E. Teugels

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.

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.

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. Int. J. Cancer 82:687–693, 1999.

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 103–104-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 (~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.

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.

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.

Abstract PD7-04: Exome based germline mutation detection in a panel of 372 cancer associated genes in BRCA1/2-negative familial breast cancer patients

Background: Ten to 20% percent of all breast cancers occur in a familial context and in 20-30% of these cases a mutation in the BRCA1 or BRCA2, CHEK2 genes or, more rarely, in PALB2 can be found. The remaining cases remain routinely undiagnosed with regard to a possible genetic cause. We have examined a cohort of undiagnosed probands using exome germline sequencing in order to identify other potential breast cancer predisposition genes. Methods: In total, 63 BRCA1/2-negative high risk familial BC cases (BRCAX) were considered for pair-end whole exome germline DNA sequencing on a HiSeq1500 (Illumina). High quality reads were mapped (BWA-MEM) to the reference genome (hg19) and variants were called according to GATK best practice guideline. The variants detected within the panel of 372 cancer associated genes were annoted with ANNOVAR. Synonymous variant as well as variants with MAF>1% were discarded. In a first phase protein truncating variants were validated using another NGS method. Subsequent validation of non-synonymous missense mutations and non-frameshift indels is planned. Patients signed a multilayered informed consent also covering disclosure or not of different types of incidental findings. Results: For each exome, the mean breath of coverage was about 96 % at 10X or more and the mean depth of coverage for targeted region was about 126X. In total, 3570709 SNPs (∼56678 SNPs/sample) and 477801 INDELs (7584 INDELs/sample) were called. Of them, 20829 SNPs (331 SNPs/sample) and 16071 INDELs (255 INDELs/sample) passed quality filter. In total, 445 variants were found in the publicly available cancer genes panel. Twenty-seven stop-gain/loss, frame-shift insertion/deletion and splice site variants were considered for validation with 454 Roch Junior, among which 22 variants in genes ABCC11, AFP, BARD1, BBS10, CD96, CYP1A1, DNAH11, ESCO2, EXO1, FANCI, FLCN, FLT4, HPS6, MYH8, NME8, PALB2, PDE11A, RECQL4, TTC8 were validated as true positive. Some of these genes have been found earlier to be associated with breast cancer and/other cancer types. Functional prioritization of the remaining 416 non-synonymous and non-frameshift insertion/deletions was also done in-silico before further sequencing validation, which is ongoing. The mutations found are further clinically validated by examining other affected and non-affected family members and mining the literature. Some of the mutations in known cancer predisposing genes are considered for prudent application in clinical counseling. Genotype-phenotype correlations are being examined. Conclusion: Next-generation sequencing enabled us to detect variants with high/low penetrance in known cancer predisposing genes in > 35% of BRAX families, in addition to many novel variants in many other genes not yet tied to cancer predisposition occurring singly or in combination with known cancer gene mutations. Validated variants are further examined in the families for co-segregation with the disease and potential application in counseling. Citation Format: Shahi RB, Caljon B, De Brakeleer S, Decoster L, Fontaine C, Vanacker L, Vanhoeij M, Pauwels I, Bonduelle M, Vandooren S, Croes D, Teugels E, De Greve J. Exome based germline mutation detection in a panel of 372 cancer associated genes in BRCA1/2-negative familial breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD7-04.

MC13-0040 A novel HER3-V855A driver mutation homologous to EGFR-L858R in lung cancer

Results: Postoperative relapse was significantly correlated with overexpression of four genes including EVI2B (P=0.001, OR=4.622), ATP2A2 (P=0.006, OR=4.688), S100B (P=0.001, OR=11.521), TM4SF3 (P=0.001, OR=6.756), and OLFM4 (P=0.008, OR=3.545). Sensitivity, specificity, and accuracy of WENCA operation platform were 94.7%, 93.5%, and 97%, respectively. Conclusions: Detection of CTC-related multiple biomarkers by WEnCA operation platform can significantly improve the early prediction rate of postoperative CRC relapse.

206 The cis/trans effect of the T790M drug resistant mutation in non-small cell lung cancer

multiple myeloma, breast, renal, and liver tumor cell lines as indicated by combination indices below 0.7. In the colon cancer cell line SW620 cell cycle analysis revealed G2M arrest as mechanisms of action for Perifosine, whereas two representative antimetabolites, i.e. 5-Fluorouracil and 6-Thioguanine, induced S-phase arrest as expected. In combination, synergistic effects were observed in terms of apoptosis, e.g. caspase activation. In summary, these results demonstrate potent synergistic activity of Perifosine with various antimetabolites in human colon, multiple myeloma, breast, renal, and liver tumor cell lines. Synergism seems to be based on combining G2M arrest by Perifosine and S-phase arrest by the antimetabolite resulting in synergistic induction of cellular apoptosis. Further experiments addressing Perifosine’s mechanism of action in combination with antimetabolites are ongoing. Currently, Perifosine is in a phase III clinical trial in combination with Capecitabine in patients with refractory advanced colorectal cancer.

Identification of NPM1 as a potential high-penetrance gene in hereditary breast cancer.

Abstract #1042 Background: In a major proportion of breast cancer patients with a strong familial cancer history no BRCA1 or BRCA2 mutations are detected which prompted us to explore other proteins on the BRCA1/2 pathway. Previously we have identified BARD1 (De Brakeleer et al, SABC 2006) as a rare high penetrance breast cancer predisposing gene. Nucleophosmin (NPM1) is a multifunctional protein that is ubiquitylated and stabilized by the BRCA1-BARD1 complex and thus is part of the BRCA1 pathway. Somatic protein truncating mutations in NPM1 are frequent events in acute myeloid leukemia (AML).
 Aim: This study was aimed at determining whether NPM1 germline mutations may contribute to the breast cancer predisposition in families where no BRCA1 or BRCA2 mutation is found.
 Methods: NPM1 mutation analysis was performed on genomic DNA extracted from blood of 198 breast cancer patients with strong indications for a hereditary form of the disease and 110 controls. All the 11 exons of the NPM1 gene were separately amplified by PCR. Since a high number of processed NPM1 pseudogenes are dispersed throughout the human genome, PCR primers had to be designed carefully. The PCR products were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE) and when an abnormal migration pattern occurred, sequencing analysis was performed to determine the exact nucleotide-sequence alteration which was verified against the pseudogene sequences.
 Results: Out of the six novel sequence variations, only one occurred in an exon: a duplication of thymidine located in the 39 non-coding sequence of exon 12 (c.1042dupT). The remaining 5 variants were located in intron sequences close to the exon borders. Two variants flanking exon7 (c.460-31G>A and c.524+42G>A) were always linked, and occurred at the same high frequency in breast cancer patients and controls. Two intron sequence variants were only found in one patient. One was a duplication of a sequence in intron 8 (c.582+52dupT) and the other a sequence deletion in intron 3 (c.139-9delT). One sequence variation located close to exon 11 (c.771-46C>A) was found in 7 familial breast cancer patients in 4 high incidence families but not in unaffected family members and in none of the control samples. This mutation leads to an alternative splicing of the NPM1 mRNA and is predicted to modify the carboxyterminal end of the protein, removing a nucleolar locating signal.
 Discussion: A germline mutation in NPM1 was found only in familial breast cancer cases. This mutation may result in the synthesis of a C-terminal truncated NPM1 protein conferring high susceptibility to breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1042.