Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a childhood cancer predisposition syndrome involving biallelic germline mutations of MMR genes, poorly recognised by clinicians so far. METHODS: Retrospective review of all 31 patients with CMMRD diagnosed in French genetics laboratories in order to describe the characteristics, treatment and outcome of the malignancies and biological diagnostic data. RESULTS: 67 tumours were diagnosed in 31 patients, 25 (37%) Lynch syndrome-associated malignancies, 22 (33%) brain tumours, 17 (25%) haematological malignancies and 3 (5%) sarcomas. The median age of onset of the first tumour was 6.9 years (1.2-33.5). Overall, 22 patients died, 9 (41%) due to the primary tumour. Median survival after the diagnosis of the primary tumour was 27 months (0.26-213.2). Failure rate seemed to be higher than expected especially for T-cell non-Hodgkin's lymphoma (progression/relapse in 6/12 patients). A familial history of Lynch syndrome was identified in 6/23 families, and consanguinity in 9/23 families. PMS2 mutations (n=18) were more frequent than other mutations (MSH6 (n=6), MLH1 (n=4) and MSH2 (n=3)). CONCLUSIONS: In conclusion, this unselected series of patients confirms the extreme severity of this syndrome with a high mortality rate mostly related to multiple childhood cancers, and highlights the need for its early detection in order to adapt treatment and surveillance.

Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D).

Lynch syndrome (LS) is an autosomal dominant disorder caused by a defect in one of the DNA mismatch repair genes: MLH1, MSH2, MSH6 and PMS2. In the last 15 years, an increasing number of patients have been described with biallelic mismatch repair gene mutations causing a syndrome referred to as 'constitutional mismatch repair-deficiency' (CMMR-D). The spectrum of cancers observed in this syndrome differs from that found in LS, as about half develop brain tumours, around half develop digestive tract cancers and a third develop haematological malignancies. Brain tumours and haematological malignancies are mainly diagnosed in the first decade of life, and colorectal cancer (CRC) and small bowel cancer in the second and third decades of life. Surveillance for CRC in patients with LS is very effective. Therefore, an important question is whether surveillance for the most common CMMR-D-associated cancers will also be effective. Recently, a new European consortium was established with the aim of improving care for patients with CMMR-D. At a workshop of this group held in Paris in June 2013, one of the issues addressed was the development of surveillance guidelines. In 1968, criteria were proposed by WHO that should be met prior to the implementation of screening programmes. These criteria were used to assess surveillance in CMMR-D. The evaluation showed that surveillance for CRC is the only part of the programme that largely complies with the WHO criteria. The values of all other suggested screening protocols are unknown. In particular, it is questionable whether surveillance for haematological malignancies improves the already favourable outcome for patients with these tumours. Based on the available knowledge and the discussions at the workshop, the European consortium proposed a surveillance protocol. Prospective collection of all results of the surveillance is needed to evaluate the effectiveness of the programme.

Virtual genome scan: a tool for restriction landmark-based scanning of the human genome.

There is substantial interest in implementing technologies that allow comparisons of whole genomes of individuals and of tissues and cell populations. Restriction landmark genome scanning (RLGS) is a highly resolving gel-based technique in which several thousand fragments in genomic digests are visualized simultaneously and quantitatively analyzed. The widespread use of RLGS has been hampered by difficulty in deriving sequence information for displayed fragments and a lack of whole-genome sequence-based framework for interpreting RLGS patterns. We have developed informatics tools for comparisons of sample derived RLGS patterns with patterns predicted from the human genome sequence and displayed as Virtual Genome Scans (VGS). The tools developed allow sequence prediction of fragments in RLGS patterns obtained with different restriction enzyme combinations. The utility of VGS is demonstrated by the identification of restriction fragment length polymorphisms, and of amplifications, deletions, and methylation changes in tumor-derived CpG islands and the characterization of an amplified region in a breast tumor that spanned <230 kb on 17q23.

CGH analysis of radon-induced rat lung tumors indicates similarities with human lung cancers.

Epidemiological studies have shown that inhalation of radon, a radioactive gas, is associated with an increased risk for lung cancer. We have developed a model of radon-induced rat lung tumors to characterize cytogenetic and molecular events involved in radon-induced lung tumorigenesis. Using comparative genomic hybridization (CGH), gains and losses of genetic material were investigated in a series of 13 carcinomas and four adenomas of the lung. Frequent losses occurred at 4q12-21, 5q11-33, and 15q, which are homologous to human chromosome (HSA) bands 7q21-36, 1p31-36/9p21-31, and 13q14.1-14.3/3p14.2, respectively. These regions are frequently (30-80%) deleted in human lung cancer and contain tumor suppressor genes or proto-oncogenes such as MET, CDKN2A/p16/MTS1, CDKN2B/p15/MTS2, FHIT, and RB1 or yet to be identified genes. Frequent gains involved 6, 7q34-qter, and 19q; chromosomes 6 and 7 being homologous to human 2p21-25 and 8q21-24 where the MYCN and MYC oncogenes are located. The genetic similarities between rat and human lung cancer suggest common underlying mechanisms for tumor evolution in both species. Moreover, cytogenetic and molecular genetic analyses of radon-induced rat lung tumors could help to better understand the development and progression of radon-induced lung cancer in man.

TP53 status and gene amplification in human colorectal carcinomas.

Gene amplification is one of the characteristics of cancer cells. In vitro studies suggested that alterations of the TP53 gene might be responsible for gene amplification. We have examined the presence of TP53 mutations and looked for cytogenetic evidence of gene amplification in a series of 79 primary colorectal carcinomas. Other parameters such as the pattern of cytogenetic alterations, microsatellite instability, tumor site, and histological staging were also considered. A multiparametric study supported by statistical analyses suggests the existence of two major pathways of colorectal carcinogenesis. No relationships could be established between the presence of TP53 alterations and gene amplification.

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas.

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q3.1. We report here the amplification and over-expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over-expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno-histochemical analysis showed that expression was restricted to tumour cells. Thus, high-level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour.

Distinct chromosomal alterations associated with TP53 status of LoVo cells under PALA selective pressure: a parallel with cytogenetic pathways of colorectal cancers.

This study investigates the chromosomal alterations involved in the acquisition of PALA resistance of LoVo colorectal cancer cells homozygous for wild-type TP53 before and after transfection with a 143Ala-mutated TP53 gene. PALA resistance was always associated with an increased number of CAD gene copies, but gene amplification sensu stricto was rarely observed. Interestingly, distinct chromosome patterns were found in relation to the TP53 status of the cells. In parental LoVo cells, the CAD copy number was increased through gains of normal chromosome 2 whereas in transfectant clones, resistance mostly occurred through chromosome rearrangements. The relationship with the two different cytogenetic patterns described in colorectal tumors is discussed.

MLL2, the second human homolog of the Drosophila trithorax gene, maps to 19q13.1 and is amplified in solid tumor cell lines.

The Mixed Lineage Leukemia (MLL) gene is commonly involved in translocations in infantile leukemia and is amplified in some cases of adult myeloid leukemia. A homolog of MLL denoted MLL2, which represents the second human homolog of the Drosophila trithorax gene, was characterized by assembling ESTs, the KIAA0304 cDNA clone, RT - PCR fragments and a new clone isolated from a cDNA phage library and compared to the available genomic sequence. The MLL2 gene maps to 19q13.1, a region of frequent rearrangement or amplification in solid tumors. MLL2 consists of an 8.5 - 9 kb transcript and spans 20 kb of genomic DNA. The predicted MLL2 protein possesses all of the major domains defined in MLL and the two genes have a similar genomic structure. We find that MLL2 is amplified in two of 14 pancreatic carcinoma cell lines and one of five glioblastoma cell lines and is a likely critical gene in 19q13.1 amplifications. It is also a candidate for chromosomal rearrangements involving this chromosome locus. MLL2 is one additional mammalian trithorax-group gene with involvement in human cancer.

Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Growth dependency of human colon cancer xenograft on organ environment is related with their original clinical stage.

The ability of cancer cells to metastasize might depend on their reduced dependency on the originating tissue. To test this hypothesis, 10 human colorectal carcinomas (CRC) were implanted either onto nude mouse caecum wall (orthotopic site), or into the subcutaneous tissue (ectopic site), and their growth in these sites compared. Prognostic factors were studied: Astler-Coller modified Dukes's stage, loss of chromosome 17p and/or 18q, and their TP53 gene. Early stage CRC [B2 (n = 3) and C1 (n = 1)] were found to grow 1.7 to 3.6 times (p < 0.05, p < 0.008 respectively) more rapidly in the caecum than in subcutaneous tissue. Metastatic stage CRC [C1 (n = 1), C2 (n = 2) or D (n = 3)] grew similarly in both sites, and more slowly than those of the first group. No relationship was found between growth rates, TP53 mutations or karyotypes. Growth rate of non metastatic cancers was slowed down by implantation in a foreign tissue whereas growth of metastatic tumours was similar in both sites, indicating that they do not recognize or need tissue growth factors.

GAC1, a new member of the leucine-rich repeat superfamily on chromosome band 1q32.1, is amplified and overexpressed in malignant gliomas.

We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is amplified. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Amplification and overexpression of GAC1 was demonstrated in two of eight tumors where amplifications were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.

Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype.

Adaptation of HT-29 cells to increasing concentrations of methotrexate (MTX) results in the selection of differentiated populations which show sequential dose-dependent changes of their differentiated phenotype with, at the highest concentrations (0.1 and 1 mM), a shift of differentiation from a mucus-secreting to an enterocytic phenotype coinciding with an amplification of the DHFR gene. We show here that DHFR gene amplification itself does not play a role in the shift of differentiation. An alternative explanation is the presence, within the mucus-secreting population, of an undetectable minor population of cells committed to enterocytic differentiation and able to develop resistance to higher concentrations of MTX. This was confirmed by cloning the population of cells resistant to 10 microM MTX. Out of 19 isolated clones, 17 were found to be mucus-secreting and 2 enterocytic. We tested 9 of these clones for their ability to develop resistance to 0.1 mM MTX: only 1 of enterocytic phenotype, was found to develop resistance to this higher concentration and to amplify the DHFR gene. The ability of enterocytic cells to develop resistance to elevated MTX concentration through amplification of the DHFR gene was demonstrated in another enterocytic HT-29 population selected by glucose deprivation. Enterocytic cells resistant to 10 microM MTX were also found, unlike mucus-secreting cells, to be readily adaptable to 5-fluorouracil, this occurring without amplification of the thymidylate synthase gene. Together these results highlight a previously uncharacterized relationship between commitment to enterocytic differentiation of colon-cancer cells and their ability to develop resistance to MTX and 5-fluorouracil.

Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization.

Using chromosome painting, a study of chromosomal abnormalities has been performed in two prostatic carcinoma cell lines, PC-3 and DU145. In PC-3, this analysis revealed a highly rearranged hypotriploid karyotype with 54 to 61 chromosomes and numerous rearrangements of chromosomes 1, 3, 5, 8, 10, and 14. At passage 73, DU145 had a hypotriploid karyotype with few rearrangements of chromosomes 1, 3, 5, 12, 13, and 20, whereas at passage 153, this cell line showed a near-tetraploid karyotype with a great number of rearrangements involving chromosomes 3, 6, 8, 10, 12, and 17. A single rearrangement was shared by the 2 cell lines, an i(5)(p10). A comparative genomic hybridization study demonstrated a noticeable amplification of bands 10q22.3-q23 and 14q22-q24 in the PC-3 cell line. No amplification signal was detected for DU145.

Colorectal carcinogenesis: from chromosomal evolution pathways to molecular pathogenesis.

Since the mid-1980s, research in the field of colorectal carcinogenesis has seen a series of breakthroughs such, as the process of loss of heterozygosity for large chromosomal segments and the consequent characterization of a series of suppressor genes considered to be the targets of the allelic deletions. More recently, a new perspective has been opened, with the discovery of germinal mutations of genes involved in mismatch repair in certain inherited forms of the disease. Through the retrospective analysis of our data on colorectal adenomas and cancers, we have tried to critically reassess a number of theoretical considerations relating to the instability of the genome viewed at the chromosome level and its consequence on tumor progression.

The accumulation and occurrence of clonal and unstable rearrangements are independent in colorectal cancer cells.

The rates of unstable and clonal rearrangements were compared in a series of colorectal cancers. These tumors were classified in relation to their karyotypes, into monosomic (MT), trisomic (TT), or normal (NT) type. MT have multiple, TT have few, and NT have no clonal, generally unbalanced, chromosome rearrangements. The rates of unstable rearrangements, i.e. chromatid and chromosome breaks, chromatid exchanges, dicentrics, and telomeric associations, were found to be very similar in these tumors, suggesting identical chromosome instability defined as the occurrence of nontransmissible rearrangements at each cell generation. Thus, the chromosomal patterns of these tumors seem to be rather driven by the specific selection of chromosome alterations than by differential rates of occurrence of rearrangements.

Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs.

The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Kruppel family of zinc-finger proteins. The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin. These binding activities are characteristic of zinc-finger proteins. Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures. Western blot analysis demonstrated the expression of a 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans-acting domain. These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins. In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis.

Identification of amplified DNA sequences in breast cancer and their organization within homogeneously staining regions.

A modified comparative genomic hybridization (mCGH) technique was used to identify and map amplified DNA sequences in six homogeneously staining regions (hsr) from three primary breast carcinomas. Five different chromosomal regions and bands were identified as sites of amplification: 8p1, 17q21.1, 17q23 (two cases), 19q13.3, and 20q13.3. The mCGH site located on 17q21.1 was demonstrated to correspond to a 50-100-fold amplification of ERBB2. Further in situ hybridization experiments were used to confirm the mCGH results and to characterize the organization of the amplified sequences within the hsr. In five of six instances, two or more chromosomal regions were found amplified in the same hsr. In the tumor with the less modified karyotype, the two hsr comprised DNA sequences from three different chromosomes and showed different patterns of amplification. In the tumor with the most rearranged karyotype, the hsr-carrying chromosomes were formed by the translocation and amplification of sequences from three or four different chromosomal sites. This illustrates the complexity of the amplification process in breast cancers.