Marco Montagna

Color bar coding the BRCA1 gene on combed DNA: A useful strategy for detecting large gene rearrangements

Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast and ovarian cancers. BRCA1 germline mutations, however, are found less frequently than expected. Mutation detection strategies, which are generally based on the polymerase chain reaction, therefore focus on point and small gene alterations. These approaches do not allow for the detection of large gene rearrangements, which also can be involved in BRCA1 alterations. Indeed, a few of them, spread over the entire BRCA1 gene, have been detected recently by Southern blotting or transcript analysis. We have developed an alternative strategy allowing a panoramic view of the BRCA1 gene, based on dynamic molecular combing and the design of a full four‐color bar code of the BRCA1 region. The strategy was tested with the study of four large BRCA1 rearrangements previously reported. In addition, when screening a series of 10 breast and ovarian cancer families negatively tested for point mutation in BRCA1/2, we found an unreported 17‐kb BRCA1 duplication encompassing exons 3 to 8. The detection of rearrangements as small as 2 to 6 kb with respect to the normal size of the studied fragment is achieved when the BRCA1 region is divided into 10 fragments. In addition, as the BRCA1 bar code is a morphologic approach, the direct observation of complex and likely underreported rearrangements, such as inversions and insertions, becomes possible.

Lack of association between androgen receptor CAG polymorphism and familial breast/ovarian cancer.

The human androgen receptor (AR) gene contains a highly polymorphic CAG repeat in exon 1 that is inversely correlated with AR transcriptional activity in vitro. Several studies have shown that fewer CAG repeats are associated with an increased risk as well as more aggressive forms of prostate cancer. More recently, AR allele length was also inversely correlated with the histological grade of breast cancer, but no association was found between the AR-CAG polymorphism and the risk of either breast or ovary cancer. On the contrary, it was proposed that a longer CAG repeat sequence might be associated with an increased risk of breast cancer in BRCA1 mutation carriers, thus suggesting a different role of the AR-CAG polymorphism in sporadic and inherited breast cancers. With the intent of better understanding the role of the AR-CAG polymorphism as a cancer risk modifier, we defined the AR genotype of 151 patients (101 with breast and 50 with ovary cancer) belonging to high-risk breast/ovary cancer families. No difference in CAG repeat length was found between either breast and ovary cancer patients or age at diagnosis of both tumors. These results were also confirmed in a sub-group of 47 breast cancer cases, that either carried a BRCA gene mutation (11 cases) or were identified by very stringent operational criteria as hereditary breast cancers. Even though a substantially larger sample size would be required to reach conclusive evidence, our findings suggest that the AR-CAG polymorphism does not act as a modifier of tumor onset or tumor phenotype in breast/ovarian cancer families.

Epstein-Barr virus-associated post-transplant lympho-proliferative disease of donor origin in liver transplant recipients

BACKGROUND/AIMS Post-transplant lymphoproliferative disease, a potential complication of solid organ transplantation, occurs in about 3% of orthotopic liver transplant recipients. We report the genetic and virological characterization of two cases of post-transplant lymphoproliferative disease that occurred early (4 and 6 months) after orthotopic liver transplant as large-cell non-Hodgkins lymphomas located at the hepatic hilum. METHODS Lymphomatous tissues were analyzed for clonality and presence of Epstein-Barr virus (EBV) sequences by Southern blot, polymerase chain reaction, and in situ hybridization techniques. RESULTS The tumors in both cases were sustained by a clonal proliferation of B lymphocytes containing type A EBV DNA. Moreover, in situ hybridization with a digoxigenin-labeled EBV-specific probe evidenced a strong nuclear signal in most of the neoplastic cells. DNA microsatellite analysis at three different loci detected alleles of donor origin in both tumor samples, suggesting that the neoplastic B cells were of donor origin. CONCLUSIONS EBV-infected donor B lymphocytes might be responsible for intragraft post-transplant lymphoproliferative disease in orthotopic liver transplant recipients. As 20 to 30% of post-transplant lymphomas involve the graft itself, donor-derived post-transplant lymphoproliferative disease might be more frequent than presently appreciated. Prospective studies are needed to assess its real incidence and identify possible risk factors.

Anomalous Transcripts and Allelic Deletions of the FHIT Gene in Human Esophageal Cancer

The fragile histidine triad (FHIT) gene is localized on chromosome 3p14 and spans the common fragile site FRA3B. Even though its role in carcinogenesis is still unclear, this gene is frequently inactivated by carcinogen-induced intragenic deletions in many types of cancers, and FHIT abnormal transcripts are found in many primary tumors and tumor-derived cell lines. We evaluated FHIT gene involvement in 39 esophageal carcinomas (18 adenocarcinomas [AC¿, 21 squamous cell carcinomas [SCC]) by both reverse transcriptase-polymerase chain reaction (RT-PCR) amplification and loss of heterozygosity analysis (LOH). Thirty cases (77%) displayed either aberrant FHIT transcripts (12 cases) and/or LOH (24 cases); among these, only 6 samples displayed both aberrant transcripts and LOH, thus suggesting that the two events are probably independent. Moreover, LOH was significantly higher in SCC (80%) than in AC (44%), and because most of our patients are heavy smokers and/or alcohol consumers, these results suggest that the FHIT gene might be a common target for carcinogens also in the esophagus.

Performance of BOADICEA and BRCAPRO genetic models and of empirical criteria based on cancer family history for predicting BRCA mutation carrier probabilities: A retrospective study in a sample of Italian cancer genetics clinics

PURPOSE To evaluate in current practice the performance of BOADICEA and BRCAPRO risk models and empirical criteria based on cancer family history for the selection of individuals for BRCA genetic testing. PATIENTS AND METHODS The probability of BRCA mutation according to the three tools was retrospectively estimated in 918 index cases consecutively undergone BRCA testing at 15 Italian cancer genetics clinics between 2006 and 2008. RESULTS 179 of 918 cases (19.5%) carried BRCA mutations. With the strict use of the criteria based on cancer family history 173 BRCA (21.9%) mutations would have been detected in 789 individuals. At the commonly used 10% threshold of BRCA mutation carrier probability, the genetic models showed a similar performance [PPV (38% and 37%), sensitivity (76% and 77%) and specificity (70% and 69%)]. Their strict use would have avoided around 60% of the tests but would have missed approximately 1 every 4 carriers. CONCLUSION Our data highlight the complexity of BRCA testing referral in routine practice and question the strict use of genetic models for BRCA risk assessment.

Identification of BRCA1 and BRCA2 carriers by allele‐specific gene expression (AGE) analysis

Mutations in BRCA1 and BRCA2 genes confer a high risk of breast and ovarian cancer. As such, their identification is essential to reduce the risk of disease in healthy carriers, as well as in carriers who have already developed the disease because they are at increased risk for a second malignancy; moreover, noncarriers of BRCA1 and BRCA2 mutated families can be spared anxiety and unnecessary medical interventions. A number of problems, including large gene size, complex mutational spectra and genetic heterogeneity of the disease, however, make genetic testing labor intensive and often inconclusive. We devised a new mutation detection strategy called AGE (allele‐specific gene expression) analysis that relies on the detection of a “functional effect” of the mutation at the RNA level known as “nonsense‐mediated RNA decay,” thus avoiding several of the problems of BRCA1 and BRCA2 genetic testing. In particular, (i) AGE analysis discriminates among the predisposing genes and identifies mutation carriers with a single RT‐PCR reaction; (ii) it relies on the effect of truncating mutations, which represent the large majority of cases and thus identifies mutation carriers regardless of the specific genomic alteration; and (iii) it is specific for cis‐regulatory mutations that are missed at present by most of the methods. As AGE analysis has the potential to identify most of the BRCA1 and BRCA2 mutation carriers, it can be used as a preliminary screening method, thereby accelerating and increasing the sensitivity of the genetic testing process. Notably, other hereditary diseases whose genetic analysis is hampered by similar problems could benefit from this kind of approach.

Co-existence of cutaneous T-cell lymphoma and B hairy cell leukemia.

A primary cutaneous form of peripheral T‐cell lymphoma (PTCL) and a low grade B‐cell non‐Hodgkins lymphoma that was classified as a variant of hairy cell leukemia (HCL) were simultaneously diagnosed in a 79‐year‐old woman by both phenotypic and genotypic analyses. The coexistence of a T‐ and B‐cell lymphoma in the same patient is rare, and, to our knowledge, this particular association has not been previously described. The patient was referred to our Department for evaluation of multiple cutaneous itchy, reddish plaques; laboratory analyses disclosed a lymphocytosis, that presented 6 years earlier. A bone marrow aspirate showed a 50% B‐cell interstitial infiltrate, while a skin biopsy surprisingly revealed a PTCL. Clonality of both neoplastic processes was assessed by Southern blot analysis. The indolent clinical course of the cutaneous disease, and the low and stable number of circulating neoplastic T cells supported the diagnosis of a mycosis fungoides (MF)‐like PTCL. Possible oncogenic events and/or putative underlying viral infections which could have played a role in the occurrence of B‐ and T‐cell non‐Hodgkins lymphomas in the same patient are discussed. Am. J. Hematol. 64:197–202, 2000.

Dominance of a single Epstein-Barr virus strain in SCID-mouse tumors induced by injection of peripheral blood mononuclear cells from healthy human donors

Severe Combined Immune Deficiency mouse tumors, induced by inoculating peripheral blood mononuclear cells from 11 healthy human donors (hu-PBMC-SCID tumors), were used to analyse Epstein-Barr virus (EBV) type and strain variations. PCR analysis of EBNA 2- and EBNA 3C-specific sequences showed that EBV type A was present in SCID-mouse tumors induced by PBMC from all donors but one, while, using amplimers for a highly polymorphic region within the latent membrane protein (LMP) coding sequence, 5 different strains could be detected among the samples examined. The same LMP fragment was present in different tumors arising in the same animal, as well as in different mice injected with PBMC from any donor. Compared to B95.8 and AG876 prototype viruses, sequence analysis of LMP variants disclosed a higher homology to the latter, with 33 bp additional repetitions and a few point mutations in specific sites. This study confirms and extends previous data on the presence of a single EBV type and strain in the peripheral blood of most normal healthy subjects using the SCID-mouse system.