Phenotype analysis of families with TP53 germline variants at the Center for Familial Breast and Ovarian Cancer, Cologne.

PURPOSE: Tumor protein p53 (TP53) pathogenic variant (PV) carriers are identified during genetic testing for hereditary causes of cancer. PVs in TP53 are associated with the Li-Fraumeni syndrome (LFS), and thus, surveillance and preventive measures are important for TP53 PV carriers. However, the penetrance of TP53 PVs can be low if the Chompret criteria are not fulfilled. In this study, we compared the phenotypic characteristics of families that did and did not fulfill the LFS criteria according to Chompret. METHODS: The German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) database was used to identify index patients with a likely pathogenic/pathogenic TP53 variant and their family members. The study investigated the type of variant, pedigree, age of onset, number of primary tumors, and histological type of BC. RESULTS: TP53 PV were present in the index cases of 35 families, 57% (20/35) of which fulfilled the Chompret criteria. The median age of onset at first BC diagnosis was lower in families that fulfilled the Chompret criteria compared to those who did not. Four of all diseased individuals were minors (4%; 4/105) when malignancy was first diagnosed. Sarcomas and brain tumors occurred in 10% (10/105) and in 7% (7/105) of all diseased persons, respectively. BC was the most frequently occurring first tumor (60%; 62/105) and additional malignancy (45%; 20/44) in this cohort. Subsequent malignancies developed in 31% (20/65) of the individuals who fulfilled the Chompret criteria compared with 15% (6/40) of those who did not. CONCLUSION: The tumor spectrum and age of onset found in this study showed that tumors other than BC had low disease penetrance in TP53 PV carriers identified using the GC-HBOC criteria for genetic testing.

Platinum sensitivity in a BRCA1 mutation carrier with advanced breast cancer.

Although BRCA1-associated breast carcinomas are frequently detected in nodal-negative stage, they typically present with an aggressive histopathological phenotype that is reflected by a poor prognosis and an increased risk for distant metastatic spread. Recent in vitro data suggest a high sensitivity of BRCA1-associated carcinomas to platinum-based chemotherapy and a lower sensitivity to anthracyclines and taxanes. This is explained by the key role of BRCA1 in DNA double-strand repair via homologous recombination, thereby leading to a higher sensitivity to DNA intercalating agents, such as platinum. Here we present the case of a woman suffering from BRCA1-associated metastatic breast carcinoma that was resistant to docetaxel, but responded strongly to cisplatin-containing chemotherapy. This supports the rationale of ongoing clinical studies.

Strong evidence that the common variant S384F in BRCA2 has no pathogenic relevance in hereditary breast cancer.

INTRODUCTION: Unclassified variants (UVs) of unknown clinical significance are frequently detected in the BRCA2 gene. In this study, we have investigated the potential pathogenic relevance of the recurrent UV S384F (BRCA2, exon 10). METHODS: For co-segregation, four women from a large kindred (BN326) suffering from breast cancer were analysed. Moreover, paraffin-embedded tumours from two patients were analysed for loss of heterozygosity. Co-occurrence of the variant with a deleterious mutation was further determined in a large data set of 43,029 index cases. Nature and position of the UV and conservation among species were evaluated. RESULTS: We identified the unclassified variant S384F in three of the four breast cancer patients (the three were diagnosed at 41, 43 and 57 years of age). One woman with bilateral breast cancer (diagnosed at ages 32 and 50) did not carry the variant. Both tumours were heterozygous for the S384F variant, so loss of the wild-type allele could be excluded. Ser384 is not located in a region of functional importance and cross-species sequence comparison revealed incomplete conservation in the human, dog, rodent and chicken BRCA2 homologues. Overall, the variant was detected in 116 patients, five of which co-occurred with different deleterious mutations. The combined likelihood ratio of co-occurrence, co-segregation and loss of heterozygosity revealed a value of 1.4 x 10-8 in favour of neutrality of the variant. CONCLUSION: Our data provide conclusive evidence that the S384F variant is not a disease causing mutation.

PTEN mutations do not cause nuclear beta-catenin accumulation in endometrial carcinomas.

PTEN: and beta-catenin mutations constitute the predominant genetic alterations in endometrioid carcinomas of the endometrium. PTEN encodes a dual-specificity phosphatase with lipid phosphatase and protein tyrosine phosphatase activities that regulate both apoptosis and interactions with the extracellular matrix. Recent studies have associated PTEN mutations with tumorigenesis of prostate carcinoma via the Wnt signaling pathway, leading to nuclear beta-catenin accumulation. To elucidate the potential interaction of PTEN and beta-catenin in endometrial cancer, we performed mutation analyses of the entire PTEN gene and of exon 3 of the beta-catenin gene that is most frequently targeted by mutations. A total of 82 endometrial carcinomas comprising 62 type I endometrioid carcinomas and 20 type II high-grade carcinomas were investigated. In addition in a subset of 22 carcinomas, the intracellular beta-catenin distribution was analyzed by immunohistochemistry. Overall, 20 (24.4%) of 82 tumors revealed mutations in the PTEN gene, and 16 (19.5%) of 82, in the beta-catenin gene. Six tumors (7.3%) showed mutations in both the PTEN and beta-catenin gene. Mutations were mainly detected in endometrioid carcinomas of the endometrium. As expected, a striking nuclear accumulation of beta-catenin could be shown in tumors with beta-catenin mutations. In the vast majority of tumors with PTEN mutations, a regular staining pattern of the cytoplasmic and membranous compartments was found. We therefore conclude that, in contrast to prostate cancer, mutations in the PTEN gene seem not to affect cellular distribution of the beta-catenin protein in endometrial carcinomas.

Interaction of cisplatin, paclitaxel and adriamycin with the tumor suppressor PTEN.

Due to its pivotal role in signal transduction, the universal tumor suppressor PTEN (also termed MMAC or TEP) is one of the putative candidates for involvement in tumorigenesis of several tissues. Although involvement of PTEN in tumorigenesis was shown in different tissues, no data are available concerning PTEN activity in response to antineoplastic agents. Therefore, we assayed the PTEN activity exposed to either blank medium or the commonly used anti-cancer drugs cisplatin, adriamycin or paclitaxel, respectively, in three different concentrations. PTEN activity was determined using the Malachite Green assay basing upon dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by the PTEN enzyme and subsequent determination of inorganic phosphate released. Although the three different anti-cancer drugs assayed act with different cellular modes, the antineoplastics influenced PTEN activity in a similar manner: at low concentrations tested all three antineoplastics significantly increased PTEN activity. However, increasing drug concentrations exhibited a decline but not a total loss of PTEN activity. The data indicate that PTEN activity is increased following cytotoxic drug exposure and, thereby, exhibits its suppressive function. However, the decrease of PTEN activity in response to increasing drug concentrations suggests an aberration of total functional activity. As far as the regulative checkpoint PTEN is abolished, tumor cells might evade cell death pathways resulting in increased proliferation of cancer cells. This might be a general event in refractory tumor cells surviving chemotherapy.

Exclusion of a major role for the PTEN tumour-suppressor gene in breast carcinomas.

PTEN is a novel tumour-suppressor gene located on chromosomal band 10q23.3. This region displays frequent loss of heterozygosity (LOH) in a variety of human neoplasms including breast carcinomas. The detection of PTEN mutations in Cowden disease and in breast carcinoma cell lines suggests that PTEN may be involved in mammary carcinogenesis. We here report a mutational analysis of tumour specimens from 103 primary breast carcinomas and constitutive DNA from 25 breast cancer families. The entire coding region of PTEN was screened by single-strand conformation polymorphism (SSCP) analysis and direct sequencing using intron-based primers. No germline mutations could be identified in the breast cancer families and only one sporadic carcinoma carried a PTEN mutation at one allele. In addition, all sporadic tumours were analysed for homozygous deletions by differential polymerase chain reaction (PCR) and for allelic loss using the microsatellite markers D10S215, D10S564 and D10S573. No homozygous deletions were detected and only 10 out of 94 informative tumours showed allelic loss in the PTEN region. These results suggest that PTEN does not play a major role in breast cancer formation.

Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites.

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.

Large linkage analysis in 100 families with autosomal recessive spinal muscular atrophy (SMA) and 11 CEPH families using 15 polymorphic loci in the region 5q11.2-q13.3.

The autosomal recessive proximal spinal muscular atrophy (SMA) gene was mapped to the region 5q11.2-q13.3 in 1990. Here, we present a large genetic linkage study of 100 SMA families and 11 CEPH families using 14 polymorphic simple sequence repeats (SSRs) and one RFLP in the region 5q11.2-q13.3. The genetic interval between the closest SMA flanking loci D5S435 and D5S557 comprises 1 cM at zmax = 27.94. Two recombinants were identified between the SMA gene and the closest telomeric marker D5S557 (theta = 0.02 at zmax = 8.63). The first places the SMA gene centromeric to this marker; the second suggests a double recombinant at D5S557, which is very unlikely. More likely explanations are discussed in the paper. No recombinant was found between D5S435 and the SMA gene (theta = 0.00 at zmax = 25.36). We localized a recently described polymorphic marker, D5S351 (Hudson et al., 1992), close to the SMA (theta = 0.00 at zmax = 19.01) and the 3'MAP1B gene (theta = 0.01 at zmax = 38.76). Due to its high PIC value of 0.70, it represents a very useful marker for prenatal diagnosis. In addition, we developed a new reverse primer for the nearest centromeric locus D5S435 (Soares et al., 1993), a useful marker for prenatal diagnosis, which has been very difficult to amplify in the past. Three of the markers presented here are newly developed polymorphic SSRs (one tetranucleotide repeat, D5S507/W15CATT, and two dinucleotide repeats, D5S544/C88.2GT and D5S682/C88.3GT). These markers are too far from the SMA gene to be relevant for cloning; nevertheless, as part of the human genome project, they are contributing to the fine genetic mapping of the region 5q11.2-q13.3. The most likely order of the loci based on two-point and multipoint linkage analyses as well as on specific recombination events and physical mapping studies is D5S76-D5S507- D5S6-D5S125-D5S680-D5S435-SMA-D5S557- D5S351-5'MAP1B-3'MAP1B-JK53CA1/2-(D5S127- D5S39)-(D5S544-D5S682). In general, the genetic distances obtained from the SMA and CEPH families are comparable.

Efficiency of various strategies and materials to generate new markers: saturating the region 5q11.2-q13.3 with 30 new randomly distributed clones.

Several different strategies and materials were used for saturating the region 5q11.2-q13.3 with new, randomly distributed markers: isolation of human clones from three chromosome-5-specific libraries (a BssHII endclone phage library from the somatic cell hybrid H64 and two total genomic phage libraries from radiation hybrids IH12 and IH132), as well as Alu-PCR from chromosome-5-specific radiation hybrids with overlapping fragments in the region around the spinal muscular atrophy locus, followed either by direct isolation of Alu-PCR products or hybridization of Alu-PCR products to chromosome-5-gridded cosmid libraries. 253 human phage and cosmid clones were mapped to various parts of chromosome 5 by deletion mapping to somatic cell hybrid panels. 30 of these clones were mapped into the region 5q11.2-q13.3, 9 of which are flanking rate cutting BssHII-sites, known to be, often, starting points for genes. They represent excellent starting material for the development of new polymorphic markers and sequence-tagged sites, for YAC screening and building of contigs, as well as for direct isolation of genes.