Multicentric neoadjuvant phase II study of panitumumab combined with an anthracycline/taxane-based chemotherapy in operable triple-negative breast cancer: identification of biologically defined signatures predicting treatment impact.

BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors for some of which the epithelial growth factor receptor (EGFR) pathway may play an important role. We investigated the efficacy and toxicity of an anti-EGFR antibody (panitumumab) combined with a standard neoadjuvant anthracycline-taxane-based chemotherapy in patients with operable, stage II-III, TNBC. PATIENTS AND METHODS: Treatment in this multicentric neoadjuvant pilot study consisted of panitumumab (9 mg/kg) for eight cycles q.3 weeks combined with four cycles of 5-fluorouracil, epidoxorubicin and cyclophosphamide (FEC100: 500/100/500 mg/m(2)) q.3 weeks, followed by four cycles of docetaxel (T: 100 mg/m(2)) q.3 weeks. Following therapy, all patients underwent surgical resection. Pathologic complete response (pCR) in assessable patients was the main end point while clinical response, toxicity and ancillary studies were secondary end points. Paraffin-embedded and frozen tumor samples were systematically collected with the aim to identify predictive biomarkers of efficacy and resistance in order to select biologically defined subpopulations for potential further clinical development of the anti-EGFR antibody. RESULTS: Sixty patients were included with 47 assessable for pathologic response. The pCR rates were 46.8% [95% confidence interval (CI): 32.5% to 61.1%] and 55.3% [95% CI: 41.1% to 69.5%] according, respectively, to Chevallier and Sataloff classifications. The complete clinical response (cCR) rate was 37.5%. Conservative surgery was carried out in 87% of cases. Toxicity was manageable. The association of high EGFR and low cytokeratin 8/18 expression in tumor cells on one hand and high density of CD8+ tumor-infiltrating lymphocytes on the other hand were significantly predictive of pCR. CONCLUSIONS: Panitumumab in combination with FEC100 followed by docetaxel appears efficacious, with acceptable toxicity, as neoadjuvant therapy of operable TNBC. Several biomarkers could help define large subsets of patients with a high probability of pCR, suggesting a potential interest to further develop this combination in biologically defined subgroups of patients with TNBC. CLINICAL TRIAL NUMBER: NCT00933517.

[Analysis of gene expression data regulated by clock-genes: methodological approach and optimization]. / Analyse de données d'expression transcriptomiques rythmées par des gènes-horloge: approche méthodologique et optimisation.

BACKGROUND: In microarray data, wide-scale correlations are numerous and increase the number of genes correlated to a test condition (phenotype, mutation status, etc.) either positively or negatively. Several methods have been developed to limit the effect of such correlations on the false discovery rate, but these may reject too many genes that have a mild or indirect impact on the studied condition. We propose here a simple methodology to correct this spurious effect without eliminating weak but true correlations. RESULTS: This methodology was applied to a microarray dataset designed to distinguish heterozygous BRCA1 mutation carriers from non-carriers. As our samples were collected at different times in the morning, we evaluated the effect of correlations due to circadian rhythm. The circadian system is a well-known correlation network, regulated by a small number of period genes whose expression varies throughout the day in predictable ways. The downstream effects of this variation on the expression of other genes, however, are incompletely characterized. We used two different strategies to correct this correlation bias, by either dividing or multiplying the expression of correlated genes by the expression of the considered period gene according to the sign of the correlation between the period gene and correlated gene (respectively positive or negative). CONCLUSIONS: We observed a linear relationship between the number of false-positive/negative genes and the strength of the correlation of the candidate gene to the test condition. BRCA1 was highly correlated to the period gene Per1; our correction methodology enabled us to recover genes coding for BRCA1-interacting proteins which were not selected in the initial direct analysis. This methodology may be valuable for other studies and can be applied very easily in case of well-known correlation networks.

Rare germline large rearrangements in the BRCA1/2 genes and eight candidate genes in 472 patients with breast cancer predisposition.

Hereditary breast cancers account for up to 5-10 % of breast cancers and a majority are related to the BRCA1 and BRCA2 genes. However, many families with breast cancer predisposition do not carry any known mutations for BRCA1 and BRCA2 genes. We explored the incidence of rare large rearrangements in the coding, noncoding and flanking regions of BRCA1/2 and in eight other candidate genes--CHEK2, BARD1, ATM, RAD50, RAD51, BRIP1, RAP80 and PALB2. A dedicated zoom-in CGH-array was applied to screen for rearrangements in 472 unrelated French individuals from breast-ovarian cancer families that were being followed in eight French oncogenetic laboratories. No new rearrangement was found neither in the genomic regions of BRCA1/2 nor in candidate genes, except for the CHEK2 and BARD1 genes. Three heterozygous deletions were detected in the 5' and 3' flanking regions of BRCA1. One large deletion introducing a frameshift was identified in the CHEK2 gene in two families and one heterozygous deletion was detected within an intron of BARD1. The study demonstrates the usefulness of CGH-array in routine genetic analysis and, aside from the CHEK2 rearrangements, indicates there is a very low incidence of large rearrangements in BRCA1/2 and in the other eight candidate genes in families already explored for BRCA1/2 mutations. Finally, next-generation sequencing should bring new information about point mutations in intronic and flanking regions and also medium size rearrangements.

Report of a family segregating mutations in both the APC and MSH2 genes: juvenile onset of colorectal cancer in a double heterozygote.

BACKGROUND AND AIMS: Mutations in DNA mismatch repair genes are associated with high risk of digestive malignancies [hereditary non-polyposis colorectal cancer (HNPCC); Lynch syndrome]; mutations of APC and MYH are associated with classic and attenuated familial adenomatous polyposis (FAP). Although the early onset of tumors in both syndromes is characteristic of their genetic origin, pediatric malignancies remain rare. Certain reports have found familial colorectal cancer (CRC) occurring in very young patients associated with mutations in more than one gene. MATERIALS AND METHOD: A family corresponding to the Amsterdam criteria for HNPCC, including two cases of colorectal cancer before the age of 25 years, was analyzed for mutations in the MSH2 genes by sequencing. Because polyposis was observed in a patient who developed CRC at age 16, the APC gene was also sequenced. RESULTS: A truncating mutation in the MSH2 gene, c.258_259delTG, was carried by patients developing cancer of the colon (two patients), uterus, kidney, bladder, and/or small intestine at ages 16, 24, 43, 44, 45, and 57, respectively. A patient with CRC at age 16 was found to carry the APC c.3183_3187del5 mutation as well as the MSH2 mutation, and it is inferred that her father, deceased of CRC at age 24, was also a double heterozygote. INTERPRETATION: These results confirm that vigilance is required when interpreting molecular results for families with very young patients, as more than one gene may contribute to the genetic risk. Cancer screening measures must also be adapted to the earlier and more penetrant risk to double heterozygotes.

Complete mutation screening and haplotype characterization of BRCA1 gene in Tunisian patients with familial breast cancer.

Breast cancer, the most commonly diagnosed cancer in women, is the second leading cause of cancer death in women worldwide. To investigate the contribution of BRCA1 gene mutations to familial breast cancer in Tunisia, 32 unrelated patients who had at least one first degree relative affected with breast and/or ovarian cancer were analysed. BRCA1 mutation analysis was performed by DNA sequencing of all BRCA1 exons. We identified four different BRCA1 frameshift mutations: c.4041delAG, c.2551delG and c.5266dupC already been described and one novel mutation, c.211dupA, observed in two unrelated families. C.5266dupC has previously been found among Jewish Ashkenazi and Eastern European populations. Our study describes it in Arabic/Berber population. Five out of thirty two familial cases had deleterious BRCA1 mutations. Fifteen additional cases carried unclassified variants (UV) or single nucleotide polymorphisms (SNPs). Our study is the first molecular investigation on the role of BRCA1 in hereditary breast cancer in North Tunisia.

Immunolocalization of BRCA1 protein in tumor breast tissue: prescreening of BRCA1 mutation in Tunisian patients with hereditary breast cancer?

BRCA1 is a tumor suppressor gene which is inactivated by mutation in familial breast and ovarian cancers. Over 300 different disease causing germ-line mutations have been described; 60% are unique to an individual family. This diversity and the large size of the gene lead us to search for a prescreening method for BRCA1 mutations. Since BRCA1 is a nuclear protein in normal cells, but reported by some authors to be cytoplasmic in breast tumor cells of patients with BRCA1 mutation, we evaluated immunohistochemistry as a prescreening technique to identify BRCA1 mutations in patients with familial presentation of breast cancer. Using a monoclonal antibody against the carboxy-terminal region of BRCA1, we performed immunohistochemistry on 18 tumor samples from patients with hereditary breast cancer. Cytoplasmic staining of BRCA1 was observed in 10 cases. Of the 18 tumors, 12 (66%) showed either BRCA mutation or BRCA1 accumulation or both, indicating that BRCA1 function might be lost in breast tumor cells not only through mutation, but also via abnormal cytoplasmic location. The immunohistochemical test used in this study would not be efficient as a pre-screening method of deleterious mutations, but it appeared useful to investigate tumor physiology.

Contribution of the BRCA1 and BRCA2 mutations to breast cancer in Tunisia.

Hereditary breast cancer accounts for 3-8% of all breast cancers, with mutations in the BRCA1 and BRCA2 genes responsible for up to 30% of these. To investigate the prevalence of BRCA1 and BRCA2 gene mutations in breast cancer patients with affected relatives in Tunisia, we studied 36 patients who had at least one first degree relative with breast and/or ovarian cancer Thirty-four 34 patients were suggestive of the BRCA1 mutation and two were suggestive of the BRCA2 mutation, based on the presence of male breast cancer detected in their corresponding pedigrees. Four mutations in BRCA1 were detected, including a novel frame-shift mutation (c.211dupA) in two unrelated patients and three other frameshift mutations--c.4041delAG, c.2551delG and c.5266dupC. Our study is the first to describe the c.5266dupC mutation in a non-Jewish Ashkenazi population. Two frameshift mutations (c.1309del4 and c.5682insA) were observed in BRCA2. Nineteen percent (7/36) of the familial cases had deleterious mutations of the BRCA1 or BRCA2 genes. Almost all patients with deleterious mutations of BRCA1 reported a family history of breast and/or ovarian cancer in the index case or in their relatives. Our data are the first to contribute to information on the mutation spectrum of BRCA genes in Tunisia, and we give a recommendation for improving clinical genetic testing policy.

[Werner's syndrome (adult onset progeria)]. / Syndrome de Werner (progeria de l'adulte).

Werner's syndrome (adult onset progeria) is a rare form of autosomal recessive genodermatosis associated in almost 80% of cases with mutation of the WRN gene. This prototype of rapid ageing syndromes is characterized by short stature with skin and hair anomalies (early graying of the hair, alopecia, depilation, sclerosed skin), orthopedic complications (flat foot, hallux valgus and other joint deformations) as well as systemic signs (early cataract, premature and diffuse atherosclerosis, endocrinopathies) and high risk of certain types of cancer (sarcomas, myeloid blood dyscrasias). Death occurs around the age of 40 - 50 years mainly as a result of cardiovascular accident or development of a malignant tumour. Signs of early aging should evoke this basic diagnosis and arrangements should be made for appropriate follow-up with screening for and treatment of systemic complications.

[p53 activation by PI-3K family kinases after DNA double-strand breaks]. / Activation de p53 par les kinases de la famille PI-3K après cassures double brin de l'ADN.

p53 plays a central role in the cellular response to DNA double-strand breaks (DSBs), and to DNA damage in general. The protein kinases ATM, ATR and DNA-PK detect DSBs and transmit this information to p53 by phosphorylation. This phosphorylation dissociates p53 from its negative regulator, mdm2. p53 then undergoes further modification and activates transcription of the genes responsible for cell cycle arrest. In certain circumstances, p53 also activates transcription of the genes responsible for apoptosis. The dysfunction of this cascade of events is oncogenic, with P53 itself being the most commonly mutated gene in malignant cells, although mutations in both the DNA damage sensors and cell cycle checkpoint and apoptosis effectors are frequent. A more complete understanding of p53 and the proteins it interacts with may allow the development of new cancer treatments.

Allelic imbalance at NBS1 is frequent in both proximal and distal colorectal carcinoma.

Nijmegen breakage syndrome (NBS) is a hereditary disorder involving chromosomal instability, cancer risk and radiosensitivity. NBS carriers have an increased risk of cancer, though the significance of mutations in the NBS1 gene in sporadic cancer has not yet been investigated. Because the loss of NBS1 is associated with increased chromosomal re-arrangements, and tumors of the colon are particularly prone to chromosomal anomalies, we have begun to study the NBS1 locus in colorectal cancer (CRC). DNA was isolated from 99 microdissected colorectal tumors, and microsatellite markers flanking the NBS1 locus at 8q21.3 as well as elsewhere on 8q were analyzed. Normal lymphocyte DNA from each patient served to normalize the amplification of each allele, and a reduction of at least 35% in the intensity of one allele was taken as evidence of allelic imbalance (AI). In proximal and distal CRCs we found 25.9 and 36.2% with AI at 8q21.3, respectively. AI in proximal CRC tended not to extend to marker D8S555 at 8q24.1, whereas in distal CRC the region of AI frequently included all the informative markers. AI of 8q21.3 was not associated with any clinical variable. These results suggest that 8q21.3 contains a tumor suppressor gene involved in proximal CRC, possibly NBS1. The large regions of AI make it difficult to determine the importance of AI at the NBS1 locus in distal CRC.

Physical map of the region surrounding the ataxia-telangiectasia gene on human chromosome 11q22-23.

Ataxia telangiectasia (A-T) is an autosomal recessive disease affecting multiple systems, including the development of the cerebellum and thymus. This results in a progressive cerebellar ataxia with onset between 1-3 years, telangiectasia occurs within the subsequent 3-5 years. We localized the A-T gene by linkage analysis to chromosome 11q22-23, between the markers D11S384, and D11S535, and constructed a series of contigs using three BACs and twelve cosmids, spanning a region of approximately 400 kb. We developed a set of sequence-tagged site (STS) markers from the ends of the BACs and cosmids. The A-T gene was isolated from within this region. It is now possible to precisely orient specific BACs, cosmids, and STSs with respect to the exons of the A-T gene (ATM). We anticipate that this information will be useful for further studies of functional domains and regulatory elements within the ATM gene, as well as for other genes in this region. In addition, these clones can be used for FISH studies of deletions, translocations and for loss of heterozygosity in various tumors.

High incidence of cancer in a family segregating a mutation of the ATM gene: possible role of ATM heterozygosity in cancer.

ATM mutations predispose cells to malignancy by promoting chromosomal instability. We have identified a family with multiple cancers that segregates a mutant allele of ATM, IVS61+2insTA, which causes skipping of exon 61 in the mRNA, as well as a previously undescribed polymorphism, IVS61+104C(54):T(46). The mutation was inherited by two sisters, one who developed breast cancer at age 39 and the second at age 44, from their mother, who developed kidney cancer at age 67. Molecular studies were undertaken to determine the role of the ATM gene in the development of cancer in this family. Studies of irradiated lymphocytes from both sisters revealed elevated numbers of chromatid breaks, typical of A-T heterozygotes. Studies on lymphoblastoid cell lines established from these individuals revealed abnormal p53 induction and apoptosis after DNA damage. Loss of heterozygosity (LOH) in the ATM region of chromosome 11q23.1 showed that the normal ATM allele was lost in the breast tumor of the older sister. LOH was not seen at the BRCA1 or BRCA2 loci. BRCA2 is not likely to be a cancer-predisposing gene in this family because each sister inherited different chromosomes 13 from each parent. The sisters share their maternal BRCA1 allele, although no mutation in this gene was detected in the family. Our findings suggest that haploinsufficiency at ATM may promote tumorigenesis, even though LOH at the locus supports a more classic two-hit tumor suppressor gene model.

ATM heterozygote cells exhibit intermediate levels of apoptosis in response to streptonigrin and etoposide.

Ataxia-telangiectasia (A-T) is a rare recessive disease characterised by cerebellar ataxia, immunodeficiency, sensitivity to ionising radiation and increased cancer risk. Heterozygotes have an increased risk of cancer and may comprise 1% of the population. In vitro, A-T heterozygote cell lines show radiosensitivity intermediate between normal and A-T homozygotes. Furthermore, in A-T homozygotes, hypersensitivity to chemical agents which cause DNA damage, similar to that produced by ionising radiation, has been observed. To investigate the chemosensitivity of A-T heterozygote cell lines, we used TUNEL to analyse the level of apoptosis after drug treatment with etoposide and streptonigrin. Our samples included four normal, eight A-T heterozygote and 10 A-T homozygote lymphoblastoid cell lines. All cell lines were exposed to drugs for 24 h, then cultivated in fresh media for 0 and 72 h. The levels of apoptosis increased significantly in all cell lines, with the greatest increase in homozygote cells and an intermediate increase in heterozygote cells (P values of < 0.01 for etoposide treatment and < 0.02 for streptonigrin treatment were obtained using the Kruskal-Wallis H-test). Our results indicate that A-T heterozygotes express intermediate sensitivity to etoposide and streptonigrin similar to that observed in response to ionising radiation.

Human fibroblasts transfected with an ATM antisense vector respond abnormally to ionizing radiation.

ATM, the gene mutated in ataxia-telangiectasia (A-T), mediates multiple cellular responses to DNA damage. A-T homozygotes have a high risk of cancer and exhibit spontaneous chromosomal instability, and cultured A-T cells react abnormally to ionizing radiation. We have developed an ATM antisense vector that confers an A-T phenotype on normal cells. An episomal antisense vector was created that contained a 1.3 kb segment of the ATM cDNA, and was transfected into normal human fibroblasts. Intracellular levels of ATM protein were typically reduced 10-fold in antisense-expressing (GM639-46alpha) clones. GM639-46alpha clones exhibited the low threshold for radiation-induced apoptosis, low clonogenic survival, and cell cycle defects normally seen in A-T cells. Transfection with the corresponding ATM sense strand vector had no effect on the behavior of normal cells, and neither vector affected the behavior of A-T cells. Our results demonstrate that interference with ATM gene expression recreates the A-T phenotype in normal cells, and provide functional evidence linking the ATM gene to cellular DNA damage responses. The ATM antisense vector should prove a useful tool for studying ATM function in a variety of normal, mutant, and malignant cell lines.

Loss of heterozygosity at the ATM locus in colorectal carcinoma.

Patients homozygous for mutation of the ATM gene exhibit constitutional genetic instability and have a high risk of cancer. A-T heterozygotes also have an increased tendency to develop adenocarcinomas. Colorectal cancer (CRC) is the second most common cancer in western populations, and tumors of the right colon are typically highly genetically unstable. The DNA mismatch repair genes mutated in most familial and some sporadic CRCs account for one route by which cells acquire additional oncogenic mutations during the progression of malignancy. Mismatch repair defects, however, do not seem to account for the majority of CRCs. Because of its role in maintaining genomic stability, and the high risk of cancer to homozygotes, ATM is a candidate gene for inactivation in the evolution of chromosomal instability in tumor cells. We have examined 114 CRC patients for loss of heterozygosity (LOH) using six microsatellite markers tightly linked to the ATM locus. Our data suggest that LOH of this region is not associated with cancer of the proximal colon. In the distal colon, LOH was found in 23-31% of cases, which is moderately elevated above the non-specific LOH reported in tumors of this tissue. No correlations were found with regard to clinicopathological variables aside from tumor location.

Seventh International Workshop on Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is a rare hereditary syndrome involving cerebellar degeneration, immunodeficiency, cancer risk, and radiosensitivity. Since the cloning of the A-T gene, ATM, in 1995, research on this pleiotropic disease and its molecular basis has expanded tremendously. ATM is a large protein kinase that appears to be one of the primary sensors of DNA strand-break damage. The vast majority of mutations in ATM result in truncation and destabilization of the protein, but certain missense and splicing errors have been shown to result in a less severe phenotype. A-T heterozygotes have been shown to have a slightly increased risk of cancer, but their increased in vitro radiosensitivity does not seem to result in any in vivo sensitivity. ATM does seem to act as a classic tumor suppressor gene in T-prolymphocytic leukemia, and LOH at the ATM locus is a common event in some tumor types, suggesting a general role for ATM in cancer. Recent work has shown the interaction of ATM with proteins involved in cell cycle control, and the direct phosphorylation of some of these interactors by ATM. ATM knockout mice have been created by several groups, and recapitulate the immunodeficiency, radiosensitivity, cancer risk, and fertility defects of A-T, although the effect on the cerebellum is slight. These diverse topics, and their integration into a global understanding of A-T, were the basis of the 7th International A-T Workshop.

No evidence for constitutional ATM mutation in breast/gastric cancer families.

Ataxia-Telangiectasia (A-T) is a rare autosomal recessive disease characterised by cutaneous telangiectasia, cerebellar ataxia, immunodeficiency, high sensitivity to ionising radiation, chromosomal instability and an increased risk of cancer. The gene mutated in A-T patients, ATM, is located on chromosome 11q22-23. ATM heterozygotes are thought to have a high tendency to develop malignancies, such as breast cancer. In order to determine the contribution of heterozygous ATM mutation to cancer, studies of cancer-affected patients have been undertaken in non site-specific cancer families and sporadic breast cancer cases. No evidence of an important role of ATM heterozygous mutations has been shown. In order to give another contribution to these results, we tried to define a specific family phenotype according to the most common cancers observed in ATM heterozygotes. Breast and gastric cancers appear to be the most frequent malignancies in A-T carriers and one ATM germ-line mutation has been described in a breast/gastric cancer family. Therefore we further investigated the role of ATM mutation in additional breast/gastric cancer families. In eighteen families associating these two malignancies, we used the protein transcription/translation test to detect ATM mutations in the index case from each family. We found one case of ATM mutation which did not cosegregate with the gastric cancer in the family.

CAND3: a ubiquitously expressed gene immediately adjacent and in opposite transcriptional orientation to the ATM gene at 11q23.1.

Using a magnetic beads-mediated cDNA selection procedure and a fetal brain expression library, we identified a transcriptional unit within a cosmid positive for the marker D11S384. Pursuit of its full-length cDNA led to the cloning of the third candidate gene (CAND3) we studied in our quest for the ataxia-telangiectasia (A-T) gene, ATM. CAND3 spans approximately 140 kb of genomic DNA and is located immediately centrimeric to ATM, with 544 bp of DNA separating the two genes. CAND3 encodes two ubiquitously expressed transcripts of approximately 5.8 kb and approximately 4.6 kb that are divergently transcribed from a promoter region common to ATM. Nucleotide sequence was determined for one of its alternately spliced transcripts. The predicted protein has 1175 amino acids and is novel in sequence, with only weak homologies to transcriptional factors, nucleoporin protein, and protein kinases, including members of the phosphatidylinositol 3-kinase (PI-3 kinase) family. Although neither homology to ATM nor any mutation of CAND3 in A-T patients has been found, the head-to-head arrangement of CAND3 and ATM, with expression of both housekeeping genes from a common stretch of 544 bp intergenic DNA, suggests a bi-directional promoter possibly for co-regulation of biologically related functions. YACs, BACs, cosmids, and STSs are defined to aid in further study of this gene.