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Preliminary study of tumor DNA content in resectable primary colorectal carcinoma in Taiwan
CYTOGENETICS PRELIMINARY STUDY OF TUMOR DNA CONTENT IN RESECTABLE PRIMARY COLORECTAL CARCINOMA IN TAIWAN
DNA PLOIDY ANALYSIS IN WILMS' TUMOR, WILMS' TUMOR VARIANTS AND NEPHROBLASTOMATOSIS.
W-J Chen', H-L Eng, H-A Fan. Departmentsof Pathology and Colorectal Surgery, Chang Gung Memorial Hospital (Kaohsiung),Taiwan
H.S. Preston'. R. W. Bvard. M. Donovan. L. Moore. Lab. Med. Services, Caulfield Gen. Med. Centre, Victoria: Department of Histopathology, Adelaide Children's Hospital (ACH), South Australia.
Colorectalcancer accountsfor over 90% of rnalignantneoplasrnsof the intestine and is one of the leading causes of cancer death in the Western world. The incidence in Southeast Asia has increased over the years. It is one of the leading causes of cancer death in Taiwan. Several published reports suggested that tumor DNA content is an important prognostic indicatorof colorectalcarcinoma. During the period from Aug '91 to Mar '92, operation of 166 cases of colorectal carcinoma was performed in our hospital. There are 85 males with mean age of 58.91 12.55 and 81 femaleswith mean ageof 54.41 5 12.55. Analysisof tumorDNArevealsthat thereare 50 diploid tumorsand 116 aneuploidturnors. Among the aneuploid tumors, 12 are hypodipbid,97 hyperdiploid,Z tetraploidand 5 multiploid. No. of Patients (%) Dukes' Slage
A
01 82
C D
Mulliploid
(n=12)
Telrapbid
(n=50)
(n=97)
(n=2)
(n=5)
3 (6.0) 8 (16.0) 22 (44.0) 10 (20.0) 7 (14.0)
1 (8.3) 0 3 (25.0) 5 (41.7) 3 (25.0)
4 (4.1) 7 (7.2) 38 (39.2) 37 (38.2) 11 (11.3)
0 0 1 (50.0) I(50.0) 0
Olpbid
Hypodploid
Hypldploic
~
0 1 (20.0) 1 (20.0) 3 (60.0) 0
~~
With correlation to Dukes' staging, it shows that 48% of aneuploid tumors are in Stage C. whereas only 20% diploid tumor in Stage C. The finding is in consistent with literature reports that aneuploid colorectal cancertended to invade the serosaor extend beyond. Furtherclinicalfollow up is necessary in order to evaluate the overall survival with respect to DNA content. Presen1er:Dr. W-J Chen
p53 EXPREsSICld I N SDJAkOUS CELL CAXINOMAS OF SKIN, E R A T Q A ~ SOLAR ~ , KERWXE AND SEBORIWOEIC KE-ES.
G.S. B a l a s u b r d a m * , R. O'Keefe, J. Catimel m u J. M e . D,"partrrent of Anatmica Pathology, St. Vincent's Hospital and university of mlbourne, V i c t o r i a .
p53 is a suppressor oncogene whicn i s t n o q h t to play an inportant role i n t m r i g e n e s i s . W e investigated the expression of p53 i n 1 2 squan-ous cell ,carcinarnas 15 solar of skin (SCCs), 1 4 k e r a t o a c a n t h m (a), keratoses (SKs) and 11 seborrhceic keratoses (seb KS). -rate to intense nuclear staining f o r p53 w a s observed in 93% (14/15) of SKs. The l a c e l l i n g w a s diffuse in about half the cases and patchy o r focal i n others. Forty one percent (5/12) of SCCS s h a d nuclear p o s i t i v i t y . Strong staining w a s observed in the single p r l y d i f f e r e n t i a t e d carcinm w h i l s t in the 4 w e l l d i f f e r e n t i a t e d carcinomas the s t a i n i n g w a s focal and of m d e r a t e intensity. O f the 1 4 cases of KAs, only L s h m d mild focal nuclear p o s i t i v i t y . Seborrfioeic keratoses were largely negative except for 2 which shcwed occasional positive nuclei. 'Iko SCCs a r i s i n g frcm SKs were h m o n e g a t i v e . Increased imnunopsit;lvity was observed i n the adjacent c t y o l q i c a l l y nolmal epithelium i n both SCCs and SKs but not i n KAS or Seb KS. O u r r e s u l t s suggest t h a t pS3 plays an irrportant role i n t h e evolution of SCCs of skin with m a x h d expression i n the i n s i t u lesion SK. The presence of p53 i n the adjacent cytologically n o d epithelium may represent a marker of early malignant t r a n s f o m t i a n . The absence of such increased expression i n the epitneliun adjacent to KAs supports the v i e w that KAs and SCCs are ism d i s t i n c t neoplasms r a t h e r than a spectnim of t h e sam neo@stic process.
Wilms' tumor is a complex embryonic tumor of the kidney occurring most commonly in children under 10 years of age. Stage and histology are currently considered to be the best predictors of outcome. In approximately 25% of cases Wilms' tumor is associated with nephroblastomatosis in the same or contralateral kidney. Much less common are cystic variants of Wilms' tumor; cystic nephroma (CN), also known as multilocular cyst of the kidney and cystic partially differentiated nephroblastoma (CFDN). In general these cystic variants have an excellent prognosis when treated by nephrectomy alone although CPDN may rarely be locally aggressive. Since methods for analysing the DNA content of nuclei extracted from paraffin blocks have become available, flow cytometry has been performed on a range of tumors and has been shown to correlate with outcome. We wished to determine if there was any difference in DNA content between typical favourable histology Wilms' tumor and the less common cystic variants and precursor lesions. Flow cytometry was used to analyse the DNA content of Wilms' (4 cases), CPDN with Wilms' tumor (1 case), CPDN (1 case), CN (2 cases) and nephroblastomatosis (1 casel. The 9 cases were processed by a modified Hedley technique. The specimens were stained with propidium iodide and analysed on a Coulter EPICS Profile II flow cytometer using a 488nm air-cooled laser. A minimum of 15000 nuclei were counted in each case. In all cases a diploid DNA content was found. The mean half peak C.V. value for the major peaks was 3.34 and the median half-peak C.V. was 3.22. The S phase fraction varied from 0.19% with a mean of 7.1%. It appears from these preliminary data that DNA ploidy does not distinguish between Wilms' tumor and variants although it is possible that S phase fraction may prove to be of benefit in determining malignant potential. We are currently undertaking a more extensive study to determine if DNA content or S phase fraction can be used to predict treatment failure in favourable histology Wilms' tumor.
STABILITY OF PROPlDlUM IODIDE STAINED SAMPLES BY M E COULT€RR~ DNA PREP WORKSTATION FOR ANALYSIS BY FLOW CYTOMElRY J. Drew, M. Donovan and H. Preston'. Laboratory Medicine Services, Caulfield General Medical Centre, Melbourne, Victoria
At the Caulfield General Medical Centre, paraffin-embedded specimens analysed for DNA ploidy are prepared by a modification of the 'Hedley Technique: Nuclear preparations are stained by the COULTEW DNA Prep Reagent System. This is a commercial workstation designed to prepare cell or nuclear suspensions for quantitative determination of nuclear DNA content by Flow Cytometry (FCM). The reagents comprise non-ionic detergents for cell lysing and permeabilisation, Propidium Iodide (PI) for DNA staining, and avian erythrocyte nuclei as an internal reference standard. The manufacturers instructions specity that to obtain optimal coefficients of variation (CVs), samples should be incubated in PI at room temperature (RT) for 15 - 17 minutes prior to analysing by FCM and must bei analysed within 2 hours of staining. This investigation set out to determine the stability of the stained specimens beyond this 2 hour limit. After routine analysis of a group of stained samples, the batch was re-analysed at different intervals ranging from 24 hours lo 14 days. Samples were stored at RT and protected from light. The values from the histograms coilected and compared over this period included: the DNA Index (DI) ; percent half peak C V s (%HPCV); mean channel fluorescence of the major peak(s) in the histogram; and percent of events found in each peak(s). Examination of the histograms after 24 hours demonstrated no alteration in the Di of the specimen,an average reduction of 10% in the %HPCV, 10% increase In the mean channel fluorescence and a 5% decrease In the percent of events found in the peaks. Re-analysis over a two week period showed these values to remain stable. Less than 10% of the samples deteriorated and were uninterpretable at the end of 14 days. The data shows that stained nuclear preparations are stable beyond the time recommended by the manufacturers and allows for storage of stained specimens with minimal deterioration.
DNA PLOIDY ANALYSIS IN WILMS' TUMOR, WILMS' TUMOR VARIANTS AND NEPHROBLASTOMATOSIS.
W-J Chen', H-L Eng, H-A Fan. Departmentsof Pathology and Colorectal Surgery, Chang Gung Memorial Hospital (Kaohsiung),Taiwan
H.S. Preston'. R. W. Bvard. M. Donovan. L. Moore. Lab. Med. Services, Caulfield Gen. Med. Centre, Victoria: Department of Histopathology, Adelaide Children's Hospital (ACH), South Australia.
Colorectalcancer accountsfor over 90% of rnalignantneoplasrnsof the intestine and is one of the leading causes of cancer death in the Western world. The incidence in Southeast Asia has increased over the years. It is one of the leading causes of cancer death in Taiwan. Several published reports suggested that tumor DNA content is an important prognostic indicatorof colorectalcarcinoma. During the period from Aug '91 to Mar '92, operation of 166 cases of colorectal carcinoma was performed in our hospital. There are 85 males with mean age of 58.91 12.55 and 81 femaleswith mean ageof 54.41 5 12.55. Analysisof tumorDNArevealsthat thereare 50 diploid tumorsand 116 aneuploidturnors. Among the aneuploid tumors, 12 are hypodipbid,97 hyperdiploid,Z tetraploidand 5 multiploid. No. of Patients (%) Dukes' Slage
A
01 82
C D
Mulliploid
(n=12)
Telrapbid
(n=50)
(n=97)
(n=2)
(n=5)
3 (6.0) 8 (16.0) 22 (44.0) 10 (20.0) 7 (14.0)
1 (8.3) 0 3 (25.0) 5 (41.7) 3 (25.0)
4 (4.1) 7 (7.2) 38 (39.2) 37 (38.2) 11 (11.3)
0 0 1 (50.0) I(50.0) 0
Olpbid
Hypodploid
Hypldploic
~
0 1 (20.0) 1 (20.0) 3 (60.0) 0
~~
With correlation to Dukes' staging, it shows that 48% of aneuploid tumors are in Stage C. whereas only 20% diploid tumor in Stage C. The finding is in consistent with literature reports that aneuploid colorectal cancertended to invade the serosaor extend beyond. Furtherclinicalfollow up is necessary in order to evaluate the overall survival with respect to DNA content. Presen1er:Dr. W-J Chen
p53 EXPREsSICld I N SDJAkOUS CELL CAXINOMAS OF SKIN, E R A T Q A ~ SOLAR ~ , KERWXE AND SEBORIWOEIC KE-ES.
G.S. B a l a s u b r d a m * , R. O'Keefe, J. Catimel m u J. M e . D,"partrrent of Anatmica Pathology, St. Vincent's Hospital and university of mlbourne, V i c t o r i a .
p53 is a suppressor oncogene whicn i s t n o q h t to play an inportant role i n t m r i g e n e s i s . W e investigated the expression of p53 i n 1 2 squan-ous cell ,carcinarnas 15 solar of skin (SCCs), 1 4 k e r a t o a c a n t h m (a), keratoses (SKs) and 11 seborrhceic keratoses (seb KS). -rate to intense nuclear staining f o r p53 w a s observed in 93% (14/15) of SKs. The l a c e l l i n g w a s diffuse in about half the cases and patchy o r focal i n others. Forty one percent (5/12) of SCCS s h a d nuclear p o s i t i v i t y . Strong staining w a s observed in the single p r l y d i f f e r e n t i a t e d carcinm w h i l s t in the 4 w e l l d i f f e r e n t i a t e d carcinomas the s t a i n i n g w a s focal and of m d e r a t e intensity. O f the 1 4 cases of KAs, only L s h m d mild focal nuclear p o s i t i v i t y . Seborrfioeic keratoses were largely negative except for 2 which shcwed occasional positive nuclei. 'Iko SCCs a r i s i n g frcm SKs were h m o n e g a t i v e . Increased imnunopsit;lvity was observed i n the adjacent c t y o l q i c a l l y nolmal epithelium i n both SCCs and SKs but not i n KAS or Seb KS. O u r r e s u l t s suggest t h a t pS3 plays an irrportant role i n t h e evolution of SCCs of skin with m a x h d expression i n the i n s i t u lesion SK. The presence of p53 i n the adjacent cytologically n o d epithelium may represent a marker of early malignant t r a n s f o m t i a n . The absence of such increased expression i n the epitneliun adjacent to KAs supports the v i e w that KAs and SCCs are ism d i s t i n c t neoplasms r a t h e r than a spectnim of t h e sam neo@stic process.
Wilms' tumor is a complex embryonic tumor of the kidney occurring most commonly in children under 10 years of age. Stage and histology are currently considered to be the best predictors of outcome. In approximately 25% of cases Wilms' tumor is associated with nephroblastomatosis in the same or contralateral kidney. Much less common are cystic variants of Wilms' tumor; cystic nephroma (CN), also known as multilocular cyst of the kidney and cystic partially differentiated nephroblastoma (CFDN). In general these cystic variants have an excellent prognosis when treated by nephrectomy alone although CPDN may rarely be locally aggressive. Since methods for analysing the DNA content of nuclei extracted from paraffin blocks have become available, flow cytometry has been performed on a range of tumors and has been shown to correlate with outcome. We wished to determine if there was any difference in DNA content between typical favourable histology Wilms' tumor and the less common cystic variants and precursor lesions. Flow cytometry was used to analyse the DNA content of Wilms' (4 cases), CPDN with Wilms' tumor (1 case), CPDN (1 case), CN (2 cases) and nephroblastomatosis (1 casel. The 9 cases were processed by a modified Hedley technique. The specimens were stained with propidium iodide and analysed on a Coulter EPICS Profile II flow cytometer using a 488nm air-cooled laser. A minimum of 15000 nuclei were counted in each case. In all cases a diploid DNA content was found. The mean half peak C.V. value for the major peaks was 3.34 and the median half-peak C.V. was 3.22. The S phase fraction varied from 0.19% with a mean of 7.1%. It appears from these preliminary data that DNA ploidy does not distinguish between Wilms' tumor and variants although it is possible that S phase fraction may prove to be of benefit in determining malignant potential. We are currently undertaking a more extensive study to determine if DNA content or S phase fraction can be used to predict treatment failure in favourable histology Wilms' tumor.
STABILITY OF PROPlDlUM IODIDE STAINED SAMPLES BY M E COULT€RR~ DNA PREP WORKSTATION FOR ANALYSIS BY FLOW CYTOMElRY J. Drew, M. Donovan and H. Preston'. Laboratory Medicine Services, Caulfield General Medical Centre, Melbourne, Victoria
At the Caulfield General Medical Centre, paraffin-embedded specimens analysed for DNA ploidy are prepared by a modification of the 'Hedley Technique: Nuclear preparations are stained by the COULTEW DNA Prep Reagent System. This is a commercial workstation designed to prepare cell or nuclear suspensions for quantitative determination of nuclear DNA content by Flow Cytometry (FCM). The reagents comprise non-ionic detergents for cell lysing and permeabilisation, Propidium Iodide (PI) for DNA staining, and avian erythrocyte nuclei as an internal reference standard. The manufacturers instructions specity that to obtain optimal coefficients of variation (CVs), samples should be incubated in PI at room temperature (RT) for 15 - 17 minutes prior to analysing by FCM and must bei analysed within 2 hours of staining. This investigation set out to determine the stability of the stained specimens beyond this 2 hour limit. After routine analysis of a group of stained samples, the batch was re-analysed at different intervals ranging from 24 hours lo 14 days. Samples were stored at RT and protected from light. The values from the histograms coilected and compared over this period included: the DNA Index (DI) ; percent half peak C V s (%HPCV); mean channel fluorescence of the major peak(s) in the histogram; and percent of events found in each peak(s). Examination of the histograms after 24 hours demonstrated no alteration in the Di of the specimen,an average reduction of 10% in the %HPCV, 10% increase In the mean channel fluorescence and a 5% decrease In the percent of events found in the peaks. Re-analysis over a two week period showed these values to remain stable. Less than 10% of the samples deteriorated and were uninterpretable at the end of 14 days. The data shows that stained nuclear preparations are stable beyond the time recommended by the manufacturers and allows for storage of stained specimens with minimal deterioration.