<0,0001 ).Positive expression of p120, Ki-67 and PCNA was detected in 64 (91.4%), 63 (90%) and 60 (85.7%) of the prostate adenocarcinoma smears, respectively. The expression of proliferative markers was statistically correlated with Gleason score (p<0.001 ), tumour differentiation (p<0.001 ) and pretreatment PSA serum levels (>10ng/ml ) (p<0.001). There was not correlation between P 120, ki-67 and pathological stage (p:0,22 and p=0,69 for p120 and ki67 respectively). In contrast PCNA immunostaining values were statistically significant (p<0.001).Positive expression of p53 and bcl-2 was detected in 50 (71,4%), 16 (22,85%) prostate adenocarcinoma smears, respectively. The expression of markers p 53 and bcl-2, was correlated with Gleason score (p<0.001 for p53 and p=0,005 for bcl-2)), with tumour differentiation (p<0.001 for p53 and bcl-2) and pretreatment PSA serum levels (>10ng/ml ) (p<0.001 for p53 and bcl-2). The distribution of p 53 and bcl-2 expression in prostate carcinomas according to histopathological staging has no statistical difference for T2a (p:0,61 for p53) and T2b (p:0,24 for bcl-2). In contrast p53 and bcl-2 immunostaining values were statistically significant (p<0.001) for T2c and T3a stages.Long rank test and Cox's Model demonstrated that Telomerase (p=0,009), PCNA (p=0,001), Ki-67 (p=0,003), p120 (p=0,009), p53 (p=0,005) and bcl-2 (p=0,007) expression and DNA index, had significant prognostic values for disease free recurrence. Recurrence function was calculated according to the Kaplan-Meier method with Telomerase expression, proliferate and apoptotic markers and DNA index.P120, Ki-67, PCNA, p53 and bcl-2 expression had significant prognostic value for the disease free recurrence. However, a higher recurrence was noted for those patients (5) who had high (++ or +++) expression in compared with those (65) who had low (+) expression for a period of 6-36 months (mean 21 months)Conclusions: Our results suggest that Telomerase, p120, ki67,PCNA, p53 and bcl-2 expression and DNA ploidy appear to be additional.

Διερευνήθηκε η βιολογική συμπεριφορά του καρκίνου του προστάτη, σε επιχρίσματα αποτυπωμάτων χειρουργικά εξαιρεθέντων καρκινωμάτων με τεχνικές ανοσοκυτταροχημείας, in situ υβριδισμού και ανάλυσης εικόνας. Για τον σκοπό αυτό εχρησιμοποιήθησαν νεοπλασματικοί δείκτες όπως Τελομεράση, δείκτες κυτταρικού πολλαπλασιασμού (PCNA, ki-67, ρ120), και δείκτες απόπτωσης (ρ53 και bcl-2) καθώς επίσης εκτιμήθηκε η πλοειδία του DNA των καρκινικών κυττάρων. Τα ευρήματα που προέκυψαν, συσχετίσθηκαν με τους κλασικούς προγνωστικούς παράγοντες και με την κλινική πορεία των ασθενών.Το υλικό της παρούσης μελέτης προέρχεται από 70 ασθενείς με εντοπισμένο αδενοκαρκίνωμα του προστάτου, οι οποίοι υποβλήθηκαν σε ριζική οπισθοηβική προστατεκτομή. Η ηλικία των ασθενών κυμαινόταν από 59 έως και 75 έτη (μέση ηλικία 67,11 έτη).Αντικείμενο της μελέτης αποτέλεσαν επιχρίσματα αποτυπωμάτων των ως άνω εξαιρεθέντων καρκινωμάτων. Τα αποτυπώματα ελήφθησαν από διαφορετικά σημεία του χειρουργικού παρασκευάσματος, τα οποία μακροσκοπικά θεωρήθηκαν ύποπτα για κακοήθεια.Σε όλους τους ασθενείς είχε προηγηθεί λεπτομερής κλινικός και εργαστηριακός έλεγχος (διορθρικό υπερηχοτομογράφημα και βιοψία του προστάτη, αξονική τομογραφία, σπινθηρογράφημα οστών, τιμές PSA του ορού).Η ιστοπαθολογική διάγνωση πραγματοποιήθηκε με την χρήση τομών από τα ίδια δείγματα τα οποία χρησιμοποιήθηκαν και για τα αποτυπώματα. Η σταδιοποίηση της νόσου, η οποία στηρίζεται στα χειρουργικά και ιστολογικά ευρήματα έγινε σύμφωνα με το σύστημα ΤΝΜ (ΤΝΜ systeri American Joint Committee of Cancer). Κανείς από τους ασθενείς δεν αντιμετωπίσθηκε με ανδρογονικό αποκλεισμό ή ακτινοβολία πριν από την χειρουργική επέμβαση.Σύμφωνα με την παθολογοανατομική εξέταση η νόσος ήταν σε αρχικά στάδια (T2a-T2b) σε 61 (87,2%) και σε προχωρημένα (T2c,T3a) σε 9 (12,8%) ασθενείς. Το άθροισμα κατά Gleason ήταν (2-6) σε 49 (70%) και >7 σε 21 (30%) περιπτώσεις. Η ιστολογική διαφοροποίηση των καρκινωμάτων ήταν υψηλή και μέτρια σε 60 (85,7%) και χαμηλή σε 10 (14,3%) περιπτώσεις. Οι προεγχειρητικές τιμές του PSA των ασθενών μας είχε ως εξής 0-9,9ng/ml σε 49 (70%) και >10 ng/πιΙ σε 21 (30%) ασθενείς.Η κατάδειξη της έκφρασης της Τελομεράσης μελετήθηκε με τη μέθοδο του in situ υβριδισμού. Η χρωστική έκφραση της Τελομεράσης στους πυρήνες των κυττάρων εκτιμήθηκε ως εξής: χρώση <5% αρνητική (-), 5-25% ασθενώς θετική (+), 26-74% μετρίως θετική (++) και 75-100% εντόνως θετική (+++). Η έκφραση των δεικτών κυτταρικού πολλαπλασιασμού και απόπτωσης κατεδείχθη με τη μέθοδο της ανοσοκυτταροχημείας και ειδικότερα με την τεχνική αβιδίνης βιοτίνης (ABC complex tecnique). Η πυρηνική έκφραση των δεικτών PCNA, ki-67, ρ-120, ρ53 και η κυτταροπλασματική έκφραση του bcl-2 εκτιμήθηκε ως εξής: έκφραση <10% αρνητική (-), 10-25% ασθενώς θετική (+), 26-74% μετρίως θετική (++) και 75-100% εντόνως θετική (+++). Η περιεκτικότητα των κυττάρων σε DNA (δείκτης DNA) εκτιμήθηκε με τη μέθοδο της ανάλυσης εικόνας (χρώση Feulgen και σύστημα SAMBA 2005 με υπολογιστή Compaq).Τα αποτελέσματα που προέκυψαν από τη μελέτη των μετρήσεων του δείκτη DNA και της έκφρασης της Τελομεράσης, των δεικτών κυτταρικού πολλαπλασιασμού και απόπτωσης συσχετίσθηκαν με το στάδιο της νόσου, το βαθμό διαφοροποίησης των καρκινωμάτων, το άθροισμα κατά Gleason και τις τιμές του PSA του ορού προεγχειρητικά.Θετική έκφραση της Τελομεράσης διαπιστώθηκε σε 62(88,6%) των επιχρισμάτων του προστάτου ενώ αρνητική σε οκτώ επιχρίσματα 8(11,4%). Στατιστικά σημαντική συσχέτιση παρατηρήθηκε μεταξύ της έκφρασης της Τελομεράσης και του αθροίσματος κατά Gleason (ρ<0,001), με την ιστολογική διαφοροποίηση του καρκινώματος (ρ<0,001) και με τα αυξημένα επίπεδα του PSA (PSA>10ng/ml) του ορού προεγχειρητικά. (ρ<0,002). Αντίθετα, δεν υπήρξε συσχέτιση μεταξύ της εκφράσεως της Τελομεράσης και του σταδίου της νόσου (ρ=0,56). Η πολυπαραγοντική ανάλυση της μελέτης μας για την έκφραση της Τελομεράσης έδειξε ότι η πιο σημαντική μεταβλητή ήταν το άθροισμα κατά Gleason (ρ=0,001) και οι τιμές του PSA του ορού (ρ=0,001).