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Epithelial ovarian neoplasms can be divided into three distinct clinicopathological groups: benign, malignant and borderline tumours. Borderline tumours are less aggressive than epithelial carcinomas, with an indolent clinical course and delayed recurrence. However, a subset of these cases can progress to malignancy and relapse, and death from recurrent disease can occasionally occur. Telomerase activation is a critical element in cellular immortalisation and cancer. The enzyme telomerase comprises a catalytic subunit (TERT) expressed in various types of cancers and regulated by promoter methylation mainly in epithelial tumours.

In 2004, a 56-year-old woman was diagnosed with Stage IA follicular lymphoma in a cervical lymph node biopsy. The patient experienced total remission after local radiation therapy. In 2009, a control computed tomography scan evidenced a pelvic mass, prompting total hysterectomy. The latter harbored a 4.8-cm intramural uterine tumor corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells, with extensive, periodic acid-Schiff negative, signet ring cell changes, and a pan-keratin negative, CD20, CD10, Bcl2, Bcl6 immunophenotype. Molecular genetic studies showed the same clonal IGHgene rearrangement in the lymph node and the uterus, establishing the uterine tumor as a relapse of the preceding follicular lymphoma, although no signet ring cells were evidenced at presentation. Uterine localization of lymphomas is rare, and lymphomas with signet ring cell features are uncommon. This exceptional case exemplifies a diagnostically challenging situation and expands the differential diagnosis of uterine neoplasms displaying signet ring cell morphology.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

Microsatellite instability MSI testing in clinics is becoming increasingly widespread; therefore, there is an urgent need for methodology standardization and the availability of quality control. This study is aimed to assess the interlaboratory reproducibility of MSI testing in archive samples by using a panel of 5 recently introduced, mononucleotide repeats MNR. The quality control involved 8 European institutions. Participants were supplied with DNA extracted from 15 archive colon carcinoma samples and from the corresponding normal tissues. Every group was asked to assess the MSI status of the samples by using the BAT25, BAT26, NR21, NR24, and NR27 mononucleotide markers. Four institutions repeated the analysis using the NCI reference panel to confirm the results obtained with the MNR markers. The overall concordance among institutions for MSI analyses at single locus level was 97.7 when using the MNR panel and 95.0 with the NCI one. The laboratories obtained a full agreement in scoring the MSI status of each patient sample, both using the mononucleotide and the NCI marker sets. With the NCI marker set, however, concordance was lowered to 85.7 when considering the MSI-Low phenotype. Concordance between the 2 panels in scoring the MSI status of each sample was complete if no discrimination was made between MSI-Stable and MSI-L, whereas it dropped to 76.7 if MSI-L was considered. In conclusion, the use of the MNR panel seems to be a robust approach that yields a very high level of reproducibility. The results obtained with the 5 MNR are diagnostically consistent with those obtained by the use of the NCI markers, except for the MSI-Low phenotype.

Transcriptional deregulation in cancer has been shown to be associated with epigenetic alterations, in particular to tumor-suppressor-gene (TSG) promoters. In contrast, DNA methylation of TSGs is not considered to be present in normal differentiated cells. Nevertheless, we previously showed that the promoter of the tumor-suppressor gene APC is methylated, for one allele only, in normal gastric cells. Recently, RASSF1A has been shown to be imprinted in normal human placenta. To clarify putative TSG methylation in the placenta, 23 normal placental tissues from the first trimester, both decidua and villi, and four normal non-gestational endometrium were screened for DNA methylation by methylation-sensitive single-strand conformation analysis (MS-SSCA) and sequencing after bisulfite modification, on a panel of 12 genes known to be implicated in carcinogenesis. In all placental villi, 4 TSG promoters - APC, SFRP2, RASSF1A and WIF1 - were hypermethylated, whereas all decidua and normal endometrium did not show any methylation. Allele-specific methylation analysis revealed that this methylation was monoallelic. Furthermore, comparison with maternal DNA indicated that APC and WIF1 were methylated on the maternal allele, whereas SFRP2 was methylated on the paternal allele. Sequence analysis of WIF1 mRNA revealed that only the unmethylated paternal allele was transcribed. The imprinting status of these TSGs is conserved during pregnancy. These results indicate that TSG imprinting is pre-existent in normal human placenta and should not be confused with carcinogenesis or pathology-induced methylation.

Superficial primitive Ewing's sarcomas are rare and have been reported to be of favorable prognosis compared to conventional deep-seated tumors. In the skin and subcutis, the diagnosis is often difficult, and performing molecular cytogenetic techniques may be helpful. We performed a retrospective analysis of 14 cases of superficial Ewing's sarcomas, all confirmed by molecular cytogenetics. Clinical, histological, immunohistochemical, molecular cytogenetic, therapeutic, and follow-up data are reported. There were 11 female and 3 male patients aged from 12 to 77 years (median: 17 years). Seven tumors occurred in the extremities, five in the trunk wall, and two in the head. Tumor size ranged from 1 to 5 cm (median, 3 cm). They were all small round-cell proliferations with a strong membranous positivity for CD99. Ewing's sarcoma translocations/fusion gene transcripts were detected in eight cases, both by FISH and reverse transcriptase (RT)–PCR. Four tumors were positive by RT–PCR alone (FISH not done in three cases and not interpretable in one case), and two cases were positive by FISH alone (RT–PCR not done). Surgical resection was performed in all patients. Chemotherapy was given in ten patients and radiotherapy in six. At last medical examination (median follow-up, 47 months), two patients who underwent surgical resection alone had died from the tumor. Our results confirm that superficial Ewing's sarcomas are of good prognosis. Given the difficulty of the diagnosis and the importance of an adapted treatment, a confirmation of the diagnosis by molecular or cytogenetic techniques is recommended when dealing with a superficial tumor.

The diagnosis of sarcomatoid mesothelioma is still a worldwide challenge and it is often difficult, both clinically and by morphological analysis, to differentiate sarcomatoid mesothelioma from synovial sarcoma, the most frequent intrathoracic sarcoma. To confirm the absence of the synovial sarcoma translocation t(X; 18) (SYT-SSX) in sarcomatoid mesothelioma, and to test its usefulness differentiating sarcomatoid mesothelioma from synovial sarcoma, 28 tumours were examined using the reverse transcriptase-polymerase chain reaction. RNA was extracted from paraffin blocks using standard methods, reverse-transcribed and PCR performed. Molecular analysis completed in two independent laboratories showed that sarcomatoid mesothelioma samples were negative for the t(X-18). This result confirms the usefulness of this analysis in differentiating sarcomatoid mesothelioma from synovial sarcoma.Modern Pathology (2007) 20, 617–621; doi:10.1038/modpathol.3800775

The diagnosis of sarcomatoid mesothelioma is still a worldwide challenge and it is often difficult, both clinically and by morphological analysis, to differentiate sarcomatoid mesothelioma from synovial sarcoma, the most frequent intrathoracic sarcoma. To confirm the absence of the synovial sarcoma translocation t(X; 18) (SYT-SSX) in sarcomatoid mesothelioma, and to test its usefulness differentiating sarcomatoid mesothelioma from synovial sarcoma, 28 tumours were examined using the reverse transcriptase-polymerase chain reaction. RNA was extracted from paraffin blocks using standard methods, reverse-transcribed and PCR performed. Molecular analysis completed in two independent laboratories showed that sarcomatoid mesothelioma samples were negative for the t(X-18). This result confirms the usefulness of this analysis in differentiating sarcomatoid mesothelioma from synovial sarcoma.

Barretts esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). Recently, and similarly to other human malignancies, the Wnt signaling pathway and its key component -catenin have been implicated in the carcinogenesis of BE. Although mutations in adenomatous polyposis coli (APC) or -catenin are rare in EAC, alterations of upstream components, such as overexpression of Wnt2 ligand or downregulation of Wnt antagonists may play dominant roles in the activation of the Wnt pathway. Increasing evidence suggests that inhibiting the Wnt pathway may be a new targeted therapy for the treatment of cancers and could, therefore, be promising for the cure of EAC, which remains a highly lethal disease.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene rearrangements could be helpful in the evaluation of B-cell lymphoma (BCL) involvement of bone marrow (BM) biopsy specimens. We evaluated 83 paraffin-embedded BM biopsy specimens from 26 patients with BCL. When BM biopsy specimens considered positive, "suspicious," or negative by morphologic and immunohistochemical examination were evaluated by PCR, a monoclonal B-cell population was detected in 81% (39/48), 64% (9/14), and 11% (2/18), respectively. In most cases, a reproducible monoclonal IgH gene rearrangement was observed from BM and extramedullary sites. Nevertheless, in 4 cases, a different and independent monoclonal IgH rearrangement was observed during the disease course. PCR is efficient and complementary to morphologic and immunohistochemical examination for the evaluation of BCL involvement of BM biopsy specimens, especially when a reproducible rearrangement is found in 2 different samples.

Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is composed of two main components: hTERT and hTERC. hTERT seems to be the rate-limiting factor for telomerase activity, although hTERC expression was also shown to correlate to a certain extent with telomerase reactivation. To determine whether the absence of hTERC expression could be the consequence of DNA methylation, we quantified hTERC RNA in 60 human samples (19 telomerase-negative normal tissues, nine telomerase-positive and 22 telomerase-negative tumor tissues, eight telomerase-positive and two telomerase-negative cell lines) using a quantitative dot blot on RT-PCR products. Most of the normal tissues did not express hTERC whereas, in telomerase-positive cell lines and in telomerase-positive tumor tissues, a strong up-regulation was observed, suggesting that hTERC transcription is up-regulated during tumorigenesis. The two telomerase-negative cell lines did not express hTERC. In a series of 22 telomerase-negative soft tissue sarcomas (STS), half did not express hTERC at all, or only weakly, whereas a wide range of expression was observed in the other half. As methylation might be involved in hTERC silencing, we examined the methylation pattern in all samples by direct sequencing and methylation-specific single stand conformation analysis after bisulfite modification. hTERC methylation was never observed, neither in normal nor in tumor tissues. Furthermore, there was no correlation between hTERC expression and proliferation, telomere length or hTERT expression in telomerase-negative STS. In contrast, three of eight telomerase-positive cell lines and the two telomerase negative cell lines were found to be hypermethylated, suggesting that the methylation observed may occur during cell line establishment. In conclusion, this study shows that hTERC expression is indeed regulated during carcinogenesis, but this regulation is unlikely to depend on hTERC methylation, cell proliferation rate, telomere length or hTERT expression.

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p &lt; 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p &lt; 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression. © 2002 Wiley-Liss, Inc.

In a previous study, we showed that telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA expression were undetectable in benign mesenchymal lesions and low-grade soft tissue sarcomas (STSs), but detectable in about 50% of intermediate-/high-grade STSs. We wondered if this lack of TA or hTERT mRNA expression could be related to the tumor sample examined and if there was a relationship between the former 2 parameters and telomere length. Two separate tumor samples from 37 STSs were examined for telomerase activity, using the telomerase repeat amplification protocol (TRAP) assay and for hTERT mRNA expression, using RT-PCR. Telomere length was determined in each tumor sample, using the terminal restriction fragments (TRF) technique. Significant variations in telomere length, TA and hTERT mRNA expression between 2 samples of the same tumor were observed in 27%, 11% and 27% of STSs, respectively. Telomere length did not correlate with TA or hTERT mRNA expression. Despite great intratumoral heterogeneity in telomere length, short and long telomeres were more often seen in the low/intermediate-grade and high-grade STS categories, respectively. Few STSs that showed a TRF pattern suggestive of alternative lengthening of telomeres (ALT) may contain ALT subpopulations. © 2002 Wiley-Liss, Inc.

The t(X;18) translocation is known to be a useful marker for the diagnosis of synovial sarcoma. In this study, the authors describe a new real-time reverse transcriptase–polymerase chain reaction (RT-PCR) method to detect SYT/SSXfusion transcripts using paraffin-embedded and frozen tumor specimens. A series of 38 soft tissue sarcomas were analyzed. Diagnosis was based on clinical, histologic, and immunohistochemical examination. The fusion transcripts were detected in 16 of 17 synovial sarcoma samples (the 17th sample was not suitable for molecular analysis). No t(X;18)-fusion transcript was PCR-amplified in the 21 nonsynovial sarcoma mesenchymal tumors. Therefore, real-time PCR amplification appears to be a powerful, rapid, specific, and sensitive technique that can be used routinely to diagnose the synovial sarcoma t(X;18) translocation. In addition, the t(X;18) can be detected not only on frozen but also on paraffin-embedded tumor samples.

The majority of the adenocarcinomas arising in Barrett esophagus manifest clinically at an advanced stage and have a poor prognosis. As a result of this poor prognosis, much attention has been directed toward the exploration of markers for neoplastic progression in Barrett esophagus. The objective of the present study was to determine the expression of beta-catenin by immunohistochemical analysis in 70 adenocarcinomas developed in Barrett esophagus and to examine its relationship to various prognostic factors currently in use. Abnormal beta-catenin expression, consisting of the loss of membranous staining and the appearance of the nuclear staining, was found in 43 cases (61%). Of patients with the 43 tumors showing abnormal beta-catenin expression, 25 (58%) survived more than 1 year. In contrast, only 7 (26%) of 27 patients with tumors showing normal beta-catenin expression survived longer than 1 year. Most of the superficial (Tis-T1) tumors (83% [10/12]) exhibited abnormal beta-catenin expression compared with only 53% (31/58) in the T2-T3 group. These results suggest a possible correlation among beta-catenin expression, tumor stage, and length of survival as prognostic factors in patients with adenocarcinoma in Barrett esophagus.

Colorectal carcinogenesis is widely accepted as one of the best-characterized examples of stepwise progression. The existing colorectal carcinogenesis model assumes genetic homogeneity of individual tumors for the main known genetic alterations: K-ras and p53 genes point mutations and loss of heterozygosity (LOH) of chromosome 5q and 18q. The object of the present study was to demonstrate the existence of an intratumor genetic heterogeneity in advanced sporadic colorectal carcinoma for these genetic alterations. Using improved tissue microdissection and DNA extraction, for each tumor, amplifiable DNA was obtained from 15 to 20 areas, of which 1 to 2 concerned lymph node metastases (LNM). This study revealed that 10 of 15 (67%) analyzed tumors were heterogeneous for at least 1 genetic alteration, with between 2 and 6 genotypically different clones detected per tumor. No correlation was observed between the genotype of these subclones and histological differentiation or invasive propensity. Intratumor heterogeneity was more frequently observed for LOH than for point mutations, 67% and 58% for LOH at APC and DCC locus, and 20% for mutation of either the K-ras or p53 gene. In 5 of the 9 (56%) heterogeneous cases with available LNM, the genotype observed in the LNM was different from that of the main clone in the primary tumor, and moreover, 2 of the LNM displayed a genotype undetected in the primary tumor. In conclusion, intratumor genetic heterogeneity was demonstrated in advanced sporadic colorectal carcinoma and was represented as topographically distinct genotypic subclones. Taking into account such a significant genetic heterogeneity of colorectal tumors, the use of genetic markers for prognosis management should be reconsidered. © 2001 Wiley-Liss, Inc.

The human telomerase enzyme is composed of two essential components, hTR, which acts as a template for reverse transcription, and hTERT, which is the putative catalytic subunit for the enzyme. Recent studies have demonstrated a good correlation between hTERT expression and telomerase activation, whereas RT‐PCR results seemed to reveal that hTR is ubiquitously expressed in all cells. These observations left unclear the role of hTR, and to a lesser extent hTERT, in the regulation of telomerase activation. In the present study, the correlation of telomerase activity and the expression of these genes was examined in a total of 70 colorectal tissues (25 adenocarcinomas, 30 adenomas, and 15 samples of normal colorectal mucosa). Total RNA for RT‐PCR analysis and cell extracts for TRAP assay were obtained from consecutive sections and histological control was simultaneously performed. To avoid false‐positive results, due to the fact that hTR cDNA and genomic hTR DNA are identical (the gene has no introns), extensive DNase digestion was performed before cDNA synthesis. RT‐PCR analysis revealed that hTERT mRNA was expressed in all cancers and in 13 of 14 telomerase‐positive adenomas, but never in telomerase‐negative colorectal tissues. hTR transcripts were observed in all telomerase‐positive samples but also in three telomerase‐negative samples, two adenomas, and one normal colonic mucosa. It is concluded that hTERT and hTR expression is strongly correlated with telomerase activity. hTR transcripts, however, also occur in some telomerase‐negative tissues and these results are in keeping with the concept that hTERT expression is a major regulator of telomerase activity. Copyright © 2000 John Wiley & Sons, Ltd.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up-regulated in the majority of cancers. The colorectal adenoma–carcinoma sequence is one of the best-characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false-negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty-five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia ( p&lt; 0·0001). All adenomas which contained high-grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low-grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low-grade to high-grade dysplasia in adenomas. Furthermore, in the adenoma–carcinoma sequence, telomerase activation seems to occur later than K- ras mutation but earlier than p53 mutation. Copyright © 1999 John Wiley & Sons, Ltd.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up‐regulated in the majority of cancers. The colorectal adenoma–carcinoma sequence is one of the best‐characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false‐negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty‐five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia ( p< 0·0001). All adenomas which contained high‐grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low‐grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low‐grade to high‐grade dysplasia in adenomas. Furthermore, in the adenoma–carcinoma sequence, telomerase activation seems to occur later than K‐ rasmutation but earlier than p53mutation. Copyright © 1999 John Wiley & Sons, Ltd.

Background:Mutations at codons 12, 13, and 61 of the ras genes have been found in a variety of human tumors and may have prognostic significance. K-rasmutations have been shown in 40%–50% of colorectal cancers. Methods:Using a simple nonradioactive polymerase chain reaction-based technique, we have investigated the possible prognostic significance of point mutations of the K-rasgene in patients with human colorectal carcinomas. The prevalence and the type of rasmutations were compared between a group of 35 patients having recurrent disease within 5 years and a group of 64 patients who were disease free 5 years following surgery. Results:First, we found that the overall prevalence of mutations within codons 12 and 13 of the K-rasgene was 25% in the nonrecurring group vs. 71% in the patients with recurrent disease (P< 0.0001) and, second, that mutations other than GGT to GAT occurred, with one exception, exclusively in recurring tumors. Conclusions:In Dukes' B and C primary tumors, mutations other than GGT to GAT identify patients at very high risk for recurrence. Our results indicate that determining the K-rasmutations provides a good prognostic factor in patients with advanced colorectal carcinoma.

The majority of colorectal cancers develop from adenomatous polyps under the influence of factors that are still poorly understood. Tumourigenesis is generally considered a multistep process in which multiple genetic alterations occur, eventually reflected in abnormalities of the cellular DNA content. Macroscopical features such as tumour size and tumour architecture (tubular, tubulovillous, or villous) are correlated wit the chance of malignancy in the lesion. Grade of dysplasia can be considered an indicator for the level of progression of the adenoma towards invasive carcinoma. These characteristics were correlated with the presence or absence of K‐rasmutations and the DNA ploidy in a prospective study performed on 46 large sporadic colorectal adenomas resected by endoscopy. DNA ploidy and K‐rasmutations were analysed in two samples taken at distant sites in the adenomas. Aneuploidy was present in 12 adenomas (26 per cent) and K‐rasmutations occurred in 26 (57 per cent). A highly significant correlation was found between aneuploidy and adenoma size, architecture, and grade of dysplasia. The presence of K‐rasmutations was significantly correlated only with the size of the adenomas. The proportion of adenomas with aneuploidy and/or a K‐rasmutation increased when two samples were analysed instead of one. This observation suggests that the prevalence of genetic mutations and of aneuploidy is probably underestimated, as generally only one sample is investigated. No correlation was observed between K‐rasmutations and ploidy. This study demonstrates the presence of genetic heterogeneity in colorectal adenomas and supports the notion that K‐rasmutation is an early event, while aneuploidy is a late event in the adenoma–carcinoma sequence. © 1997 John Wiley & Sons, Ltd.

We have developed and tested a rapid and sensitive method of detecting expansion of T-cell clones using the polymerase chain reaction (PCR) and a single set of consensus primers for the V and J regions to amplify rearranged T-cell receptor-γ (TCR-γ) genes. Monoclonality was continued in all of the 18 cases of T-cell neoplasms tested, but not in reactive lymphadenopathy, non-Hodgkin's B lymphomas, and Hodgkin s disease. PCR analysis, using the primer sequence outlined in this study, had an overall specificity of 100 when compared with Southern blot analysis. No false-negative results were observed, certainly owing to the choice of consensus primers and to the control of PCR reactions on agarose gels before testing for clonality by separation of PCR products on polyacrylamide gels. This method for the detection of T-cell monoclonality can be especially useful in cases that are diagnostically problematic with standard histological and immunological analysis and in cases where the material available is limited.

In both the primary tumor and associated lymph-node metastases of 40 cases of Dukes' C colorectal adenocarcinomas, exons 5 to 9 of the p53 tumor-suppressor gene were examined by PCR amplification and single-strand-conformation-polymorphism(SSCP) analysis. Mobility shifts indicating p53 mutations, which were confirmed by direct sequencing, were identified in 14 primary cancers (35%) and in 19 of the 40 lymph-node metastases (48%). In 12 cases (30%), the p53-mutation status in the primary cancer and its lymph-node metastases was identical. This result is compatible with the hypothesis that when a p53 mutation occurs before the establishment of lymph-node metastasis, it subsequently persists in the metastatic nodes. In 7 cases (18%), p53 mutations were identified in lymph-node metastases that were not concordant with the p53 status in the primary tumor. This finding can be explained by assuming that (1) p53 heterogeneity existing in the primary tumor is not reflected in all metastases and/or (2) new p53 mutations may occur during the development of metastatic lesions. Int. J. Cancer 70:674–678, 1997. © 1997 Wiley-Liss, Inc.

The majority of colorectal cancers develop from adenomatous polyps under the influence of factors that are still poorly understood. Tumourigenesis is generally considered a multistep process in which multiple genetic alterations occur, eventually reflected in abnormalities of the cellular DNA content. Macroscopical features such as tumour size and tumour architecture (tubular, tubulovillous, or villous) are correlated wit the chance of malignancy in the lesion. Grade of dysplasia can be considered an indicator for the level of progression of the adenoma towards invasive carcinoma. These characteristics were correlated with the presence or absence of K-ras mutations and the DNA ploidy in a prospective study performed on 46 large sporadic colorectal adenomas resected by endoscopy. DNA ploidy and K-ras mutations were analysed in two samples taken at distant sites in the adenomas. Aneuploidy was present in 12 adenomas (26 per cent) and K-ras mutations occurred in 26 (57 per cent). A highly significant correlation was found between aneuploidy and adenoma size, architecture, and grade of dysplasia. The presence of K-ras mutations was significantly correlated only with the size of the adenomas. The proportion of adenomas with aneuploidy and/or a K-ras mutation increased when two samples were analysed instead of one. This observation suggests that the prevalence of genetic mutations and of aneuploidy is probably underestimated, as generally only one sample is investigated. No correlation was observed between K-ras mutations and ploidy. This study demonstrates the presence of genetic heterogeneity in colorectal adenomas and supports the notion that K-ras mutation is an early event, while aneuploidy is a late event in the adenoma–carcinoma sequence. © 1997 John Wiley & Sons, Ltd.

Genetic instability related to defective DNA mismatch repair genes may be involved in the pathogenesis of carcinoma in Hereditary Non-Polyposis Colorectal Cancer (HNPCC). To test that the targets of genetic instability could include critical transforming genes involved in colon tumor progression, we examined 23 colorectal carcinomas in patients with HNPCC in order to detect somatic mutations in K-ras and p53 genes. Using single strand conformation polymorphism followed by direct DNA sequencing, we detected 4 mutations in K-ras gene (17%) and 3 in p53 gene (13%) which change the aminoacid sequence of the protein p53. This is significantly lower than in sporadic cancer. Our data suggest that colon cancer in HNPCC might partly involve a distinct pathogenetic mechanism that involves other genes than those altered in sporadic tumors. Int. J. Cancer 74:94–96. © 1997 Wiley-Liss, Inc.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain (IgH) gene rearrangements could be helpful in the evaluation of B-cell lymphoma (BCL) involvement of bone marrow (BM) biopsy specimens. We evaluated 83 paraffin-embedded BM biopsy specimens from 26 patients with BCL. When BM biopsy specimens considered positive, “suspicious,” or negative by morphologic and immunohistochemical examination were evaluated by PCR, a monoclonal B-cell population was detected in 81% (39/48), 64% (9/14), and 11% (2/18), respectively. In most cases, a reproducible monoclonal IgH gene rearrangement was observed from BM and extramedullary sites. Nevertheless, in 4 cases, a different and independent monoclonal IgH rearrangement was observed during the disease course. PCR is efficient and complementary to morphologic and immunohistochemical examination for the evaluation of BCL involvement of BM biopsy specimens, especially when a reproducible rearrangement is found in 2 different samples.