Frequent incidence of BARD1-truncating mutations in germline DNA from triple-negative breast cancer patients.

Triple-negative breast cancer (TNBC) accounts for 10-20% of all breast cancers (BCs), and conventional chemotherapy is the only effective systemic treatment. Germline BRCA1/2 mutations are found in approximately 15% of TNBC patients. In the past, we have documented pathogenic mutations in BARD1, a BRCA1 interacting protein, in families at high risk for BC. In this study, we have analyzed germline DNA from 61 estrogen receptor negative patients (of which 42 were TNBC) for the presence of mutations in the BRCA1, BRCA2 and BARD1 gene. BRCA1/2 mutations were found in 8 out of 42 (19%) TNBC patients, but not in the ER-/HER2+ cohort. We also found four good candidate pathogenic BARD1 mutations in the TNBC cohort, including two protein-truncating mutations (p.Gln564Ter and p.Arg641Ter). Our data suggest that TNBC patients are enriched for pathogenic BARD1 germline mutations as compared to control samples and high BC risk families. Ten of the 42 investigated TNBC patients carry a BRCA pathway mutation (in BRCA1, BRCA2 or BARD1) rendering them susceptible to homologous recombination deficiency. These patients should become eligible for exploring the efficacy of poly (ADP-ribose) polymerase (PARP) inhibitors.

Geographical distribution of Slovenian BRCA1/2 families according to family origin: implications for genetic screening.

Knowledge of the geographical distribution of highly recurrent mutations may be useful for efficient screening in cancer families. Since the cloning of the BRCA1/2 genes, it is known that the wide spectrum of deleterious mutations shows high ethnic and geographic heterogeneity. In this study, we have tested probands from 582 breast/ovarian cancer families and positioned all 156 BRCA1/2 families on the map according to the family origin. We observed that high-risk families with the same recurrent mutation present a typical geographical distribution and that different recurrent mutations may show different distribution patterns. We then evaluated the genetic screening implications of this heterogeneous prevalence of the most recurrent mutations found [300T>G(c.181T>G), 1806C>T(c.1687C>T), 969ins7(c.844_850dupTCATTAC), 5382insC(c.5266dupC), 235G>A(c.116G>A) in BRCA1 and IVS16-2A>G(c.7806-2A>G) in BRCA2]. On the basis of these results, specific testing procedures for new incident cases may be offered according to their family origins and, according to the information regarding clusters revealed in this study, the individuals (especially those at low risk), originating from regions with clusters, might be screened preferentially for cluster mutations and analysis may be simplified according to the family origin.

Clinical activity of afatinib (BIBW 2992) in patients with lung adenocarcinoma with mutations in the kinase domain of HER2/neu.

Human epidermal growth factor receptor (HER)2/neu kinase domain mutations are found in approximately 1-4% of lung adenocarcinomas with a similar phenotype to tumors with epidermal growth factor receptor (EGFR) mutations. Afatinib is a potent irreversible ErbB family blocker. We determined the tumor genomic status of the EGFR and HER2 genes in non- or light smokers with lung adenocarcinoma in patients who were entered into an exploratory Phase II study with afatinib. Five patients with a non-smoking history and metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified, three of which were evaluable for response. Objective response was observed in all three patients, even after failure of other EGFR- and/or HER2-targeted treatments; the case histories of these patients are described in this report. These findings suggest that afatinib is a potential novel treatment option for this subgroup of patients, even when other EGFR and HER2 targeting treatments have failed.

BRCA1/2 status and clinicopathologic characteristics of patients with double primary breast and ovarian cancer.

The aim of the study was to analyse the results of BRCA1/2 testing in a group of patients with double primary breast and ovarian cancer (DPBOC) in Slovenia. Additionally, the family history and the clinicopathologic characteristics of BRCA1/2 mutation positive and negative patients with DPBOC were analysed, comparing them to a group of untested patients with DPBOC. For these groups of patients, survival analysis was also performed. From the 52 patients who were invited to genetic counselling and testing, 20 responded positively (38% compliance). BRCA1/2 mutations were found in 60% (12/20): 45% BRCA1 and 15% BRCA2 (9 and 3 patients, respectively). There was significantly higher grade of ovarian cancer and significantly higher rate of multiple primary breast cancer in BRCA1/2 positive group. Additionaly, there was a trend towards higher rate of first-degree family history of breast cancer, a trend towards higher stage of ovarian cancer, and a trend towards breast cancer being the first cancer in BRCA1/2 positive group. According to survival analysis, the tested group was not a representative sample of the larger untested group (of 51 patients), so we estimate that the rate of BRCA1/2 mutations in DPBOC patients is probably less than 60%.

Accurate KRAS mutation testing for EGFR-targeted therapy in colorectal cancer: emphasis on the key role and responsibility of pathologists.

Patients with metastatic colorectal cancer and KRAS mutation are unlikely to benefit from treatment with anti-EGFR antibodies, and testing for KRAS mutation in this setting is recommended. Pathologists have a crucial role in accurate testing for KRAS mutations, whether or not testing is performed in their own laboratory, as mutation analysis is performed on paraffin embedded tissue selected by the pathologists. The type of fixative used is a very important issue, as some fixatives do not allow molecular testing. Pathologists must select the most appropriate tumoral tissue block for KRAS mutation analysis and hence, must know the sensitivity of the KRAS mutation detection methodology utilized in their reference laboratory. It is essential that they select a tissue block that contains enough percentage of viable tumour cells, as false negative results will occur when the sample is contaminated with high levels of nontumour elements. Pathologists not only have to recognize the area of invasive carcinoma and distinguish it from non-invasive neoplastic components, but also have to estimate the percentage of necrotic debris and nontumoural elements. For tests that require a high percentage of tumour cells, macrodissection before extraction of nucleic acids is often indicated. The primary pathologists in addition are responsible for preparation of the pathology report for the tissue block on which the KRAS mutation analysis was performed and should transmit the results to the requesting clinician. Pathologists should participate in a multidisciplinary oncologic consult to achieve correct interpretation of the results e.g. in case of potential false negative results.

Loss of nuclear BRCA1 localization in breast carcinoma is age dependent.

Abstract. Patients carrying a germ line mutation in the BRCA1 gene are predisposed to breast cancer. Somatic BRCA1 mutations were almost never reported in sporadic breast tumors, but several authors have described a decrease in BRCA1 mRNA and protein. In order to further investigate the possible role of BRCA1 in sporadic breast cancer, an improved immunohistochemical protocol was developed and applied on tissue sections obtained from 102 cancer patients belonging to two nonoverlapping age groups (less than 40 and more than 60 years). Based on the obtained BRCA1 specific nuclear staining we could distinguish two categories of breast cancer. The staining was present in almost all the nuclei of the normal and the cancer cells in about 50% of young (less than 40 years old) as well as older patients (more than 60 years old). Thus, BRCA1 does not seem to be involved in the genesis of these cancers. In the second category of patients, either a fraction of, or all tumor cells showed no nuclear staining. In this category, no nuclear BRCA1 staining could be observed in the tumor cells of 14 (27%) young and 3 (6%) older patients. Among six young patients bearing a breast tumor showing no BRCA1 nuclear staining at all, one was found to carry a BRCA1 germline mutation. Taken together, our results suggest that the molecular pathway in which the BRCA1 protein participates is disturbed in about 50% of sporadic breast cancer, this effect being more pronounced in tumors of young patients.

Transduction of ovarian cancer cells: a recombinant adeno-associated viral vector compared to an adenoviral vector.

Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 10(3)-10(4)-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (approximately 15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.

Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells.

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.

Characterization of permanent cell lines that contain the AAV2 rep-cap genes on an Epstein-Barr-virus-based episomal plasmid.

Recombinant adeno-associated virus (rAAV) has emerged as a promising gene therapy vector. Its development, however, has been hampered by the lack of a readily available efficient production method. We investigated the possibility of establishing permanent cell lines for the production of rAAV with a new Epstein-Barr-virus (EBV)-based episomal AAV rep-cap plasmid (pCEP-rep/cap). HeLa and 293 cells were stably transfected with plasmids that carry the AAV2 rep/cap genes under transcriptional control of their endogenous promoters (p5, p19 and p40) either on the pCEP-rep/cap or an integrated (pIM45) plasmid. For the ease of monitoring transgene expression in live cells, a rAAV vector expressing gfp (the green fluorescent protein gene, rAAV-gfp/neo) was used. Establishment of stable transfected cell lines with these plasmids proved feasible but their usefulness was limited because of their instability. Within 8-12 weeks after their establishment, stably transfected rep-cap cell lines invariably lost their function. In addition, the rAAV-gfp/neo vector we used was susceptible to mutation in stably transfected HeLa cells. Our observations demonstrate specific problems both at the level of rep/cap gene function and the rAAV genome that can occur with the establishment of rAAV production cell lines. These experiments should aid the further development of efficient rAAV production protocols.

Alteration of jun proto-oncogene status by plasmid transfection affects growth of human ovarian cancer cells.

The AP-1 transcription factor (the Jun and Fos proteins) is suspected of playing an important role in the biology of human cancer. Human epithelial ovarian tumors and cancer cell lines express the c-jun and jun-B proto-oncogenes at a high level, in contrast with the jun-D gene. We have investigated here the functional relevance of these observations for the growth of ovarian cancer cells. Transient constitutive expression of a dominant negative c-jun mutant (TAM67) in human AZ224, SKOV3 and OVCAR3 ovarian cancer cells inhibited the outgrowth of selection marker-resistant colonies by at least 75% as opposed to a control plasmid. Transfection of jun-B did not affect these cell lines, while jun-D transfection had a cell line-specific effect. In comparison, transfection of the tumor-suppressor gene p53 had a less important inhibitory effect on OVCAR3 cells and no effect on SKOV3 and AZ224 cells when compared to TAM67. Regulated TAM67 expression in AZ224 cells, from plasmids containing the mouse metallothionein or the MMTV promoter, suppressed cancer cell growth in vitro and in nude mice without evidence of increased cell death. Our observations support a role for the c-jun proto-oncogene as a positive mediator of human ovarian cancer cell growth and make it a potential therapeutic target.

Insufficient TGF-beta 1 production inactivates the autocrine growth suppressive circuit in human ovarian cancer cell lines.

TGF-beta 1 is a secreted polypeptide that elicits an antiproliferanve response in many cell types. However, many epithelial cancer cell lines are resistant to TGF-beta 1 growth inhibition. We investigated the in vitro growth suppressive effect of TGF-beta 1 on five ovarian cancer cell lines. Two of these (OVCAR-3 and AZ364) were growth inhibited by TGF-beta 1. The other three cell lines (SKOV-3, AZ224 and AZ547), were resistant to the antiproliferative action of the cytokine. All five cell lines produce TGF-beta 1 mRNA at very different levels and also secrete the TGF-beta 1 polypeptide, but mainly in a biologically latent form as tested by ELISA; this probably explains the fact that the TGF-beta 1 autocrine growth inhibition circuit is not active, even in sensitive cell lines. Even complete activation of the in vitro secreted latent form would be insufficient to induce growth arrest when compared to the levels of exogenous TGF-beta 1 needed to induce growth arrest in sensitive cell lines. The TGF-beta 1 receptor type I mRNA is expressed by all five ovarian cancer cell lines, but two of them (AZ224 and AZ547) lack detectable TGF-beta 1 receptor type II mRNA expression. Since TGF-beta 1 signaling requires both receptor types, the lack of receptor type II in two cell lines may explain their resistance to growth inhibition. Further experiments should be carried out on receptors and downstream components to pinpoint the cause of resistance in the SKOV cell line.

High frequency of BRCA1/2 germline mutations in 42 Belgian families with a small number of symptomatic subjects.

AIM: The initial risk assessments for BRCA1/2 mutation carriers and estimates of carrier frequencies were based on extended pedigrees with a large number of symptomatic subjects. When counselling based on BRCA gene mutation analysis was initiated, we faced requests for counselling mostly from members of small families with only two or three affected members. We report on the likelihood of finding a BRCA mutation in such small families. METHODS: In the first 100 families that came for oncogenetic counselling since September 1994, a BRCA1/2 gene mutation screen was initiated if there were two or more symptomatic first degree relatives, if one of them had ovarian cancer, or if one breast cancer was diagnosed before the age of 50 years. RESULTS: BRCA gene mutations were found and confirmed by sequencing in 14 out of 42 families (33%); 10 mutations were in the BRCA1 gene and four in the BRCA2 gene. Our findings indicate an increased probability of detecting a BRCA gene mutation when ovarian cancer occurred in the family. There is no increased probability of detecting a mutation with increasing numbers of breast cancers. Only 22% of the eligible presymptomatic family members opted for testing. The presymptomatic female carriers currently prefer breast surveillance rather than prophylactic surgery. CONCLUSION: BRCA1/2 gene mutation testing can be done with reasonable efficiency in the Belgian population when there are two symptomatic family members. The availability of testing does not lead to a high frequency of requests for testing by presymptomatic family members.

The alphaE-catenin gene (CTNNA1) acts as an invasion-suppressor gene in human colon cancer cells.

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the alphaE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second alphaE-catenin allele. The alphaE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.

Haplotype and phenotype analysis of nine recurrent BRCA2 mutations in 111 families: results of an international study.

Several BRCA2 mutations are found to occur in geographically diverse breast and ovarian cancer families. To investigate both mutation origin and mutation-specific phenotypes due to BRCA2, we constructed a haplotype of 10 polymorphic short tandem-repeat (STR) markers flanking the BRCA2 locus, in a set of 111 breast or breast/ovarian cancer families selected for having one of nine recurrent BRCA2 mutations. Six of the individual mutations are estimated to have arisen 400-2,000 years ago. In particular, the 6174delT mutation, found in approximately 1% of individuals of Ashkenazi Jewish ancestry, was estimated to have arisen 29 generations ago (1-LOD support interval 22-38). This is substantially more recent than the estimated age of the BRCA1 185delAG mutation (46 generations), derived from our analogous study of BRCA1 mutations. In general, there was no evidence of multiple origins of identical BRCA2 mutations. Our study data were consistent with the previous report of a higher incidence of ovarian cancer in families with mutations in a 3.3-kb region of exon 11 (the ovarian cancer cluster region [OCCR]) (P=.10); but that higher incidence was not statistically significant. There was significant evidence that age at diagnosis of breast cancer varied by mutation (P<.001), although only 8% of the variance in age at diagnosis could be explained by the specific mutation, and there was no evidence of family-specific effects. When the age at diagnosis of the breast cancer cases was examined by OCCR, cases associated with mutations in the OCCR had a significantly older mean age at diagnosis than was seen in those outside this region (48 years vs. 42 years; P=.0005).

A high proportion of novel mutations in BRCA1 with strong founder effects among Dutch and Belgian hereditary breast and ovarian cancer families.

We have identified 79 mutations in BRCA1 in a set of 643 Dutch and 23 Belgian hereditary breast and ovarian cancer families collected either for research or for clinical diagnostic purposes. Twenty-eight distinct mutations have been observed, 18 of them not previously reported and 12 of them occurring more than once. Most conspicuously, a 2804delAA mutation has been found 19 times and has never been reported outside the Netherlands. A common haplotype spanning > or = 375 kb could be identified for each of the nine examined recurrent mutations, indicating the presence of multiple BRCA1 founder mutations in the Dutch population. The 2804delAA mutation has been estimated to have originated approximately 32 generations ago. No specific breast or ovarian cancer phenotype could be assigned to any of the common mutations, and the ovarian cancer incidence among 18 families with the 2804delAA mutation was heterogeneous.

Plastic phenotype of human oligodendroglial tumour cells in vitro.

Human oligodendroglioma cells cultured in serum-supplemented media lose their oligodendrocytic antigenic markers and acquire astrocytic markers. However, after reimplantation in rodent brain, these cells re-express oligodendrocytic markers. This switch in human oligodendroglioma cell phenotype could result from the interplay of different stimuli in vitro vs in vivo The in vitro differentiation into astrocytes might result from non-physiological culture conditions. It is shown that human oligodendroglioma cells behave in a way similar to that of rodent bipotential 0-2 A progenitor cells which can be driven to differentiate into either oligodendrocytes or type 2-astrocytes depending on the culture medium. Indeed, in serum-supplemented medium, human oligodendroglioma cells proliferated and expressed the GFAP astrocytic marker. In chemically defined medium containing insulin, human oligodendroglioma cells were quiescent and expressed the OI oligodendrocyte-specific marker. In both media, human oligodendroglioma cells expressed the A2B5 membrane marker as well as the SCIP transcription factor specific of 0-2 A cells, further confirming their oligodendrocytic origin. Replacement of insulin by platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), known to maintain 0-2 A progenitors in a proliferative state, stimulated DNA replication of human oligodendroglioma cells cultured in chemically defined medium.