Carmen Rivas

Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype.

Purpose: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

Loss of heterozygosity analysis at the BRCA loci in tumor samples from patients with familial breast cancer

The BRCA1 and BRCA2 genes are responsible for a high proportion of familial breast cancer; germline mutations in these genes confer a lifetime risk of about 70% for developing breast cancer. Most of the described deleterious mutations are small deletions or insertions that originate a truncated protein; however, in many cases, they are amino acid changes whose significance is unknown. In these cases, there are some tests that can analyze the meaning of these variants, but most remain unclassified. The BRCA genes are tumor supressors and it is beleived that complete loss of the wild‐type allele is a common mechanism of inactivation in tumors from patients carrying a germline deleterious mutation in these genes; if this is true, loss of heterozygosity (LOH) analysis in the tumor sample could help to distinguish if a rare variant is either a deleterious mutation or a common polymorphism. In the present study, we performed LOH analysis at the BRCA loci in 47 tumors from patients who belonged to high‐risk breast cancer families and were carriers of any type of alteration in these genes. Our results suggest that (i) loss of the wild‐type allele is the most common mechanism of inactivation in tumors from patients who carry a deleterious mutation in any of the genes, (ii) this loss is not common when we analyze familial tumors not associated with mutations in BRCA and (iii) LOH can be used to clarify variants of unknown significance in the BRCA genes.

Immunohistochemical Expression of DNA Repair Proteins in Familial Breast Cancer Differentiate BRCA2-Associated Tumors

PURPOSEnMorphologic and immunohistochemical studies of familial breast cancers have identified specific characteristics associated with BRCA1 mutation-associated tumors when compared with BRCA2 and non-BRCA1/2 tumors, but have not identified differences between BRCA2 and non-BRCA1/2 tumors. Because BRCA1 and BRCA2 genes participate in the DNA repair pathway, we have performed an immunohistochemical study with markers related to this pathway to establish the profile of the three groups.nnnMATERIALS AND METHODSnWe have studied two tissue microarrays that include 103 familial and 104 sporadic breast tumors, with a panel of DNA repair markers including ATM, CHEK2, RAD51, RAD50, XRCC3, and proliferating cell nuclear antigen.nnnRESULTSnWe found more frequent expression of CHEK2 in BRCA1 and BRCA2 tumors than in non-BRCA1/2 and sporadic tumors. We found absence of nuclear expression and presence of cytoplasmic expression of RAD51 in BRCA2 tumors that differentiate them from other familial tumors. We validated these results with a new series of patient cases. The final study with 253 familial patient cases (74 BRCA1, 71 BRCA2, 108 non-BRCA1/2), and 288 sporadic patient cases, has allowed us to confirm our preliminary results. Because BRCA2 tumors present a specific immunohistochemical profile for RAD51 and CHEK2 markers that is different from non-BRCA1/2 tumors, we have built a multivariate model with these markers that distinguish both tumors with an estimated probability of at least 76%.nnnCONCLUSIONnOur results suggest that BRCA2 tumors demonstrate more cytoplasmic and less nuclear RAD51 staining, and increased CHEK2 staining. This pattern may distinguish BRCA2 from familial non-BRCA1/2 tumors.

Frequent inactivation of the p73 gene by abnormal methylation or LOH in non‐Hodgkin's lymphomas

p73 is a candidate tumor suppressor and imprinted gene that shares significant homology with the p53 gene. It is located on 1p36, a region frequently deleted in neuroblastoma and other tumors. To investigate the pattern of inactivation of this gene in human lymphomas, we studied 59 tumors to identify abnormal methylation in exon 1 and loss of heterozygosity (LOH) at this locus. p73 was methylated in 13/50 (26%) B cell lymphomas. There was no evidence of p73 methylation in the 9 T cell lymphomas analyzed. Burkitts lymphomas showed the highest proportion of methylated cases (36%), although this alteration also affected other aggressive lymphomas such as diffuse large cell and some marginal zone lymphomas. LOH at the p73 locus was detected in 4/34 (11%) B and 1/9 (11%) T cell lymphomas. The p73 expression analysis showed absence or low level of p73 product in methylated lymphomas, whereas p73 was always detected in unmethylated tumors. We found monoallelic expression in normal peripheral blood samples, consistent with imprinting. None of the tumors showed LOH and methylation of the remaining allele simultaneously, suggesting that alteration of the expressed allele could lead to the total inactivation of the gene. Our results show that deletion or methylation of the p73 gene could be important mechanisms in suppressing p73 expression in B cell non‐Hodgkins lymphomas.

Different Incidence and Pattern of p15INK4b and p16INK4a Promoter Region Hypermethylation in Hodgkin’s and CD30-Positive Non-Hodgkin’s Lymphomas

The p16INK4a and p15INK4b 5 CpG island hypermethylation has been described as one of the most frequent mechanisms leading to inactivation of these tumor suppressor genes in hematological malignancies. The p16 and p15 promoter regions were studied using methylation-specific polymerase chain reaction in 53 CD30 non-Hodgkins lymphomas (25 anaplastic large-cell, 13 peripheral T cell, and 15 anaplastic diffuse large B cell) and 26 Hodgkins lymphomas, with the aim of comparing the methylation status of these tumor suppressor genes in anaplastic large-cell lymphomas and other related entities. p16 and p15 methylation was detected, respectively, in 28% and 60% of CD30 non-Hodgkins lymphomas and in 38% and 42% of Hodgkins neoplasms. This confirms the p16-methylated status in Hodgkins cases described in a single previous study and adds information concerning the p15 gene that was also found to be methylated in this lymphoma subtype. Methylation incidence within cases at diagnosis and at relapse suggests that it is an early event in anaplastic large-cell lymphomas, being involved in tumor progression in Hodgkins cases. Our results show that although p16 and/or p15 methylation is involved in non-Hodgkins and Hodgkins tumors that share morphological and phenotypic features, differences in incidence, pattern of methylation, and implication in tumor progression are observed.

The detection of B-cell monoclonal populations by polymerase chain reaction : accuracy of approach and application in gastric endoscopic biopsy specimens

The recently developed strategy for the detection of monoclonal B-cell populations, based on the selective amplification of predominant immunoglobulin heavy chain gene (IgH) rearrangement, is seen to be a suitable alternative to Southern blot analysis. The new technique uses a pair of consensus primers for variable (VH) and joining (JH) regions. We first tested the accuracy of this new approach on a broad series of 67 samples that had been well characterized by both Southern blot and immunohistochemical techniques before being subjected to blind testing. Our results show that this technique gave 100% specificity (absence of false-positive results) and approximately 70% sensitivity (30% false-negative results). The only exception was the presence of an IgH polymerase chain reaction ambiguous result in a case of Sezarys syndrome. The polymerase chain reaction technique was then applied to a panel of 27 frozen gastric endoscopy biopsy specimens following previous clinical suspicion of lymphoma. Monoclonality was detected in nine of 13 samples previously diagnosed as lymphomas and in one of two carcinomas. Further examination of the gastrectomy specimen in the latter case disclosed a B-cell lymphoma associated with the carcinoma. In spite of its limited sensitivity, the high specificity attained by this technique in the detection of monoclonality makes it a useful adjunct to routine morphologic criteria, as it is sometimes capable of detecting true positive cases that conventional morphology studies show to be negative.

Phenotypic expression of histocompatibility antigens in human primary tumours and metastases

HLA class I and II expression was studied on 244 (177 primary and 67 metastatic) solid human tumours of different origin. Alkaline immunophosphatase (APAAP) and immunoperoxidase were used on cryostatic sections to stain MHC antigens. Monomorphic MoAbs were used against class I heavy chain, β2-microglobulin, DR, DQ and DP molecules.Class I expression was homogeneous on colon, melanoma and epidermoidal primitive tumours. Loss of HLA class I antigens was more frequent on basal cell carcinomas and sarcomas and was related to tumour differentiation on larynx carcinoma. Class I expression was heterogeneous on breast, larynx and stomach primitive neoplasias. Class I negative tumours were more frequent on metastatic than on primitive melanomas. Divergence of class I between primary tumours and autologous metastases was observed on melanomas, larynx and colorectal carcinomas.Class II expression was heterogeneous on all tumours and in a large number of cases was associated with high intensity of leukocytic infiltrate. HLA-DR expression was higher than HLA-DP and HLA-DQ (DR>DP>DQ) and was related to tumour progression. Four human tumour cell lines were modulated with recombinant interferon-γ for HLA class I and II antigens. Different HLA profiles were obtained: increased class I and II expression, increased class II or a low response.Finally, class I genes from 22 tumours were compared with autologous normal cells by Southern blot analysis: 12 tumours were class I positive and 10 negative. No clear differences in RFLP were observed that could be associated with class I rearrangement. The results are discussed in relation to the role that histocompatibility antigens may play in tumour progression and invasiveness.

Increased C-MYC Oncogene Copy Number Detected with Combined Modified Comparative Genomic Hybridization and FISH Analysis in a Richter Syndrome Case with Complex Karyotype

Modified comparative genomic hybridization (mCGH) was performed in a Richter syndrome case with a complex karyotype to identify and map gains of DNA sequences with possible importance in the pathogenesis and progression of the tumor. The mCGH analysis revealed a more intense signal on part of the long arm of one pair of chromosomes belonging to group C. The G-banding study showed that the increased DNA-sequence copy number originated from the 8q22-->qter chromosomal region. This increase was confirmed by performing a fluorescence in situ hybridization analysis on tumor metaphases by first using a chromosome 8-specific library and subsequently a C-MYC probe, which revealed positive staining on six different regions located on six different chromosomes, each one bearing a single copy of the C-MYC oncogene. These results show the existence of C-MYC oncogene copy-number increases and confirm the usefulness of mCGH in the genetic analysis of malignancies.

Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow-up of non-Hodgkin's lymphomas.

The hypermethylation of p16ink4a and p15ink4b genes have been described as an inactivating mechanism alternative to deletions and mutations that accounts for a relatively high proportion of cancers, including non‐Hodgkins lymphomas (NHLs). To investigate whether detection of abnormal methylation could have clinical applications in the management and follow‐up of lymphomas, we have analysed the behaviour and evolution of p16ink4a and p15ink4b methylation in 13 NHL cases undergoing chemotherapy. All cases were also analysed for the presence of monoclonal rearrangements of immunoglobulin or T‐cell receptor genes. Six patients showed methylation in at least one of these genes at diagnosis, whereas in two other cases methylation appeared during the treatment. The other five cases were always unmethylated. Methylation was detected when any histological or molecular evidence of disease was present, suggesting a good correlation between methylation and disease. In some cases, we were able to detect methylation in patients at complete remission and without evidence of monoclonal cell population, indicating a high sensitivity of the PCR to detect methylation. These results suggest that p16ink4a and p15ink4b methylation could be good markers of disease and could be helpful in identifying lymphoma patients at risk of relapse.

Frequent allelic losses of 9p21 markers and low incidence of mutations at p16(CDKN2) gene in non-Hodgkin lymphomas of B-cell lineage☆

We present an allelotype analysis of 35 cases of non-Hodgkin lymphomas and normal pairs using four microsatellite markers that flank the region occupied by the CDKN2 gene locus at 9p21. Frequent allelic losses (LOH) were detected in B-cell lineage NHLs, including Burkitt lymphoma (33.3% of total, if we only consider high grade tumors). In five of these tumors LOH did not include the CDKN2 gene. Mutational analysis of exon 1 and 2 of CDKN2 (SSGP and sequencing of abnormal bands) revealed a nonsense mutation (Arg72Ter) in one tumor (case 10), where the second hit of the Knudsons model consisted of the elimination of the wild type allele. In view of these results, the hypothesis of two different candidate tumor suppressor gene regions around the CDKN2 locus remains an intriguing possibility.