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Flow cytometric analysis of the DNA content in colorectal adenomas with focal cancers
GASTROENTEROLOGY1995;109:1098-1104
Flow Cytometric Analysis of the DNA Content in Colorectal Adenomas with Focal Cancers SEIYUU SUZUKI,* MOTOWO MIZUNO,* JUN TOMODA,* MASAKI OHMORI,* and TAKAO TSUJI* *First Department of Internal Medicine, OkayamaUniversityMedical School, Okayama; and ~Departmentof Pathology, Kagawa Medical School, Kagawa, Japan
Background & Aims: Variable results have been reported on the nuclear DNA content of colorectal polyps. The significance of DNA aneuploidy in the malignant transformation of colorectal polyps was evaluated. Methods: We analyzed by flow cytometry the nuclear DNA content of freshly frozen samples of 50 colorectal adenomas with or without focal cancers, analyzing separately the adenomatous and cancerous regions of the polyps. Results: In the adenomatous regions of the 50 polyps, the DNA was diploid in 43 and aneuploid in 7; the adenomas with DNA aneuploidy in the adenomatous regions were more frequently accompanied by focal cancers than were the DNA-diploid adenomas (P < 0.01). In 60% of the polyps with DNA aneuploidy in the cancerous regions, the DNA was also aneuploid in the adenomatous region and had similar DNA indices; this result suggests that the DNA aneuploidy had already occurred during the adenomatous stage, which lends support to the concept of the adenoma-carcinoma sequence. DNA aneuploidy in the adenomatous region was significantly correlated with the size of colorectal polyps (P < 0.05). Conclusions: DNA aneuploidy may be an important indicator for the early diagnosis of malignant transformation of colorectal polyps. ecause colorectal adenomatous polyps are often accompanied by focal cancers, colorectal adenomas are often endoscopically resected as precancerous lesions. Clinical and pathological parameters such as size, grade of dysplasia, and histological type of polyps are risk factors for malignant transformation from adenomas to adenocarcinomas. 1'2 Recent advances in molecular biology have enabled the initiation of carcinoma at the D N A level to be identified. 3 In particular, development of flow cytometry has facilitated D N A analysis, and the nuclear D N A content of various malignant tumors has been studled. 4-6 Studies on the nuclear D N A content of colorectal cancers have suggested that D N A aneuploidy is related to poor prognosis of colorectal carcinomas. 7-1° Moreover, several studies of the nuclear D N A content of precancerous lesions, such as long-standing ulcerative colitis, 11'.2 cervical intraepithelial neoplasia, 13 giant melanocytic nevi, 14 and Barrett's esophagus, 15 showed that D N A an-
B
euploidy could be a marker for malignant transformation of these lesions. There have been several studies performed on the nuclear D N A content of colorectal polyps, but the results have been variable. 16-22 The variations seem to be mostly due to the methods used. Many of the studies were retrospective, and paraffin-embedded archival samples were used. Moreover, these studies did not discriminate between cancerous and adenomatous regions regarding D N A ploidy in adenomas with focal cancers. Therefore, the variable frequency of aneuploidy could have been caused by concomitant cancer in the adenomatous polyp. To evaluate the significance of D N A aneuploidy in malignant transformation of colorectal adenomas to adenocarcinomas, we analyzed separately the nuclear D N A content of the adenomatous and cancerous regions of colorectal adenomas containing focal cancers.
Materials
and Methods
Specimens Fifty-one colorectal adenomatous polyps obtained by endoscopic polypectomy from 46 patients (age range, 34-90 years; mean age, 61 years) were studied. Polyps containing or not containing focal cancers were included. Informed consent was obtained from each patient. To analyze the DNA content of the adenomatous and cancerous regions of the polyps separately, we adopted the following technique (Figure 1). Polyps were cut in half vertically. From one half, a 5-mm-wide section was cut out and frozen in citrate buffer (Cycle TEST DNA Reagent Kit; Becton Dickinson, San Jose, CA) at -70°C immediately for flow cytometry of the nuclear DNA content. The remaining tissue in this half and the other half was fixed with formalin and embedded in paraffin for histological examination. These materials were marked at the upper, lower, right, and left sides. Thus, the adenomatous and cancerous regions of the fresh-frozen section could be identified based on the histological examination of the corresponding paraffin-embedded tissues of both faces of the frozen section, and 3-5-ram 3 tissues were excised from the fresh-frozen section with a surgical blade separately from
© 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
DNA ANEUPLOIDY IN COLORECTAL ADENOMAS
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cancer
Table 1. DNA Index in the A d e n o m a t o u s a n d / o r Cancerous Regions of the Polyp With DNA A n e u p l o i d y
colon polyp ~ a d e n o m a
DNA index
~ endoscopic polypectomy Case no.
paraffin embedded specimens hematoxylin - eosin stained section upper rig
frozen at 1 - 70 "C
I hematoxylin - eosin
~stained section upper le
cancer \ adenoma
adenoma
DNA analysis
Adenomatous region (%)
Cancerous region (%)
1
1.1 (56)
2
1.1 (18) 1.6 (5) 1.4 (46) 1.1 (52) 1.7 (58) 1.2 (9) 1.5 (13) ----
1.1 (37) 1,5 (23) 1.1 (20)
paraffin embedded specimens
eft
cancerous region
1099
ight
cancer
adenomatous region
DNA analysis
Figure 1. A diagram of tissue preparation for the DNA analysis. Polyps
were cut in half vertically, From one half, a 5-mm-wide section was cut out and frozen in citrate buffer at -70°C immediately for flow cytometry of the nuclear DNA content. The remaining tissue in this half and the other half was fixed with formalin and embedded in paraffin for histological examination. These materials were marked at the upper, lower, right, and left sides. Thus, the adenomatous and cancerous regions of the fresh-frozen section could be identified based on the histological examination of the corresponding paraffinembedded tissues of both faces of the frozen section, and 3 - 5 mm 3 tissues were excised with a surgical blade separately from the adenomatous and cancerous regions for DNA analysis.
the adenomatous and cancerous regions for D N A analysis. In addition, dispersed specimens of the adenomatous region were examined cytologically to ensure that apparent cancer cells were not contained. The histological and cytological analysis was performed by an experienced pathologist (M.O.),
Histology The maximal diameter of each polyp was estimated from the size of histological specimens. The grade of dysplasia of adenomatous regions in the polyp was classified as mild or moderate dysplasia,2 and the histological types of adenomas were classified as tubular, tubulovillous, or villous. 2 Cancerous regions in the polyp were classified as Tis (carcinoma in situ) or T1 (tumor invades submucosa) according to the pathological staging of colorectal cancer of T N M classification.23'24
3 4 5 6 7 8 9 10 11 12
--
1 . 3 (18)
--
2.0 (79)
NOTE. Values are percentage of cells in the aneuploid peak.
using a Cycle TEST D N A Reagent Kit (Becton Dickinson) according to the manufacturer's instructions. The nuclear D N A contents were analyzed by use of a flow cytometer (FACScan equipped with a 15-mW argon-ion laser; Becton Dickinson). Inflammatory and stromal ceils contained in the tumor samples served as internal standards. The data were processed by use of cell-fit software (Becton Dickinson) for DNA analysis.
DNA Histogram Analysis We evaluated D N A histograms according to previously published recommendations and guidelines. 26-28 D N A histograms showing at least two separate Gon peaks were classified as D N A aneuploidy, and the degree of D N A content aberration was expressed according to the D N A index. 26 Peaks comprising more than 5 % of the cell events were regarded as distinct peaks. 28 Samples with G 2 M(4C) of > 1 5 % of total cells analyzed were recorded as tetraploidy. 28 The mean coefficient of variation of the diploid Go/1 peaks of the specimens was 3.8% + 1.7% (mean _ SD). Forty-five of 7 1 samples (63%) investigated had a coefficient of variation of the diploid Goa peaks < 4 % , and coefficients of variation of 68 specimens (96%) were < 6 % . One of 7 1 D N A histograms had a coefficient of variation of > 8 % . This D N A histogram was judged as inadequate 28 and excluded from the analysis.
T a b l e 2. DNA PIoidy in the A d e n o m a t o u s Region of the Polyp and the Presence of C o n c o m i t a n t Focal Cancer
Flow Cytometry The nuclear D N A contents of both the cancerous and adenomatous regions of the frozen material were analyzed by flow cytometry according to the method described by Vindel0v et al. 25 Specimens were cut into small pieces using ophthalmologic scissors, filtered through a nylon mesh, and processed
1.2 (52) 1.2 (78) 1.7 (79) 1.1 (25) -1.3 (21) 1.1 (37) 1.6 (14)
Focal cancer DNA ploidy in adenomatous region
-
+ (%)
Total
Diploid Aneuploid
31 0
12 (28) 1~ 7 (100) 1
43 7
ap < 0.01.
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SUZUKI ET AL.
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GASTROENTEROLOGY Vol, 109, No. 4
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Figure 2. (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA diploidy in both the (A and C) adenomatous and (B and D) cancerous portions. A single Go/~ peak at channel 245 and a G2/M peak at channel 490 can be seen in both portions.
For statistical analysis, the
~2 method was used.
Results O f the 51 polyps, 20 adenomas were accompanied with focal cancers; 15 were classified as Tis and 5 as T1. The other 31 adenomas did not contain focal cancers. One polyp with focal cancer that showed a D N A histogram with coefficient of variation of > 8 % in the adenomatous region was excluded from the study. Thus, the nuclear D N A contents of 69 specimens (50 from adenomatous and 19 from cancerous regions) from 50 polyps were analyzed. Aneuploidy in adenomatous and/or cancerous regions was observed in 11 cases, and tetraploidy was observed in one case (Table 1). The minimal D N A index in D N A histograms classified as D N A aneuploidy was 1.1, and the percentage of cells in the aneuploid peak ranged from 5% to 79% (mean, 35%) (Table 1). In the adenomatous regions of the 50 polyps, D N A diploidy was present in 43 specimens, and aneuploidy was found in 7 (cases 1 - 7 in Table 1). Twelve of the 43 polyps (28%) with diploidy in the adenomatous region and all of the 7 polyps with D N A aneuploidy in the adenomatous region were accompanied with focal cancers
(Table 2). Aneuploid adenomas were accompanied with focal cancers more frequently than those that were diploid (P < 0.01). Histograms of the D N A distribution and the histological appearance of representative polyps with D N A diploidy both in the cancerous and the adenomatous regions (Figure 2) or with aneuploidy both in the cancerous and the adenomatous regions (Figure 3) are presented. In the cancerous region of the 19 polyps with focal cancers, the D N A was aneuploid in 10 polyps (cases 1 6 and 8 - 1 1 in Table 1). In 6 of these 10 polyps (60%), D N A aneuploidy was also found in the adenomatous region (cases 1 - 6 in Table 1). The D N A indices in the adenomatous regions were similar to those in the cancerous regions in most cases (Table 1). In case 7 in Table 1, D N A aneuploidy was found in the adenomatous region, although the cancerous region was diploid. In this case, numerous lymphocytes infiltrated the minute cancerous region in the polyp and could have obscured aneuploidy of the cancer cells (Figure 4). In case 2 in Table 1, D N A polyploidy was found in the adenomatous region, whereas a single aneuploid peak was present in the cancerous region (Figure 3).
October 1995
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DNA ANEUPLOIDY IN COLORECTAL ADENOMAS
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Figure 3. (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA polyploidy in the (A and C) adenomatous portion and aneuploidy in the (B and D) cancerous portion (case 2 in Table 1), The DNA indices (DI) were 1.1 and 1.6 in the adenomatous region and 1.1 in the cancerous portion. In the former, two separate Go/~ peaks are evident at channels 277 and 316, and another Go/~ peak at channel 437 is present. In the cancerous region, two separate Go/1 peaks can be seen at channels 277 and 316.
The frequency of D N A aneuploidy in the adenomatous portions of the polyps was analyzed in relation to the conventional risk factors of colorectal polyps for malignant transformation (Table 3). D N A aneuploidy was present in none of 22 polyps of < 1 cm, in 4 of 20 polyps (20%) of 1 - 2 cm, and in 3 of 8 polyps (38%) of > 2 cm in diameter. These differences reached statistical significance (P < 0.05). D N A aneuploidy in the adenomatous region tended to be less common in polyps with mild dysplasia (12%) than in those with moderate dysplasia (22%), but it did not reach statistical significance. No significant correlation was observed between the D N A ploidy in the adenomatous part and the histological type of the adenoma.
Discussion In this study, we showed that colorectal adenomas that had D N A aneuploidy were more frequently accompanied by focal cancers than were DNA-diploid adenomas. Also, the frequency of D N A aneuploidy in the adenomatous region of the polyp was correlated with the size of polyps, one of the conventional risk factors of
carcinogenesis in colorectal adenomas. Our results suggest that D N A aneuploidy is an early indicator of malignant transformation of adenomas to adenocarcinomas in colorectal polyps. W e also showed that in polyps with focal cancers accompanied by D N A aneuploidy in the cancerous region, aneuploidy was often found in the adenomatous regions with similar D N A indices to those in the cancerous regions in most cases. This result suggests that the D N A aneuploidy had already occurred during the adenomatous stage and that the cancer cells arose from the cell clones with the D N A aneuploidy in these specimens. Thus, the findings support the concept of the adenoma-carcinoma sequence. In addition, although D N A polyploidy in coIonic cancer cells was observed in one case here and has been reported by o t h e r s , 6'10'12'29-32 w e described a DNA polyploid adenoma accompanied by a single aneuploid peak in the cancerous region. In this specimen, one neoplastic cell clone may have been selected during the development of the cancer, supporting a model of clonal evolution in neoplasia as proposed by Nowell. 33 Studies by Vogelstein et al. on familial adenomatous
1102
SUZUKI ET AL.
109
GASTROENTEROLOGY Vol. 109, No. 4
175
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Figure 4, (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA aneuploidy in the (A and C) adenomatous portion and diploidy in the (B and D) cancerous portion (case 7 in Table 1). An aneuploid peak with the DNA index (DI) of 1.5 is evident in the adenomatous region but cannot be seen in the cancerous region. Numerous lymphocytes infiltrate the minute cancerous region in this polyp (D).
polyposis indicated that malignant transformation of colorectal adenomas to adenocarcinomas was a multistep process 34 and that several genetic alterations have already occurred during the adenomatous stage. 35-37 Shackney et al. suggested that the activation of a growth-promoting gene was required to produce a discrete aneuploid peak, 38
Table 3. DNA Ploidy in the A d e n o m a t o u s Region and Conventional Risk Factors of the Colorectal Polyp for M a l i g n a n t T r a n s f o r m a t i o n DNA ploidy in adenomatous region
Diploid Diameter of polyp (cm)
Grade of dysplasia Histological type "P < 0.05.
<1 1-2 >2 Mild Moderate Tubular Tubulovillous
22 16 5 36 7 22 21
Aneuploid (%) 0 4 3 5 2 5 2
(0) a (20) a (38) ~ (12) (22) (19) (9)
Total 22 20 8 41 9 27 23
and Burmer et al. showed that K-ras mutations preceded ploidy alterations in the progression of tumors. 39 Other studies have indicated that D N A aneuploidy correlates with the frequency of K-ras mutations and the allelic loss of chromosome 17p. 4°'4. The D N A aneuploidy in the adenomatous portion of the polyps found here may reflect these genetic alterations that had already occurred in the adenomatous stage. In our study, D N A aneuploidy was correlated with increasing size of the polyp, which is in agreement with the results of other studies. 16-2° W e further showed that the change of D N A ploidy in the adenomatous portion of the polyp occurred in the way of growth of the polyp from < 1 cm to > 1 cm in diameter. Thus, our findings are in agreement with other studies describing that some genetic alterations such as K-ras mutations often appeared in adenomas 1 - 2 cm in size. 35 These findings show that important genetic changes likely have already occurred in colorectal polyps of 1 - 2 cm in diameter. In previous studies, 16-22 the frequency of aneuploidy in colorectal polyps varied from 5% to 32%. These variations seem to be mainly due to the methods used. Paraf-
October 1 9 9 5
fin-embedded tissues were usually studied because they are useful for retrospective investigation. However, the incidence of aneuploidy was underestimated in these studies when compared with those that used frozen or fresh materials. 19'22 Freezing with citrate buffer was found to sufficiently preserve materials for DNA analysis. 25 In this study, we used fresh specimens frozen in citrate buffer to maximize the sensitivity of the analysis. In addition, we estimated the nuclear DNA content in the adenomatous and cancerous regions separately, whereas in other studies, whole colorectal polyps, which contained various degrees of dysplasia, were analyzed. The number of adenomas with focal cancers studied in our series is larger than that in previous studies using freshly frozen samples. 18'19'32 Thus, this study reliably disclosed the frequency of changes of DNA ploidy in colorectal adenomatous polyps and provided further insight into the malignant transformation from adenoma to adenocarcinoma of colorectal polyps.
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40. Suchy B, Zietz C, Rabes HM. K-ras point mutations in human colorectal carcinomas: relation to aneuploidy and metastasis. Int J Cancer 1 9 9 2 ; 5 2 : 3 0 - 3 3 . 41. Offerhaus GJA, Feyter EPD, Cornelisse CJ, Tersmette KWF, Floyd J, Kern SE, Vogelstein B, Hamilton SR. The relationship of DNA aneuploidy to molecular genetic alterations in colorectal carcinoma. Gastroenterology 1 9 9 2 ; 1 0 2 : 1 6 1 2 - 1 6 1 9 .
Received April 8, 1994. Accepted May 29, 1995. Address requests for reprints to: Motowo Mizuno, M.D., First Department of Internal Medicine, Okayama University Medical School, 2-5-1, Shikatacyo, Okayama 700, Japan. Fax: (81) 86-225-5991. A portion of this work was presented at the 78th annual meeting of the Japanese Society of Gastroenterology in Tokyo in May 1992. The authors thank Drs. William R. Brown and Dennis J. Ahnen for assistance in preparation of the manuscript.
Flow Cytometric Analysis of the DNA Content in Colorectal Adenomas with Focal Cancers SEIYUU SUZUKI,* MOTOWO MIZUNO,* JUN TOMODA,* MASAKI OHMORI,* and TAKAO TSUJI* *First Department of Internal Medicine, OkayamaUniversityMedical School, Okayama; and ~Departmentof Pathology, Kagawa Medical School, Kagawa, Japan
Background & Aims: Variable results have been reported on the nuclear DNA content of colorectal polyps. The significance of DNA aneuploidy in the malignant transformation of colorectal polyps was evaluated. Methods: We analyzed by flow cytometry the nuclear DNA content of freshly frozen samples of 50 colorectal adenomas with or without focal cancers, analyzing separately the adenomatous and cancerous regions of the polyps. Results: In the adenomatous regions of the 50 polyps, the DNA was diploid in 43 and aneuploid in 7; the adenomas with DNA aneuploidy in the adenomatous regions were more frequently accompanied by focal cancers than were the DNA-diploid adenomas (P < 0.01). In 60% of the polyps with DNA aneuploidy in the cancerous regions, the DNA was also aneuploid in the adenomatous region and had similar DNA indices; this result suggests that the DNA aneuploidy had already occurred during the adenomatous stage, which lends support to the concept of the adenoma-carcinoma sequence. DNA aneuploidy in the adenomatous region was significantly correlated with the size of colorectal polyps (P < 0.05). Conclusions: DNA aneuploidy may be an important indicator for the early diagnosis of malignant transformation of colorectal polyps. ecause colorectal adenomatous polyps are often accompanied by focal cancers, colorectal adenomas are often endoscopically resected as precancerous lesions. Clinical and pathological parameters such as size, grade of dysplasia, and histological type of polyps are risk factors for malignant transformation from adenomas to adenocarcinomas. 1'2 Recent advances in molecular biology have enabled the initiation of carcinoma at the D N A level to be identified. 3 In particular, development of flow cytometry has facilitated D N A analysis, and the nuclear D N A content of various malignant tumors has been studled. 4-6 Studies on the nuclear D N A content of colorectal cancers have suggested that D N A aneuploidy is related to poor prognosis of colorectal carcinomas. 7-1° Moreover, several studies of the nuclear D N A content of precancerous lesions, such as long-standing ulcerative colitis, 11'.2 cervical intraepithelial neoplasia, 13 giant melanocytic nevi, 14 and Barrett's esophagus, 15 showed that D N A an-
B
euploidy could be a marker for malignant transformation of these lesions. There have been several studies performed on the nuclear D N A content of colorectal polyps, but the results have been variable. 16-22 The variations seem to be mostly due to the methods used. Many of the studies were retrospective, and paraffin-embedded archival samples were used. Moreover, these studies did not discriminate between cancerous and adenomatous regions regarding D N A ploidy in adenomas with focal cancers. Therefore, the variable frequency of aneuploidy could have been caused by concomitant cancer in the adenomatous polyp. To evaluate the significance of D N A aneuploidy in malignant transformation of colorectal adenomas to adenocarcinomas, we analyzed separately the nuclear D N A content of the adenomatous and cancerous regions of colorectal adenomas containing focal cancers.
Materials
and Methods
Specimens Fifty-one colorectal adenomatous polyps obtained by endoscopic polypectomy from 46 patients (age range, 34-90 years; mean age, 61 years) were studied. Polyps containing or not containing focal cancers were included. Informed consent was obtained from each patient. To analyze the DNA content of the adenomatous and cancerous regions of the polyps separately, we adopted the following technique (Figure 1). Polyps were cut in half vertically. From one half, a 5-mm-wide section was cut out and frozen in citrate buffer (Cycle TEST DNA Reagent Kit; Becton Dickinson, San Jose, CA) at -70°C immediately for flow cytometry of the nuclear DNA content. The remaining tissue in this half and the other half was fixed with formalin and embedded in paraffin for histological examination. These materials were marked at the upper, lower, right, and left sides. Thus, the adenomatous and cancerous regions of the fresh-frozen section could be identified based on the histological examination of the corresponding paraffin-embedded tissues of both faces of the frozen section, and 3-5-ram 3 tissues were excised from the fresh-frozen section with a surgical blade separately from
© 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
DNA ANEUPLOIDY IN COLORECTAL ADENOMAS
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cancer
Table 1. DNA Index in the A d e n o m a t o u s a n d / o r Cancerous Regions of the Polyp With DNA A n e u p l o i d y
colon polyp ~ a d e n o m a
DNA index
~ endoscopic polypectomy Case no.
paraffin embedded specimens hematoxylin - eosin stained section upper rig
frozen at 1 - 70 "C
I hematoxylin - eosin
~stained section upper le
cancer \ adenoma
adenoma
DNA analysis
Adenomatous region (%)
Cancerous region (%)
1
1.1 (56)
2
1.1 (18) 1.6 (5) 1.4 (46) 1.1 (52) 1.7 (58) 1.2 (9) 1.5 (13) ----
1.1 (37) 1,5 (23) 1.1 (20)
paraffin embedded specimens
eft
cancerous region
1099
ight
cancer
adenomatous region
DNA analysis
Figure 1. A diagram of tissue preparation for the DNA analysis. Polyps
were cut in half vertically, From one half, a 5-mm-wide section was cut out and frozen in citrate buffer at -70°C immediately for flow cytometry of the nuclear DNA content. The remaining tissue in this half and the other half was fixed with formalin and embedded in paraffin for histological examination. These materials were marked at the upper, lower, right, and left sides. Thus, the adenomatous and cancerous regions of the fresh-frozen section could be identified based on the histological examination of the corresponding paraffinembedded tissues of both faces of the frozen section, and 3 - 5 mm 3 tissues were excised with a surgical blade separately from the adenomatous and cancerous regions for DNA analysis.
the adenomatous and cancerous regions for D N A analysis. In addition, dispersed specimens of the adenomatous region were examined cytologically to ensure that apparent cancer cells were not contained. The histological and cytological analysis was performed by an experienced pathologist (M.O.),
Histology The maximal diameter of each polyp was estimated from the size of histological specimens. The grade of dysplasia of adenomatous regions in the polyp was classified as mild or moderate dysplasia,2 and the histological types of adenomas were classified as tubular, tubulovillous, or villous. 2 Cancerous regions in the polyp were classified as Tis (carcinoma in situ) or T1 (tumor invades submucosa) according to the pathological staging of colorectal cancer of T N M classification.23'24
3 4 5 6 7 8 9 10 11 12
--
1 . 3 (18)
--
2.0 (79)
NOTE. Values are percentage of cells in the aneuploid peak.
using a Cycle TEST D N A Reagent Kit (Becton Dickinson) according to the manufacturer's instructions. The nuclear D N A contents were analyzed by use of a flow cytometer (FACScan equipped with a 15-mW argon-ion laser; Becton Dickinson). Inflammatory and stromal ceils contained in the tumor samples served as internal standards. The data were processed by use of cell-fit software (Becton Dickinson) for DNA analysis.
DNA Histogram Analysis We evaluated D N A histograms according to previously published recommendations and guidelines. 26-28 D N A histograms showing at least two separate Gon peaks were classified as D N A aneuploidy, and the degree of D N A content aberration was expressed according to the D N A index. 26 Peaks comprising more than 5 % of the cell events were regarded as distinct peaks. 28 Samples with G 2 M(4C) of > 1 5 % of total cells analyzed were recorded as tetraploidy. 28 The mean coefficient of variation of the diploid Go/1 peaks of the specimens was 3.8% + 1.7% (mean _ SD). Forty-five of 7 1 samples (63%) investigated had a coefficient of variation of the diploid Goa peaks < 4 % , and coefficients of variation of 68 specimens (96%) were < 6 % . One of 7 1 D N A histograms had a coefficient of variation of > 8 % . This D N A histogram was judged as inadequate 28 and excluded from the analysis.
T a b l e 2. DNA PIoidy in the A d e n o m a t o u s Region of the Polyp and the Presence of C o n c o m i t a n t Focal Cancer
Flow Cytometry The nuclear D N A contents of both the cancerous and adenomatous regions of the frozen material were analyzed by flow cytometry according to the method described by Vindel0v et al. 25 Specimens were cut into small pieces using ophthalmologic scissors, filtered through a nylon mesh, and processed
1.2 (52) 1.2 (78) 1.7 (79) 1.1 (25) -1.3 (21) 1.1 (37) 1.6 (14)
Focal cancer DNA ploidy in adenomatous region
-
+ (%)
Total
Diploid Aneuploid
31 0
12 (28) 1~ 7 (100) 1
43 7
ap < 0.01.
1100
SUZUKI ET AL.
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GASTROENTEROLOGY Vol, 109, No. 4
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Figure 2. (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA diploidy in both the (A and C) adenomatous and (B and D) cancerous portions. A single Go/~ peak at channel 245 and a G2/M peak at channel 490 can be seen in both portions.
For statistical analysis, the
~2 method was used.
Results O f the 51 polyps, 20 adenomas were accompanied with focal cancers; 15 were classified as Tis and 5 as T1. The other 31 adenomas did not contain focal cancers. One polyp with focal cancer that showed a D N A histogram with coefficient of variation of > 8 % in the adenomatous region was excluded from the study. Thus, the nuclear D N A contents of 69 specimens (50 from adenomatous and 19 from cancerous regions) from 50 polyps were analyzed. Aneuploidy in adenomatous and/or cancerous regions was observed in 11 cases, and tetraploidy was observed in one case (Table 1). The minimal D N A index in D N A histograms classified as D N A aneuploidy was 1.1, and the percentage of cells in the aneuploid peak ranged from 5% to 79% (mean, 35%) (Table 1). In the adenomatous regions of the 50 polyps, D N A diploidy was present in 43 specimens, and aneuploidy was found in 7 (cases 1 - 7 in Table 1). Twelve of the 43 polyps (28%) with diploidy in the adenomatous region and all of the 7 polyps with D N A aneuploidy in the adenomatous region were accompanied with focal cancers
(Table 2). Aneuploid adenomas were accompanied with focal cancers more frequently than those that were diploid (P < 0.01). Histograms of the D N A distribution and the histological appearance of representative polyps with D N A diploidy both in the cancerous and the adenomatous regions (Figure 2) or with aneuploidy both in the cancerous and the adenomatous regions (Figure 3) are presented. In the cancerous region of the 19 polyps with focal cancers, the D N A was aneuploid in 10 polyps (cases 1 6 and 8 - 1 1 in Table 1). In 6 of these 10 polyps (60%), D N A aneuploidy was also found in the adenomatous region (cases 1 - 6 in Table 1). The D N A indices in the adenomatous regions were similar to those in the cancerous regions in most cases (Table 1). In case 7 in Table 1, D N A aneuploidy was found in the adenomatous region, although the cancerous region was diploid. In this case, numerous lymphocytes infiltrated the minute cancerous region in the polyp and could have obscured aneuploidy of the cancer cells (Figure 4). In case 2 in Table 1, D N A polyploidy was found in the adenomatous region, whereas a single aneuploid peak was present in the cancerous region (Figure 3).
October 1995
129
DNA ANEUPLOIDY IN COLORECTAL ADENOMAS
1101
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,~'t".
"".- : " - , - , - , . .
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Figure 3. (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA polyploidy in the (A and C) adenomatous portion and aneuploidy in the (B and D) cancerous portion (case 2 in Table 1), The DNA indices (DI) were 1.1 and 1.6 in the adenomatous region and 1.1 in the cancerous portion. In the former, two separate Go/~ peaks are evident at channels 277 and 316, and another Go/~ peak at channel 437 is present. In the cancerous region, two separate Go/1 peaks can be seen at channels 277 and 316.
The frequency of D N A aneuploidy in the adenomatous portions of the polyps was analyzed in relation to the conventional risk factors of colorectal polyps for malignant transformation (Table 3). D N A aneuploidy was present in none of 22 polyps of < 1 cm, in 4 of 20 polyps (20%) of 1 - 2 cm, and in 3 of 8 polyps (38%) of > 2 cm in diameter. These differences reached statistical significance (P < 0.05). D N A aneuploidy in the adenomatous region tended to be less common in polyps with mild dysplasia (12%) than in those with moderate dysplasia (22%), but it did not reach statistical significance. No significant correlation was observed between the D N A ploidy in the adenomatous part and the histological type of the adenoma.
Discussion In this study, we showed that colorectal adenomas that had D N A aneuploidy were more frequently accompanied by focal cancers than were DNA-diploid adenomas. Also, the frequency of D N A aneuploidy in the adenomatous region of the polyp was correlated with the size of polyps, one of the conventional risk factors of
carcinogenesis in colorectal adenomas. Our results suggest that D N A aneuploidy is an early indicator of malignant transformation of adenomas to adenocarcinomas in colorectal polyps. W e also showed that in polyps with focal cancers accompanied by D N A aneuploidy in the cancerous region, aneuploidy was often found in the adenomatous regions with similar D N A indices to those in the cancerous regions in most cases. This result suggests that the D N A aneuploidy had already occurred during the adenomatous stage and that the cancer cells arose from the cell clones with the D N A aneuploidy in these specimens. Thus, the findings support the concept of the adenoma-carcinoma sequence. In addition, although D N A polyploidy in coIonic cancer cells was observed in one case here and has been reported by o t h e r s , 6'10'12'29-32 w e described a DNA polyploid adenoma accompanied by a single aneuploid peak in the cancerous region. In this specimen, one neoplastic cell clone may have been selected during the development of the cancer, supporting a model of clonal evolution in neoplasia as proposed by Nowell. 33 Studies by Vogelstein et al. on familial adenomatous
1102
SUZUKI ET AL.
109
GASTROENTEROLOGY Vol. 109, No. 4
175
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Figure 4, (A and B) A histogram of the DNA distribution and (C and D) histological appearance of a polyp with DNA aneuploidy in the (A and C) adenomatous portion and diploidy in the (B and D) cancerous portion (case 7 in Table 1). An aneuploid peak with the DNA index (DI) of 1.5 is evident in the adenomatous region but cannot be seen in the cancerous region. Numerous lymphocytes infiltrate the minute cancerous region in this polyp (D).
polyposis indicated that malignant transformation of colorectal adenomas to adenocarcinomas was a multistep process 34 and that several genetic alterations have already occurred during the adenomatous stage. 35-37 Shackney et al. suggested that the activation of a growth-promoting gene was required to produce a discrete aneuploid peak, 38
Table 3. DNA Ploidy in the A d e n o m a t o u s Region and Conventional Risk Factors of the Colorectal Polyp for M a l i g n a n t T r a n s f o r m a t i o n DNA ploidy in adenomatous region
Diploid Diameter of polyp (cm)
Grade of dysplasia Histological type "P < 0.05.
<1 1-2 >2 Mild Moderate Tubular Tubulovillous
22 16 5 36 7 22 21
Aneuploid (%) 0 4 3 5 2 5 2
(0) a (20) a (38) ~ (12) (22) (19) (9)
Total 22 20 8 41 9 27 23
and Burmer et al. showed that K-ras mutations preceded ploidy alterations in the progression of tumors. 39 Other studies have indicated that D N A aneuploidy correlates with the frequency of K-ras mutations and the allelic loss of chromosome 17p. 4°'4. The D N A aneuploidy in the adenomatous portion of the polyps found here may reflect these genetic alterations that had already occurred in the adenomatous stage. In our study, D N A aneuploidy was correlated with increasing size of the polyp, which is in agreement with the results of other studies. 16-2° W e further showed that the change of D N A ploidy in the adenomatous portion of the polyp occurred in the way of growth of the polyp from < 1 cm to > 1 cm in diameter. Thus, our findings are in agreement with other studies describing that some genetic alterations such as K-ras mutations often appeared in adenomas 1 - 2 cm in size. 35 These findings show that important genetic changes likely have already occurred in colorectal polyps of 1 - 2 cm in diameter. In previous studies, 16-22 the frequency of aneuploidy in colorectal polyps varied from 5% to 32%. These variations seem to be mainly due to the methods used. Paraf-
October 1 9 9 5
fin-embedded tissues were usually studied because they are useful for retrospective investigation. However, the incidence of aneuploidy was underestimated in these studies when compared with those that used frozen or fresh materials. 19'22 Freezing with citrate buffer was found to sufficiently preserve materials for DNA analysis. 25 In this study, we used fresh specimens frozen in citrate buffer to maximize the sensitivity of the analysis. In addition, we estimated the nuclear DNA content in the adenomatous and cancerous regions separately, whereas in other studies, whole colorectal polyps, which contained various degrees of dysplasia, were analyzed. The number of adenomas with focal cancers studied in our series is larger than that in previous studies using freshly frozen samples. 18'19'32 Thus, this study reliably disclosed the frequency of changes of DNA ploidy in colorectal adenomatous polyps and provided further insight into the malignant transformation from adenoma to adenocarcinoma of colorectal polyps.
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14. Stenzinger W, Suter L, Schumann J. DNA aneuploidy in congenital melanocytic nevi: suggestive evidence for premalignant changes. J invest Dermatol 1 9 8 4 ; 8 2 : 5 6 9 - 5 7 2 . 15. Fennerty MB, Sampliner RE, Way D, Riddell R, Steinbronn K, Garewal HS. Discordance between flow cytometric abnormalities and dysplasia in Barrett's esophagus. Gastroenterology 1989; 97:815-820. 16. Quirke P, Fozard JBJ, Dixon MF, Dyson JED, Giles GR, Bird CC. DNA aneuploidy in colorectal adenomas. Br J Cancer 1986; 5 3 : 4 7 7 - 4 8 1 . 17. Giaretti W, Sciallero S, Bruno S, Geido E, Aste H, Vinci AD. DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas. Cytometry 1 9 8 8 ; 9 : 2 3 8 - 2 4 4 . 18. Van den Ingh HF, Griffioen G, Cornelisse CJ. Flow cytometry detection of aneuploidy in colorectal adenomas. Cancer Res 1985; 4 5 : 3 3 9 2 - 3 3 9 7 . 19. Perrin-Cottier M, Jouffre C, Sabido O, Maubon I, Barthelemy C, Pasteur X, Audigier JC, Laurent JL. Flow cytometric analysis of DNA content in colorectal polyps resected at endoscopy. Anal Quant Cytol Histo] 1991; 1 3 : 1 3 2 - 1 3 8 . 20. Gob HS, Jass JR. DNA content and adenoma-carcinoma sequence in the colorectum. J Clin Pathol 1 9 8 6 ; 3 9 : 3 8 7 - 3 9 2 . 21. Weiss H, Wilter GP, Jacobasch KH, Heinz U, Schaelicke W. Characterization of human adenomatous polyps of the colorectal bowel by means of DNA distribution patterns. Oncology 1985; 42:33-41. 22. Murad T, Bauer K, Scarpelli DG. Histopathologic and flow cytometric analysis of adenomatous colonic polyps. Arch Pathol Lab Med 1989; 1 1 3 : 1 0 0 3 - 1 0 0 8 . 23. International Union Against Cancer. TNM-atlas illustrated guide to the TNM/pTNM-classification of malignant tumors. In: Spiess] B, Hermanek P, Scheibe O, Wagner G, eds. 2rid ed. Berlin: Springer-Verlag, 1985. 24. American Joint Committee on Cancer. Manual for staging of cancer. in: Beahrs OH, Henson DE, Hutter RVP, Myers MH, eds. 3rd ed. Philadelphia:Lippincott, 1988. 25. Vindel#v LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 1 9 8 3 ; 3 : 3 2 3 - 3 2 7 . 26. Hiddemann W, Schumann J, Andreeff M, Barlogie B, Herman CJ, Leif RC, Mayall BH, Murphy RF, Sandberg AA. Convention on nomenclature for DNA cytometry. Cytometry 1984; 5 : 4 4 5 - 4 4 9 . 27. Bauer KD, Bagwell CB, Giaretti W, Meiamed M, Zarbo RJ, Witzig TE, Rabinovitch PS. Consensus review of the clinical utility of DNA flow cytometry in colorectal cancer. Cytometry 1993;14: 486-491. 28. Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, Mayall BH, Wheeless L. Guidelines for implementation of clinical DNA cytometry. Cytometry 1993; 1 4 : 4 7 2 - 4 7 7 . 29. Hamada S, Namura K, Itch R, Fujita S. Characteristics of colerectal epithelia and adenomas as revealed by DNA cytofluorometry. Jpn J Cancer Res 1 9 8 7 ; 7 8 : 8 2 6 - 8 3 2 . 30. Hiddemann W, Von Bassewitz DB, Kleinemeier HJ, SchulteBrochterbeck E, Hauss J, Lingemann B, Buchner T, Grundmann E. DNA stemline heterogeneity in colorectal cancer. Cancer 1986; 5 8 : 2 5 8 - 2 6 3 . 31. Wersto RP, Liblit RL, Deitch D, Koss LG. Variability in DNA measurements in multiple tumor samples of human colonic carcinoma. Cancer 1991; 6 7 : 1 0 6 - 1 1 5 . 32. Fischbach W, Zidianakis Z, Luke G, Kirchner T, Mossner J. DNA mapping of colorectal neoplasms: a flow cytometric study of DNA abnormalities and proliferation. Gastroenterology 1993; 105: 1126-1133. 33. Nowell PC. The clonal evolution of tumor cell populations. Science 1976; 1 9 4 : 2 3 - 2 8 . 34. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AMM, Bos JL. Genetic
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40. Suchy B, Zietz C, Rabes HM. K-ras point mutations in human colorectal carcinomas: relation to aneuploidy and metastasis. Int J Cancer 1 9 9 2 ; 5 2 : 3 0 - 3 3 . 41. Offerhaus GJA, Feyter EPD, Cornelisse CJ, Tersmette KWF, Floyd J, Kern SE, Vogelstein B, Hamilton SR. The relationship of DNA aneuploidy to molecular genetic alterations in colorectal carcinoma. Gastroenterology 1 9 9 2 ; 1 0 2 : 1 6 1 2 - 1 6 1 9 .
Received April 8, 1994. Accepted May 29, 1995. Address requests for reprints to: Motowo Mizuno, M.D., First Department of Internal Medicine, Okayama University Medical School, 2-5-1, Shikatacyo, Okayama 700, Japan. Fax: (81) 86-225-5991. A portion of this work was presented at the 78th annual meeting of the Japanese Society of Gastroenterology in Tokyo in May 1992. The authors thank Drs. William R. Brown and Dennis J. Ahnen for assistance in preparation of the manuscript.