Comparative genomic hybridization analysis of primary colorectal carcinomas and their synchronous metastases

We have analyzed 26 tumors from 12 patients with metastatic colorectal adenocarcinoma by comparative genomic hybridization (CGH). Primary tumors and their lymph node metastases from five Dukes' C patients as well as primary tumors and their liver metastases from seven Dukes' D patients were used to assess the extent of genetic differences between primary and secondary colorectal carcinomas from the same patients, to calculate the degree of clonal divergence and genetic heterogeneity in metastatic colorectal cancer, and to determine the differences in genetic imbalances between Dukes' C and D stage tumors. We show that the same genetic aberrations were frequently found in the primary tumors and their metastases. However, metastases often contained genetic aberrations not found in the corresponding primary tumors. The comparison of Dukes' stages C and D revealed genetic aberrations common to both. However, reduced copy number of chromosome arm 17p (5/5 vs. 0/7; P = 0.001) was significantly associated with Dukes' stage C and lymph node metastases, while increased copy number of chromosome arms 6p (6/7 vs. 0/5; P = 0.007) and 17q (5/7 vs. 0/5; P = 0.027) was associated more with Dukes' stage D and liver metastases. Our results established a repertoire of chromosomal alterations associated with metastatic colorectal cancer and suggest that Dukes' C (lymph node metastasis) tumors are not always simply an earlier stage of Dukes' D (liver metastasis) tumors and, thus, in some instances at least, they are distinct forms of the disease.     

Clonal chromosome abnormalities in human breast carcinomas I. Twenty-eight cases with primary disease

Cytogenetic analysis was performed on a selected series of short-term cultures of primary breast carcinomas from 28 patients. All patients had histopathologically confirmed malignancies, with the majority (25/28 cases) demonstrating infiltrating ductal carcinoma. All 28 cases evidenced clonal chromosome abnormalities, with 10/28 displaying only numeric aberrations, whereas 18/28 displayed clonal structural alterations. In near-diploid tumors the most common numeric changes were — 17 and — 19. However, trisomy 7 was the only numeric change in two near-diploid tumors. Structural chromosome alterations were primarily isochromosomes, apparent terminal deletions, and unbalanced non-reciprocal translocations. Chromosomes 1 (10/18–56%) and 6 (8/18–44%) were most frequently altered in this series. Breakpoints of clonal structural abnormalities were shown to cluster to several chromosome segments, including 1p22-q11, 3p11, 6p11–13, 7p11-q11, 8p11-q11, and 19q13. Analysis of the gain or loss of specific chromosome segments revealed that the most consistent tendency was over-representation of 1q, 3q, and 6p. © 1993 Wiley-Liss, Inc.     

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric -satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis.     

Direct chromosome analysis from neonatal cord blood

Direct chromosome preparations of neonatal cord blood provides the unique opportunity for rapid chromosome analysis (turnaround time; 6 hr), without the necessity of bone marrow aspiration. Based on 42 samples we confirm the finding of Garnham and Sutherland [1987] for suitability of cord blood for direct chromosome preparation. Procedural modifications are provided for higher yield of cells for chromosome analysis. The procedure may well be of major significance for rapid diagnosis of neonates who suffer from aneusomy.     

Direct cytogenetic analysis of primary neuroblastoma

In this paper the results of cytogenetic analysis of a metastatic neuroblastoma from a 14-month-old boy are described. Direct cytogenetic analysis was performed on tumor pieces obtained from surgery prior to therapy. Consistent numerical and structural chromosome aberrations were identified. The modal chromosome number was 48, with 9.4% of the cell population being in the near-tetraploid range. In all karyotyped cells, the Y chromosome was missing and additions of chromosomes 7 and 14 were identified. Two rearranged #1 were observed: del(1)(p22 or p31) and t(1;18)(p22 or p31;q11-12), resulting in monosomy of the distal segment of the short arm and trisomy of the long arm. In two cells, single minutes were found; this chromosomal aberration has been previously described in a case of metastatic neuroblastoma.     

原发性胃癌的比较基因组杂交研究

目的　研究胃癌发生和发展过程中是否涉及其它未知的癌基因和抑癌基因。方法　采用比较基因组杂交法(com parative genom ic hybridization,CGH)分析了43 例原发性胃癌。结果　3p(8/43)、8q(8/43)、20 号染色体[20(9/43),20p(7/43)、20q(4/43)]、12q(16/43)、13q(12/43)的扩增,19 号染色体[19(15/43),19p(13/43)]、17 号染色体[17(8/43),17p(10/43)]、16 号染色体(10/43)和1p(11/43)区域的缺失为胃癌的特征性变化。结论　提示3p、8q、12q、13q、20 号染色体、17 号染色体、16 号染色体和1p 区域可能存在一些与胃癌形成相关的未知基因。     

Cytogenetic analysis of three primary Bellini duct carcinomas

Three Bellini duct carcinomas (BDC) of the kidney were cytogenetically analyzed after short-term culture. All three had clonal chromosome abnormalities: 91–92,XXY,-Y, +12, +12, −15, −16, −18, +mar (case I); 53, XY, +2,t(2;7)(p22;q11), +der(2)t (2;7)(p22;q11), +3, +r(3), add(5)(p15), +7, −8, +12, +17, +r(17), +20, −21 (case 2); and 44–47,X,-Y, +9, +16, −21/46,XY. Some of the numerical abnormalities are shared with papillary renal cell carcinomas (PRCC)(+7, +12, +16, +17, and +20) but not with transitional renal cell carcinomas. The present findings support the previous notion that BDC are different from other types of RCC. Genes Chromosom Cancer 15:170–172 (1996). © 1996 Wiley-Liss, Inc.     

结直肠癌临床及病理免疫组织化学多因素分析

目的探讨结直肠癌的生长方式,间质淋巴细胞浸润,Ｄｕｋｅｓ分期,ｐ５３和ｃ－ｅｒｂＢ－２蛋白,增殖细胞核抗原（ＰＣＮＡ）增殖指数,Ｐ２１蛋白,上皮生长因子受体（ＥＧＦＲ）、间质纤维组织增生及组织学类型１０种因素对患者预后的影响。方法运用Ｃｏｘ模型对６８例结直肠癌上述十种因素与预后的关系进行分析。结果单因素分析显示上述前六种因素对患者的预后有影响;经多因素分析显示Ｐ２１和ｃ－ｅｒｂＢ－２蛋白表达情况,Ｄｕｋｅｓ分期以及ＰＣＮＡ指数与病人预后有关,两种癌基因蛋白共同表达时病人的死亡相对危险度更高,ＤｕｋｅｓＡ期Ｐ２１或ｃ－ｅｒｂＢ－２蛋白阳性病人的死亡相对危险度比ＤｕｋｅｓＢ期相应蛋白阴性的病人高。结论Ｐ２１、ｃ－ｅｒｂＢ－２蛋白、ＰＣＮＡ增殖指数及Ｄｕｋｅｓ分期可作为估计结直肠癌患者预后的独立指标,Ｐ２１及ｃ－ｅｒｂＢ－２蛋白同时表达是预后更差的指征。     

Recurrent chromosome changes in 62 primary gastric carcinomas detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been applied to detect recurrent chromosome alterations in 62 primary gastric carcinomas. Several nonrandom chromosomal changes, including gains of 8q (31 cases, 50%), 20q (29 cases, 47%) with a minimum gain region at 20q11. 2-q12, 13q (21 cases, 34%) with a minimum gain region at 13q22, and 3q (19 cases, 31%) were commonly observed. The regions most frequently lost included: 19p (23 cases, 37%), 17p (21 cases, 33%), and 1p (14 cases, 23%). High copy number gain (DNA sequence amplification) was detected in 6 cases. Amplification of 8q23-q24.2 and 20q11.2-q12 were observed in 3 cases. Gain of 20q and loss of 19p were confirmed by fluorescence in situ hybridization using corresponding bacterial artificial chromosomes (BAC) clones from those regions. The gain and loss of chromosomal regions identified in this study provide candidate regions involved in gastric tumorigenesis.     

Interstitial deletion of the short arm of chromosome 3 as a primary chromosome abnormality in carcinomas of the breast

Interstitial deletions of the short arm of chromosome 3 were found in short-term cultures of five breast carcinomas (of 41 breast cancers with clonal aberrations analyzed by us during the same period). They were the only clonal structural change in three tumors; in the remaining two, the clone with 3p– coexisted with seemingly unrelated clones that had other structural and numerical aberrations. The deletions were identical, del(3)(p12p14), in four cases. The fifth tumor seemed to have a smaller deletion, interpreted as del(3)(p13p14). Our findings constitute karyotypic evidence that 3p deletions are relatively common in breast carcinomas and concur with the molecular genetic detection of loss of heterozygosity in this chromosome arm. The fact that the deletions were found as solitary changes indicates that loss of genetic information from 3p loci is an early, possibly primary, event in tumorigenesis. © 1993 Wiley-Liss, Inc.     

Mutational analysis of FLASH and PTPN13 genes in colorectal carcinomas

AIMS: The Fas-Fas ligand system is considered a major pathway for induction of apoptosis in cells and tissues. FLASH was identified as a pro-apoptotic protein that transmits apoptosis signal during Fas-mediated apoptosis. PTPN13 interacts with Fas and functions as both suppressor and inducer of Fas-mediated apoptosis. There are polyadenine tracts in both FLASH (A8 and A9 in exon 8) and PTPN13 (A8 in exon 7) genes that could be frameshift mutation targets in colorectal carcinomas. Because genes encoding proteins in Fas-mediated apoptosis frequently harbor somatic mutations in cancers, we explored the possibility as to whether mutations of FLASH and PTPN13 are a feature of colorectal carcinomas. METHODS: We analysed human FLASH in exon 8 and PTPN13 in exon 7 for the detection of somatic mutations in 103 colorectal carcinomas by a polymerase chain reaction (PCR)- based single-strand conformation polymorphism (SSCP). RESULTS: We detected two mutations in FLASH gene, but none in PTPN13 gene. However, the two mutations were not frameshift (deletion or insertion) mutations in the polyadenine tracts of FLASH. The two mutations consisted of a deletion mutation (c.3734-3737delAGAA) and a missense mutation (c.3703A>C). CONCLUSION: These data indicate that frameshift mutation in the polyadenine tracts in both FLASH and PTPN13 genes is rare in colorectal carcinomas. Also, the data suggest that both FLASH and PTPN13 mutations in the polyadenine tracts may not have a crucial role in the pathogenesis of colorectal carcinomas.     

Low chromosome number in chromophobe renal cell carcinomas.

Cytogenetic analysis revealed low chromosome number, telomeric association, and pulverisation of chromosomes in three chromophobe renal cell carcinomas. One fully karyotyped and a previously published case showed the common loss of chromosomes 1, 2, 6, 10, 13, 17, and 21.     

Alpha-antitrypsin deposition in primary hepatic carcinomas.

With the use of immunoperoxidase methods, paraffin-embedded hepatic tissue from 21 patients with primary hepatic carcinoma was tested for alpha-antitrypsin (AAT) deposition and was compared for sensitivity with the PAS reaction. Specific AAT immunoreactivity was present in tumor cells in half of the test cases, either alone or in combination with positive nonneoplastic hepatocytes. While the PAS findings generally parralleled the specific immunohistochemical studies, the latter technique exhibited greater sensitivity in AAT detection. Cytoplasmic tumor cell inclusions of three types were identified, but only one type, the solid, was found to represent immunoreactive AAT deposits. These findings confirm as association between hepatic deposition of AAT and the occurrence of primary liver carcinoma. Further prospective serum and tissue studies on patients with hepatoma are needed to extend these observations.     

Spectral karyotyping and chromosome banding studies of primary breast carcinomas and their lymph node metastases

Three primary breast tumors and their lymph node metastases were characterized by G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). In each case, the karyotypic abnormalities detected were similar in the primary tumor and its matched metastasis. Two of the pairs had near-diploid karyotypes with three to four chromosomal aberrations, whereas the third pair had a near-pentaploid chromosome content and many marker chromosomes in the primary tumor and a near-tetraploid chromosome number with almost the same marker chromosomes in the metastasis. SKY and FISH confirmed the karyotypic similarities between the primary tumors and their metastases and, in addition, improved the identification and characterization of marker chromosomes. One of the tumor pairs with near-diploid karyotypes had gain of 8q, 16q, and 17q, whereas the other had gain of 1q and chromosome 8 material in the form of ring chromosomes. The third pair had more complex chromosomal translocations and numerical changes resulting in net gain of material from chromosomes X, 1, 2, 6, 7, 14, 16, 19, and 20, and chromosome arms 8q and 11q, as well as net loss of material from chromosomes 3, 13, 18, 21, and 22. The present study underscores the need to combine conventional chromosome banding and molecular cytogenetic techniques in the cytogenetic analysis of solid tumors.     

Discordance of p53 mutations of synchronous colorectal carcinomas.

It is unclear whether synchronous multiple tumors arise from multicentric or monoclonal origins. To verify the multicentric origin of synchronous colorectal carcinomas at a genetic level, immunohistochemical and molecular techniques were used to determine the p53 alterations in individual lesions of synchronous colorectal carcinomas. This study was based on a total of 32 colorectal tumors from 16 patients. Twenty-one of the 32 (66%) advanced tumors examined had positive staining for p53. Single-strand conformation polymorphism and polymerase chain reaction direct sequencing were carried out for exons 5 to 8 of p53. All cases had p53 mutations in one or more tumors of synchronous lesions. In nine patients in this series, individual lesions were found to carry a different mutated codon of the p53 gene. In the other seven patients, a p53 mutation was found in one tumor but not in another. These results indicate discordance of the mutation pattern of p53 in individual lesions of multiple colorectal carcinomas and support the idea that most synchronous colorectal carcinomas are genetically distinguishable and are multicentric in origin. We also confirmed the high frequency of p53 mutations in left-sided (71%) and rectal (91%) carcinomas, rather than right-sided (43%; P = .04) carcinomas, suggesting that the molecular mechanism of synchronous colorectal carcinomas might differ between right- and left-sided tumors in the same patient.     

Screening for specific chromosome involvement in hematological malignancies using a set of seven chromosome painting probes. An alternative approach for chromosome analysis using standard FISH instrumentation

We report the application of multi-color fluorescence in situ hydribidization (FISH) for bone marrow metaphase cell analysis of hematological malignancies using a sub-set of the human karyotype for chromosome painting. A combination of chromosome probes labeled with three haptens enabled the construction of a "painting probe" which detects seven different chromosomes. The probe was used to screen three chronic myeloid leukemia (CML) derived cell lines and ten CML patient bone marrow samples for aberrations, additional to the Ph rearrangement, that are associated with the onset of blast crisis of CML. This approach was shown to identify karyotype changes commonly seen by conventional karyotyping, and in addition revealed chromosome changes unresolved or undetected by conventional cytogenetic analysis. The seven-color painting probe provides a useful, fast, and reliable complementary tool for chromosome analysis, especially in cases with poor chromosome morphology. This is a simple approach, since the probes can be displayed in a standard red/green/blue format accessible to standard fluorescence microscopes and image-processing software. The proposed approach using panels of locus-specific probes as well as chromosome paints will be useful in all diagnostic routine environments where analysis is directed towards screening for genetic rearrangements and/or specific patterns of chromosome involvement with diagnostic/prognostic value.     

Fascin expression in colorectal carcinomas

PURPOSE

The purpose of this study was to investigate the significance of fascin expression in colorectal carcinoma.

METHODS

This is a retrospective study of 167 consecutive, well-documented cases of primary colorectal adenocarcinoma for which archival material of surgical specimens from primary tumor resections were available. We chose a representative tissue sample block and examined fascin expression by immunohistochemistry using a primary antibody against “fascin”. We calculated the “immunohistochemical score (IHS)” of fascin for each case, which was calculated from the multiplication of scores for the percentage of stained cells and the staining intensity.

RESULTS

Fascin immunoreactivity was observed in 59 (35.3%) of all cases with strong reactivity in 24 (14.4%), moderate reactivity in 25 (14.9%) and weak reactivity in 10 (6.0%) cases. Strong/moderate immunoreactivities were mostly observed in invasive fronts of the tumors or in both invasive and other areas. Fascin immunoreactivity scores were significantly higher in tumors with lymph node metastasis (p:0.002) and advanced stage presentation (p:0.007). There was no relation between fascin expression and age, gender, depth of invasion, distant metastasis or histological grade (p>0.05). There was a higher and statistically significant correlation between fascin immunoreactivity in the invasive borders of tumors and lymph node metastasis (r:0.747, p:0.005). In stage III/IV tumors, two-year survival was 92.2% in tumors without fascin immunoreactivity, and only 60.0% in tumors with a fascin IHS>10 (p:0.003).

CONCLUSION

These findings suggest that fascin is heterogeneously expressed in approximately one third of colorectal carcinomas with a significant association with lymph node metastasis, tumor stage and location. Moreover, these results indicate that fascin may have a role in the lymph node metastasis of colorectal carcinomas.