Elisabeth Turpin

High Frequency of TP53 Mutation in BRCA1 and Sporadic Basal-like Carcinomas but not in BRCA1 Luminal Breast Tumors

Breast tumors with a germ-line mutation of BRCA1 (BRCA1 tumors) and basal-like carcinoma (BLC) are associated with a high rate of TP53 mutation. Because BRCA1 tumors frequently display a basal-like phenotype, this study was designed to determine whether TP53 mutations are correlated with the hereditary BRCA1 mutated status or the particular phenotype of these tumors. The TP53 gene status was first investigated in a series of 35 BRCA1 BLCs using immunohistochemistry, direct sequencing of the coding sequence, and functional analysis of separated alleles in yeast, and compared with the TP53 status in a series of 38 sporadic (nonhereditary) BLCs. Using this sensitive approach, TP53 was found to be frequently mutated in both BRCA1 (34 of 35, 97%) and sporadic (35 of 38, 92%) BLCs. However, the spectrum of mutation was different, particularly with a higher rate of complex mutations, such as insertion/deletion, in BRCA1 BLCs than in the sporadic group [14 of 33 (42%) and 3 of 34 (9%), [corrected] respectively; P = 0.002]. Secondly, the incidence of TP53 mutations was analyzed in 19 BRCA1 luminal tumors using the same strategy. Interestingly, only 10 of these 19 tumors were mutated (53%), a frequency similar to that found in grade-matched sporadic luminal tumors. In conclusion, TP53 mutation is highly recurrent in BLCs independently of BRCA1 status, but not a common feature of BRCA1 luminal tumors.

Cyclophosphamide Dose Intensification May Circumvent Anthracycline Resistance of p53 Mutant Breast Cancers

The predictive value of p53 for the efficacy of front-line anthracycline-based chemotherapy regimens has been a matter of significant controversy. Anthracyclines are usually combined with widely different doses of alkylating agents, which may significantly modulate tumor response to these combinations. We analyzed three series of de novo stage II-III breast cancer patients treated front line with anthracycline-based regimens of various cyclophosphamide dose intensities: 65 patients with estrogen receptor (ER)(-) tumors treated with anthracyclines alone (Institut Jules Bordet, Brussels), 51 unselected breast cancer patients treated with intermediate doses of cyclophosphamide (MD Anderson Cancer Center, Houston, TX), and 128 others treated with a dose-dense anthracycline-cyclophosphamide combination (St. Louis, Paris). After chemotherapy and surgery, pathologic complete response (pCR) was evaluated. p53 status was determined by a yeast functional assay on the pretreatment tumor sample. In a multivariate analysis of the pooled results, a lack of ER expression and high-dose cyclophosphamide administration were associated with a higher likelihood of pCR. A sharp statistical interaction was detected between p53 status and cyclophosphamide dose intensity. Indeed, when restricting our analysis to patients with ER(-) tumors, we confirmed that a mutant p53 status was associated with anthracycline resistance, but found that p53 inactivation was required for response to the dose-intense alkylating regimen. The latter allowed very high levels of pCR in triple-negative tumors. Thus, our data strongly suggest that cyclophosphamide dose intensification in ER(-) p53-mutated breast cancer patients could significantly improve their response.

Allelic Loss Detection in Inflammatory Breast Cancer: Improvement with Laser Microdissection

Solid tumors are composed not only of tumor cells but also of stromal nonneoplastic cells. In whole tumor samples, stromal cells retaining their alleles may therefore obscure detection of loss of heterozygosity (LOH) in tumor cells. An increasing number of studies have used laser-assisted tissue microdissection to improve LOH detection, but the real gain in sensitivity has been poorly quantified. We studied a group of 16 inflammatory breast carcinomas that were submitted to both standard DNA extraction from frozen whole tumor samples and laser microdissection performed on paraffin-embedded tumor samples. Using PCR with fluorescence-labeled primers, we comparatively analyzed ten polymorphic markers with both sources of DNA. With the LOH detection threshold set at −25%, we showed that 25 LOHs could not be diagnosed with whole tumor samples out of 73 LOHs positively diagnosed in microdissected samples (34%). With the LOH detection threshold set at −50%, the respective figures were 39 LOHs not diagnosed out of 55 LOHs (71%). Measuring the intensity of the allelic decrease, we showed that the mean decrease of the lost allele is −34% with whole tumor samples and −67% with microdissected samples. The increase in sensitivity of LOH detection with microdissection is associated with the density of stromal cells. This strong improvement in LOH detection in this aggressive type of breast cancer indicates that many other molecular studies performed on heterogeneous solid tumors may benefit from a first step of laser microdissection.

Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers

The TSG101 gene, identified through insertional mutagenesis, is localized in a region that exhibits LOH in human cancers, suggesting that TSG101 might be a tumor suppressor gene. Numerous studies have then shown the presence of abnormal transcripts in various tumors which appear to result from aberrant splicing of the gene, rather than from intragenic deletions. Moreover, many studies demonstrated that these aberrantly spliced transcripts were not found in matched normal tissues. We have analysed TSG101 transcripts in 85 breast cancer samples and found that abnormal splicing of the gene is tightly correlated with tumor grade and p53 mutation. In addition, stress induced the appearance of these abnormal transcripts in primary lymphocytes. Hence, TSG101 splicing defects, while unrelated to the oncogenic process per se, could reflect the cellular environment of the tumor cells. The proposed role of stress and hypoxia to select p53 mutant cells could account for the tight association with p53 status.

Identification of a three-gene expression signature of early recurrence in non-muscle-invasive urothelial cell carcinoma of the bladder

OBJECTIVES To identify the expression profile of early recurring non-muscle-invasive bladder cancer (NMI-BC). MATERIALS AND METHODS We selected primary NMI-BC according to the following criteria: complete resection, primary occurrence of urothelial cell carcinoma, stage cTa or T1, low grade, no carcinoma in situ. All patients underwent a transurethral resection of the bladder and were followed according to the Guidelines of European Association of Urology. Expression of 110 target genes was studied by using real time quantitative RT-PCR. The correlation between the absolute expression and early recurrence was analyzed by using a Cox regression model. The ability of a gene to distinguish the tumors with early recurrence from those with late recurrence was determined by using a ROC-AUC test. A hierarchical classification was established by using the software of GeneANOVA and tested by a χ(2) test. RESULTS Forty-seven tumors were studied: 25 with early recurrence vs. 22 with late or null recurrence. These 2 groups of tumors have a significantly different expression profile in 3 genes among the 110-gene expression profile: peroxysome proliferator-activated receptor-γ (PPARG) (P = 0.031), stathmin-1 (STMN1) (P = 0.041), Caveolin-2 (CAV2) (P = 0.004). The combination of the expression of these 3 genes was significantly predictive for early recurrence leading to a correct hierarchical classification of the NMI-BC regarding the time-to-recurrence (P < 0.001). CONCLUSIONS This study identifies a concise 3-gene panel that is associated with time to recurrence. This 3-gene signature has to be confirmed in a prospective study and in larger cohort of patients. However, our preliminary results are promising and gene expression profiling seems to be an interesting approach in cTa-T1 tumors for recurrence prediction.

Principe et applications de la microdissection laser en histopathologie

Resume Les tissus biologiques normaux sont constitues de differentes populations de cellules adoptant une architecture souvent tres intriquee. Ceci entraine une grande heterogeneite dans la composition de chaque tissu. La microdissection laser permet, sous controle de la vue et de l’expertise du pathologiste, de selectionner une population ou une region cellulaire d’interet sur tous types de prelevements tissulaires. Elle repond ainsi au besoin de s’affranchir de l’heterogeneite cellulaire caracterisant aussi les tissus cancereux ou sont disseminees cellules stromales, cellules precancereuses, cellules cancereuses et cellules normales residuelles. Grâce aux developpements technologiques qui n’ont fait que croitre durant la decennie qui vient de s’ecouler dans le domaine de la biologie moleculaire, cette technique a permis de preciser le role important des acides nucleiques (ADN et ARN) et de certaines proteines qualifiees de marqueurs biologiques dans des mecanismes physiopathologiques. C’est en pathologie tumorale que la microdissection laser demontre a l’heure actuelle ses performances dans de nombreux champs d’application dont l’interet sera ici precise.

p53 mutations to predict efficacy of alkylating-containing regimen: a metaanalysis of four different clinical trials.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6064 Background. The predictive value of p53 mutations for efficacy of anthracycline-based chemotherapy is matter of controversy. Inconsistencies among studies could be related to the heterogeneous use of alkylating agents in combination with anthracyclines in different studies. We examined the predictive value of p53 mutations in four different series of breast cancer patients treated with preoperative anthracycline-based chemotherapy including different doses of cyclophosphamide (C). Patients and Methods. All patients had stage II-III breast cancer and received anthracycline-containing chemotherapies. A total of 352 patients were included in four different clinical studies : 65 patients with estrogen receptor (ER)-negative cancers treated with single agent epirubicine (E) (100 mg/m2/3w x 4) in the TOP trial (R1), 52 patients treated with FAC (500 mg/m2 C, 5-FU and 50 mg/m2 doxorubicin/3w x 6) (R2), 96 patients treated with EC-T (75mg/m2 E and 750 mg/m2 C/3w x 4 followed by 100mg/m2 docetaxel/3w x 4) (R3) and 139 patients treated with dose-dense EC (1200mg/m2 C and 75 mg/m2 E/2w x 6) (R4). Before therapy, p53 status was determined in all tumors by yeast functional complementation (FASAY) assay. After chemotherapy, all patients underwent surgery. Pathologic complete response (pCR) was defined as no residual invasive tumour cells in breast and lymph nodes. Results. P53 mutations were more frequently observed in the 128 ER-neg compared to ER-pos cancers (78% vs 29.4%). In p53 mutated tumours, the pCR rate increased with the dose-intensity of C. Conversely, in p53 wild type tumours, pCR rates decreased. ![][1] Focusing on ER-neg, p53 mutated tumors, the pCR rates rose from 11%, 6%, 32% to 52% in R1, R2, R3 and R4 groups. The R1 and R4 regimen, differing only in C dose intensity, showed marked differences in pCR in ERneg tumors. ![][2] Conclusions: Increasing doses of C do not seem to improve pCR rates in P53 wild type tumours, raising the possibility of antagonism with anthracycline in this group. On the other hand, in ER-neg, p53 mutant tumors, inclusion of dose-intense C seems to significantly increase pCR rates. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6064. [1]: /embed/graphic-1.gif [2]: /embed/graphic-2.gif

Consistent TP53 mutations in BRCA1 and sporadic basal-like breast tumors, while infrequent in luminal BRCA1 tumors.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4070 Breast tumors with a germline mutation of BRCA1 ( BRCA1 tumors) and Basal-Like Carcinoma (BLC) have been shown to be associated with a high rate of TP53 mutations. Because it has also been shown that BRCA1 tumors frequently display a basal-like phenotype, we sought to determine whether TP53 mutations were correlated to the hereditary BRCA1 mutated status or to the particular histological type of these tumors. We first explored the status of the TP53 gene in a series of 28 BRCA1 BLCs using immunohistochemistry, direct sequencing of the coding sequence, and functional assay in yeast (FASAY), and compared it to the status of TP53 in 27 series of sporadic (non hereditary) BLCs. With this sensitive approach, TP53 was found consistently mutated in BRCA1 (28/28) and sporadic (26/27) BLCs. However, mutation spectrum was different, in particular with a much higher rate of insertion/deletion in BRCA1 BLCs (12/27) than in the sporadic group (2/25). Secondly, we analyzed the incidence of TP53 mutations in 10 BRCA1 luminal tumors, using the same strategy. Surprisingly, only 3 of these 10 tumors were mutated, a frequency similar to that found in matched sporadic luminal tumors. In conclusion, TP53 mutation is a mandatory event in BRCA1 or sporadic BLCs, but not a common feature of BRCA1 luminal tumors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4070.