Increased incidence of pathogenic variants in ATM in the context of testing for breast and ovarian cancer predisposition.

Pathogenic Variants (PV) in major cancer predisposition genes are only identified in approximately 10% of patients with Hereditary Breast and Ovarian Cancer (HBOC) syndrome. Next Generation Sequencing (NGS) leads to the characterization of incidental variants in genes other than those known to be associated with HBOC syndrome. The aim of this study was to determine if such incidental PV were specific to a phenotype. The detection rates of HBOC-associated and incidental PV in 1812 patients who underwent genetic testing were compared with rates in control groups FLOSSIES and ExAC. The rates of incidental PV in the PALB2, ATM and CHEK2 genes were significantly increased in the HBOC group compared to controls with, respective odds ratios of 15.2 (95% CI = 5.6-47.6), 9.6 (95% CI = 4.8-19.6) and 2.7 (95% CI = 1.3-5.5). Unsupervised Hierarchical Clustering on Principle Components characterized 3 clusters: by HBOC (P = 0.01); by ExAC and FLOSSIES (P = 0.01 and 0.02 respectively); and by HBOC, ExAC and FLOSSIES (P = 0.01, 0.04 and 0.04 respectively). Interestingly, PALB2 and ATM were grouped in the same statistical cluster defined by the HBOC group, whereas CHEK2 was in a different cluster. We identified co-occurrences of PV in ATM and BRCA genes and confirmed the Manchester Scoring System as a reliable PV predictor tool for BRCA genes but not for ATM or PALB2. This study demonstrates that ATM PV, and to a lesser extent CHEK2 PV, are associated with HBOC syndrome. The co-occurrence of ATM PV with BRCA PV suggests that such ATM variants are not sufficient alone to induce cancer, supporting a multigenism hypothesis.

Localization of the gene for Cowden disease to chromosome 10q22-23.

Cowden disease (CD) (MIM 158350), or multiple hamartoma syndrome, is a rare autosomal dominant familial cancer syndrome with a high risk of breast cancer. Its clinical features include a wide array of abnormalities but the main characteristics are hamartomas of the skin, breast, thyroid, oral mucosa and intestinal epithelium. The pathognomonic hamartomatous features of CD include multiple smooth facial papules, acral keratosis and multiple oral papillomas. The pathological hallmark of the facial papules are multiple trichilemmomas. Expression of the disease is variable and penetrance of the dermatological lesions is assumed to be virtually complete by the age of twenty. Central nervous system manifestations of CD were emphasized only recently and include megalencephaly, epilepsy and dysplastic gangliocytomas of the cerebellum (Lhermitte-Duclos disease, LDD). Early diagnosis is important since female patients with CD are at risk of developing breast cancer. Other lesions include benign and malignant disease of the thyroid, intestinal polyps and genitourinary abnormalities. To localize the gene for CD, an autosomal genome scan was performed. A total of 12 families were examined, resulting in a maximum lod score of 8.92 at theta = 0.02 with the marker D10S573 located on chromosome 10q22-23.

Contribution of PTEN large rearrangements in Cowden disease: a multiplex amplifiable probe hybridisation (MAPH) screening approach.

BACKGROUND: Cowden disease is an autosomal dominant syndrome predisposing to cancer and characterised by the occurrence throughout life of hyperplastic, hamartomatous and tumoural lesions affecting various organs. In 60-80% of patients a germline intragenic point mutation of the PTEN tumour suppressor gene is identified, but at least 20% of patients with a well characterised phenotype remain without any identified mutation. METHODS: To evaluate the impact of large rearrangement involving the PTEN locus in Cowden disease, we analysed by a multiplex amplifiable probe hybridisation (MAPH) technique 80 unrelated patients referred for diagnosed or suspected Cowden disease, and in whom no PTEN point mutation was detected by a denaturing gradient gel electrophoresis (DGGE) screening. RESULTS: Four heterozygous genomic deletions involving the PTEN gene were identified. These deletions ranged from 13.6-662 kb and are restricted to the PTEN locus in two cases. In the two other cases, the deletion encompassed PTEN and either two or three contiguous genes without any obvious phenotypic effect, except a possible consequence of PAPSS2 haploinsufficiency on bone growth. Sequence analysis of the four deleted alleles did not reveal identity or sequence homology at the two breakpoints of a same allele, suggesting that a mechanism such as non-homologous recombination of the breakage and reunion type could lead to the occurrence of these deletions. CONCLUSION: Large rearrangements of the PTEN gene can be involved as causing mutation in Cowden disease and MAPH is an efficient screening methodology to detect such a genetic alteration.

Differential loss of heterozygosity in the region of the Cowden locus within 10q22-23 in follicular thyroid adenomas and carcinomas.

The susceptibility gene for Cowden disease (CD), an autosomal dominant inherited cancer syndrome, has recently been mapped to an approximately 6-cM interval on chromosome subband 10q22-23 between the markers D10S541 and D10S564. CD is characterized by hamartomas of many organ systems, including the thyroid, breast, skin, and gastrointestinal tract, as well as carcinoma of the thyroid and breast. Follicular thyroid adenomas and carcinomas are significant component tumors in CD; thus, we sought to examine their sporadic counterpart tumors for loss of heterozygosity (LOH) of microsatellite markers in the 20-cM region within and flanking the Cowden critical interval. In all, 38 sporadic thyroid tumors were analyzed. LOH within the CD interval was observed in 5 of 19 (26%) follicular thyroid adenomas and 1 of 9 (11%) Hürthle cell adenomas. Furthermore, of these adenomas with LOH, 3 of 4 (75%) were atypical follicular adenomas, whereas 2 of 15 (13%) were typical follicular adenomas. Surprisingly, no LOH was detected in this region in 10 follicular carcinomas. The shortest region of overlap includes the markers D10S1735 and D10S1739. If the LOH observed in these sporadic tumors is related to the CD gene, then the Cowden critical interval can be revised to lie within the interval defined by D10S579 and D10S564. LOH in this narrow interval implicates the CD gene, or another gene in that interval, in follicular thyroid tumorigenesis. However, this does not explain the lack of LOH in follicular carcinomas. Taken together, it may instead be evidence against a stepwise progression from atypical adenomas to carcinomas. Alternatively, sporadic thyroid adenoma formation may be independent of that locus, but loss of this region could prevent carcinoma formation, thus implying that the CD gene may be an oncogene or growth promoter.

Germ line mutation at BRCA1 affects the histoprognostic grade in hereditary breast cancer.

Histoprognostic grade is a determinant parameter to select the initial therapeutic strategy in breast cancer. Our aim was to analyze the grade repartition in BRCA1-associated breast cancer (BC) and to explore the possible connections between grade and the BRCA1 gene function. We first compared 27 BRCA1-associated BCs from 14 families with 4,461 cases from an administrative district registry and 242 cases from a hospital-based registry, matching for grade and constitutive elements, and then considered their repartition in families. We observed a prevalence of grade 3 (P < 0.0001) in BRCA1-associated BC. This was attributed mainly to nuclear polymorphism (P < 0.0001), mitotic activity (P < 0.0001), and tubular differentiation (P = 0.0004), implying that BRCA1-associated BCs are highly proliferating tumors. Moreover, it is suggested that grade segregates as a genetic trait within families (P = 0.0015), and this was attributed to the mitotic index segregation only (P = 0.0005). Therefore grade, through its components, could be interpreted as the morphological translation of the BRCA1 germ line mutation. Thus, a genotype-phenotype correlation may exist between the type of mutation and the aggressiveness of the disease. These findings are bound to have an important impact in the care management of hereditary breast cancer at the individual and at the familial level and in the comprehensive approach of breast cancer development.

Genome-wide search for loss of heterozygosity shows extensive genetic diversity of human breast carcinomas.

Deletions of genomic regions involving tumor suppressor genes are thought to be important in the initiation and progression of breast cancer. We conducted a genome-wide search for deleted regions in a series of 75 human breast carcinomas by studying the allelic patterns of 184 microsatellite markers distributed over all chromosomes and looking for loss of heterozygosity (LOH). We identified 56 regions of consistent LOH. Strikingly, every tumor had a different set of deletions. To study this complexity, we applied a phylogenetic-like type of analysis. Each region was involved in a certain proportion of tumors, ranging from 20 to 62%; the most frequently involved regions were on chromosome arms 8p, 11q, 16q, and 17p. There was a correlation (P = 0.005) between the level of LOH and the size of the tumors. Tumors with a high level of LOH were also highly proliferative and had a high mitotic index.

Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors.

The majority of familial medullary thyroid neoplasms are associated with germ-line mutations of the RET proto-oncogene, yet very little is known about the mechanisms involved in the pathogenesis of familial and sporadic nonmedullary thyroid tumors. A subset of thyroid tumors have loss of heterozygosity of chromosome 10q22-23, a region harboring the gene responsible for Cowden disease, an autosomal dominant hamartoma syndrome associated with thyroid and breast tumors. PTEN/MMAC1/TEP1 codes for a dual-specificity phosphatase and is likely a tumor suppressor gene. We sought to determine the PTEN status in a series of epithelial thyroid neoplasms. We studied 95 sporadic thyroid tumors, of which 39 were papillary thyroid carcinomas (PTCs), 12 were follicular carcinomas, 9 were anaplastic carcinomas, 5 were Hürthle cell carcinomas, 21 were nonfunctioning follicular adenomas, and 9 were Hürthle cell adenomas. Direct sequencing of PCR-amplified products was performed for all nine exons of PTEN. Two polymorphic markers, one located in intron 8 and another, a dinucleotide repeat marker, AFMa086wg9, located within intron 2, were analyzed in paired blood-tumor DNA samples to assess hemizygous deletions of PTEN. We found a somatic frameshift mutation in one PTC, which was expected to generate a premature stop codon 2 amino acids downstream. Twenty-six % of informative benign tumors (four follicular adenomas and three Hürthle cell adenomas) and only 3 of 49 (6.1%) informative malignant tumors (one PTC, one follicular carcinoma, and one anaplastic carcinoma) showed evidence of hemizygous deletion of PTEN (P = 0.046). We conclude that a subset of thyroid tumors have somatic deletions of the PTEN gene, predominantly the benign forms, and that small intragenic mutations of PTEN are infrequent in thyroid tumors. We speculate that other mechanisms of PTEN inactivation, rather than small intragenic mutations, might occur in the hemizygously deleted samples and act as the "Knudson second hit." Alternatively, other tumor suppressor genes mapping to chromosome 10q22-23 could be the actual targets for such deletions and thus represent the various hits in the pathway of multistep carcinogenesis.

Mutations at BRCA1: the medullary breast carcinoma revisited.

BRCA1-associated breast cancers (BRCA1-BCs) frequently harbor a high histoprognostic grade, p53 alterations, and estrogen receptor negativity. Although these parameters predict a poor outlook, the overall survival in BRCA1-BCs is equivalent to or even better than that in sporadic cases. These features are reminiscent of what is observed for breast carcinoma of the medullary type, a high-grade tumor with a particular favorable course. To explore a possible relationship between this phenotype and BRCA1 mutations, we first compared 32 BRCA1-BCs and 200 consecutive cases of breast cancer without familial history for the prevalence of typical medullary breast carcinoma (TMC) using the criteria given by Ridolfi et al. [R. Ridolfi et al, Cancer (Phila.), 40: 1365-1385, 1977]. Second, we searched for BRCA1 mutations in a set of 18 cases of TMC, using denaturing gradient gel electrophoresis and Cleavase fragment length polymorphism scanning. Six of 32 (19%) BRCA1-BCs were of the TMC type, compared to 0 of 200 controls (P < 0.0001). Among the 18 TMCs, 2 BRCA1 nonsense mutations were found. This corresponds to almost 7 times the contribution of BRCA1 mutations in the general population. Two additional missense mutations were identified. Together, these results suggest that, although TMC and BRCA1-BCs are not strictly coincidental, an important connection between the two populations does exist.

PTEN/MMAC1/TEP1 involvement in primary prostate cancers.

The PTEN/MMAC1/TEP1 gene, located at 10q23.3, is a tumor suppressor gene responsible for the familial cancer syndromes Cowden disease and Bannayan-Zonana syndrome, and is commonly somatically mutated in several types of cancers. Mutations of the PTEN gene have been found in prostate cancer cell lines and LOH at 10q22-24 in prostate tumors have also been described with a high frequency. To determine the role of this gene in prostate tumorigenesis, we therefore analysed 22 primary tumors for loss of heterozygosity (LOH) within the 10q22-23 region such that tumors hemizygous at those loci may be examined for somatic PTEN mutations. Losses of heterozygosity of at least one locus was found in 12 (55%) of the 22 tumors DNAs. Among these, six tumors exhibited allele loss in the interval between D10S1765 and D10S541 wherein lies the PTEN gene. We searched the entire coding region of PTEN for somatic mutations in these six tumors. One somatic mutation (17%), a 1 bp deletion, was detected in exon 7 of the gene, in one tumor, indicating that somatic mutations of the PTEN gene may occur in primary prostate tumors.

WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type.

Secreted Frizzled-related protein 1 (SFRP1) encodes a member of a protein family that contains a cysteine-rich domain similar to the WNT-binding site of Frizzled receptors and regulates the WNT pathway. The WNT pathway is frequently altered in human cancers. We have defined the pattern of SFRP1 mRNA expression in the progression of breast cancer. We show that SFRP1 is expressed in the epithelial component of normal breast, in the in situ component of ductal carcinomas and is lost in more than 80% of invasive breast carcinomas except the medullary type. Loss of SFRP1 expression is correlated with the presence of hormonal receptors. Conversely, the maintenance of SFRP1 in carcinomas is correlated with the presence of lymphoplasmocytic stroma. No significant association was observed between SFRP1 status and the level of apoptosis in tumoral cells.

Loss of heterozygosity in human breast carcinomas in the ataxia telangiectasia, Cowden disease and BRCA1 gene regions.

To appreciate the involvement of known or potential susceptibility genes in sporadic breast tumors, we have searched for chromosomal deletions by studying loss of heterozygosity (LOH) at 43 microsatellite (CA)n markers from human chromosomes 10, 11 and 17, in 115 unselected consecutive samples of breast carcinoma with particular emphasis on specific regions. No site of consistent LOH was identified on chromosome 10. Five regions of LOH were contained within bands q22-24 of chromosome 11 for which nearly 50% of the tumors had LOH at at least one marker. This region is thus a major site of deletion in breast cancer and several tumor suppressor genes seem to be involved. One of them may be the ataxia telangiectasia (ATM) gene which is located in one of the affected regions. Five regions of LOH, one of which is within the BRCA1 gene area, were recognized along chromosome 17. LOH at three of these regions were found in highly proliferative tumors. When combined with a previous study of chromosome 13 with emphasis on BRCA2 and Rb1 genes, this work allowed to distinguish a total of 12 regions of LOH, variably affected in breast tumors and correlated with prognostic parameters.

Loss of heterozygosity and linkage analysis in breast carcinoma: indication for a putative third susceptibility gene on the short arm of chromosome 8.

We have analysed losses of heterozygosity (LOH) at eight markers from the p12-p22 region of human chromosome 8 in a panel of 113 breast tumors. LOH were detected in almost half of the tumors. The most frequently deleted region included microsatellite (CA)n repeats markers D8S258, D8S133 and D8S259, located at 8p12-p22, while markers NEFL and LPL appeared less frequently altered. In parallel, linkage analysis was performed using the same informative markers, to test for the involvement of chromosome 8p loci in familial breast cancer. Positive cumulative multipoint lod score of 2.51 at theta = 0.0 was obtained with markers NEFL and D8S259. These results suggest that region 8p12-p22 carries at least one tumor suppressor gene involved in sporadic and perhaps also in familial breast cancer.

Clinical presentation of PTEN mutations in childhood in the absence of family history of Cowden syndrome.

BACKGROUND: PTEN gene (MIM 601628) is a tumor suppressor gene implicated in PTEN hamartoma tumor syndromes (PHTS) including Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, and Proteus-like syndrome. Bannayan-Riley-Ruvalcaba syndrome is considered as the pediatric form of PHTS. More recently, children presenting autism spectrum disorders with macrocephaly (ASD-M) have been reported. METHODS: We report clinical data from seven patients diagnosed in childhood with a PTEN germline mutation, excluding cases of familial Cowden syndrome. RESULTS: This study underlines the variability of phenotype associated with PTEN mutations diagnosed at pediatric age. Most of the patients did not fulfill usual criteria of Bannayan-Riley-Ruvalcaba syndrome or ASD-M. CONCLUSION: PTEN testing should be considered in any child presenting with severe macrocephaly (>+4SD) and another feature of PHTS.

Acute promyelocytic leukemia with (15;17) translocation and chromosome no. 11 deletion (q23).

The patient, a 76-year-old man, was referred with fever, large ecchymotic lesions and ulcerative laryngitis. Blood counts showed a hemoglobin of 11 g/100 ml, hematocrit of 31%, red blood cell count of 3.5 X 10(12)/1, white blood cell count of 6.8 X 10(9)/1 and platelet count of 16.0 X 10(9)/1. The differential count showed 17% neutrophils, 4% lymphocytes, 40% promyelocytes and 39% myeloblasts. The sternal marrow sample showed a marked hypercellularity. Of the cells, 80-85% were hypergranular promyelocytes, some of them showing bundles of Auer rods. No granulocytic maturation was observed. A few erythroblasts were present. A disseminated intravascular coagulation was observed (fibrinogen 0.85 g/l, factor V 18%, fibrin degradation products 640 mg/l). The serum creatinin was at 217 micromol/1 and the urea at 16.8 mmol/1. The treatment (daunorubicin, heparin, platelet transfusion) was unsuccessful and the patient died three days after entering hospital. The bone marrow karyotype by direct examination showed only normal metaphases (32 photographed). All the metaphases from the unstimulated blood 48-h culture (25 photographed) were clonal, showing the pattern 47,XY,del(11) (q23),t(15;17) (q24? q22?), +mar. The marker was '16 like' in size but its origin could not be determined (Figs. 1 and 2).

Marker segregation information in breast/ovarian cancer genetic counseling: is it still useful? Groupe Génétique et Cancer de la Fédération Nationale des Centres de Lutte Contre le Cancer.

The use of mutation screening of BRCA1 and BRCA2 genes as a genetic test is still to a certain extent limited and the oncogeneticist may want to use complementary approaches to identify at-risk individuals. In a series of 23 families with at least three breast or ovarian cancer cases, screened for mutations at BRCA1 and BRCA2 and typed for markers at both loci, we investigated the usefulness of marker segregation information at two levels: 1) to what extent can the indirect approach identify the mutation carrier status of screened cases and their first-degree relatives, and 2) in what way does it help to identify the gene implicated in a family in which neither BRCA1 nor BRCA2 mutation has been detected? Using the indirect approach, the carrier status of the screened case could be determined with quasi certainty in three families and with a high probability in eight families. This status could be inferred in unaffected first-degree relatives as almost certain in one family and as highly probable in six families. Fourteen mutations were found concurrently in our series. Among the nine mutation-negative families, we were able to conclude that a BRCA1 mutation most probably segregated in one and that a mutation other than BRCA1 and BRCA2 was probably involved in two families. Our results show that, in small families, little help is to be expected from linkage data and mutation screening is the only way of identifying the origin of a genetic predisposition in a family. Marker segregation information may be useful in some large breast/ovarian cancer families in which no BRCA1 or BRCA2 mutation has been detected.

Method for the purification of tissue DNA suitable for PCR after fixation with Bouin's fluid. Uses and limitations in microsatellite typing.

Paraffin-embedded tissues are often the only available material to perform polymerase chain reaction (PCR)-based analysis in various medical purposes. Unfortunately, the use in many countries of acid fixatives such as Bouin's fluid limits the use of such a material for molecular analysis. This article reports the methodological details of a DNA purification technique from Bouin-fixed and paraffin-embedded samples based on a double washing, in an alcohol then in an aqueous medium, of the DNA, which enables PCR reactions from this material. Comparison of the results with those obtained by organic solvent purification of DNA from frozen tissue fragments showed excellent reproducibility in terms of detection of an amplification product on agarose gel. However, differences between the methods were quite frequently seen in the allelic typing profile of microsatellite sequences (CA repeats), either as neo-alleles or by the loss of normal alleles in the fixed materials that constitute a limitation in using DNA from Bouin-fixed tissue as a substrate for fine allelotyping.

Polymerase chain reaction diagnosis of t(14;18) from paraffin-embedded tissues fixed with Holland Bouin fluid.

The t(14;18) translocation and its molecular counterpart, the bcl-2/IgH gene rearrangement, are highly characteristic of follicular non-Hodgkin lymphomas. The identification of the tumor-specific t(14;18) clone is mandatory for any molecular studies on residual disease because of the existence of circulating t(14;18)-bearing benign cells. In this study, the ability to specifically polymerase chain reaction (PCR) amplify t(14;18) with DNA purified from tissues fixed with Holland Bouin fluid is demonstrated. The specificity of the PCR product was confirmed by internal probe hybridization and with comparison of the nucleotidic sequences of this PCR product with those obtained from the corresponding frozen material. Although the sensitivity of the technique is 50% to 60%, paraffin-embedded tissues fixed with bouin fluid may be a good alternative to frozen tissues to detect t(14;18) in tumors.

Chromosomal analysis of colonic adenomatous polyps.

Chromosomal analysis of 25 colonic adenomatous polyps was performed by a direct method similar to that used in prenatal diagnosis of chromosomal aberration on chorionic villi. Fourteen lesions showed an abnormal karyotype. Two changes were recurrent: trisomy 7 (observed in eight cases) and trisomy 13 (observed in seven cases). No monosomy of the short arm of chromosome 17 was observed even at the level of two polyps with in situ carcinoma lesions.

Genetic alterations in colorectal cancer, comparative analysis of deletion events, and point mutations.

Although data on genetic alterations leading to the development of colorectal cancer are abundant, no specific genetic alteration, as has been demonstrated for certain rare tumors such as lymphoma, leukemia, or sarcoma, has been shown to be responsible for the development of colorectal carcinomas. The colorectal cancer phenotype undoubtedly originates from an accumulation of different genetic alterations. The nature of these alterations, their order of appearance, and their associations vary greatly from one tumor to another, suggesting that the concept of a unique model of carcinogenesis is not applicable to these tumors. We studied a panel of 40 colorectal tumors in an attempt to identify different carcinoma subsets distinguishable by the pattern of genetic alterations. We examined a series of genetic anomalies frequently implicated in the development of colorectal cancer, including genetic material loss, demonstrated by loss of heterozygosity on chromosome arms 1p, 17p, and 18q; mutations of proto-oncogene K-RAS codons 12, 13, and 61; and gene TP53 mutations, identified by studying the accumulation of the corresponding immunohistochemically detectable protein. Our findings showed an important correlation between the genetic material loss events and an independent distribution of point mutations, which favors the hypothesis of a specific type of genetic instability characterized by the recurrent loss of chromatin fragments implicated in a subset of colorectal cancers.