M. Marty

Effect of mutated TP53 on response of advanced breast cancers to high-dose chemotherapy

TP53 activation by genotoxic drugs can induce apoptosis or cell-cycle arrest. Thus, whether the gene is mutated or wild type could affect the response of a tumour to chemotherapy. Clinical data are unclear, possibly as a result of heterogeneity of tumours, drugs, methods of assessing response, or TP53 status. We studied 50 non-inflammatory, locally advanced breast cancers that had been treated with high doses of a combination of epirubicin and cyclophosphamide. We noted eight complete responses, which all occurred in the 14 patients with tumours containing mutated TP53 (p<0.0001). In high-grade, advanced breast cancers, inactivation of the TP53 pathway could greatly improve the response to this chemotherapy regimen.

p53 mutations and overexpression in locally advanced breast cancers.

Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (chi 2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (chi 2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

Chemotherapy for metastatic breast cancer–report of a European expert panel

The anthracyclines doxorubicin and epirubicin, and the taxanes paclitaxel and docetaxel, are effective chemotherapeutic agents for the first-line and second-line treatment of metastatic breast cancer, and their clinical use is widespread. However, for women whose disease has progressed despite receiving these drugs, treatment options are limited. These women often have a good performance status, and may survive for many months or even years, so they should be given the opportunity to benefit from further chemotherapy. The goals of chemotherapy in these patients are to obtain maximum control of symptoms, prevent serious complications, and increase survival without diminishing quality of life. Several agents are used for this purpose, including fluorouracil, docetaxel (in patients who have already received paclitaxel), vinorelbine, and mitomycin c, but because data from controlled trials are limited, a standard regimen has not yet been established. Moreover, these agents may be inconvenient to administer and can be associated with adverse events requiring hospitalisation. Therefore, there is a clear need for additional therapeutic options for patients with metastatic breast cancer. Ideally, agents should have a convenient method of administration, eg, oral, and should be suitable for home-based rather than hospital-based therapy. Treatment should control disease in at least 20-30% of patients with an acceptable side-effect profile. Novel oral therapies have now been developed and are being used increasingly in patients whose disease has progressed following taxane therapy.

Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab

Background:Phosphatidylinositol 3-kinase (PI3K) pathway activation has been suggested to negatively influence response to anti-HER2 therapy in breast cancer patients. The present study focused on mutations of the PIK3CA gene, encoding one of the two PI3K subunits.Methods:PIK3CA mutations were assessed by direct sequencing in 80 HER2-positive patients treated with 1 year of trastuzumab. All patients preoperatively received four cycles of anthracycline-based chemotherapy, followed by four cycles of docetaxel and 1 year of trastuzumab, starting either before surgery with the first cycle of docetaxel and continuing after surgery (neoadjuvant trastuzumab arm, n=43), or only after surgery (adjuvant trastuzumab arm, n=37).Results:PIK3CA mutations were found in 17 tumours (21.3%). Better disease-free survival (DFS) was observed in patients with PIK3CA wild-type compared with mutated tumours (P=0.0063). By combining PIK3CA status and treatment arms, four separate prognostic groups with significantly different DFS (P=0.0013) were identified.Conclusion:These results confirm that the outcome of HER2-positive patients treated with trastuzumab is significantly worse in patients with PIK3CA-mutated compared with wild-type tumours.

HER2-overexpressing breast cancer: FDG uptake after two cycles of chemotherapy predicts the outcome of neoadjuvant treatment.

Background:Pathologic complete response (pCR) to neoadjuvant treatment (NAT) is associated with improved survival of patients with HER2+ breast cancer. We investigated the ability of interim positron emission tomography (PET) regarding early prediction of pathology outcomes.Methods:During 61 months, consecutive patients with locally advanced or large HER2+ breast cancer patients without distant metastases were included. All patients received NAT with four cycles of epirubicin+cyclophosphamide, followed by four cycles of docetaxel+trastuzumab. 18F-fluorodeoxyglucose (18F-FDG)-PET/computed tomography (CT) was performed at baseline (PET1) and after two cycles of chemotherapy (PET2). Maximum standardised uptake values were measured in the primary tumour as well as in the axillary lymph nodes. The correlation between pathologic response and SUV parameters (SUVmax at PET1, PET2 and ΔSUVmax) was examined with the t-test. The predictive performance regarding the identification of non-responders was evaluated using receiver operating characteristics (ROC) analysis.Results:Thirty women were prospectively included and 60 PET/CT examination performed. At baseline, 22 patients had PET+ axilla and in nine of them 18F-FDG uptake was higher than in the primary tumour. At surgery, 14 patients (47%) showed residual tumour (non-pCR), whereas 16 (53%) reached pCR. Best prediction was obtained when considering the absolute residual SUVmax value at PET2 (AUC=0.91) vs 0.67 for SUVmax at PET1 and 0.86 for ΔSUVmax. The risk of non-pCR was 92.3% in patients with any site of residual uptake >3 at PET2, no matter whether in breast or axilla, vs 11.8% in patients with uptake ⩽3 (P=0.0001). The sensitivity, specificity, PPV, NPV and overall accuracy of this cutoff were, respectively: 85.7%, 93.8%, 92.3%, 88.2% and 90%.Conclusion:The level of residual 18F-FDG uptake after two cycles of chemotherapy predicts residual disease at completion of NAT with chemotherapy+trastuzumab with high accuracy. Because many innovative therapeutic strategies are now available (e.g., addition of a second HER2-directed therapy or an antiangiogenic), early prediction of poor response is critical.

Evidence of chromosome regions and gene involvement in inflammatory breast cancer

Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. In contrast to noninflammatory breast cancer (non IBC), the molecular alterations underlying IBC are poorly known. We postulated that the kind and frequency of these alterations might differ between IBC and non IBC and account for its particular aggressiveness. We investigated allelic losses associated with primary breast cancer (on chromosome arms 1p, 3p, 6p, 6q, 7q, 8p, 9p, 11p, 11q, 16q, 17p and 17q) by analyzing 71 microsatellite markers in 66 cases of IBC. Loss of heterozygosity (LOH) was frequent, with a mean fractional allelic loss (FAL) index of 52%. Relative to published data on non IBC, allelic loss was particularly frequent at 3p21‐p14, 6p, 8p22, 11q, 13q14 and 17q21, suggesting the presence of genes that are markedly altered in IBC. In contrast, the DNA amplification levels of ERBB2, MYC and CCND1, as measured by real‐time quantitative PCR, did not differ between IBC and non IBC.

Long-term survival of advanced triple-negative breast cancers with a dose-intense cyclophosphamide/anthracycline neoadjuvant regimen.

Background:Triple-negative (TN) breast cancers exhibit major initial responses to neoadjuvant chemotherapy, but generally have a poor outcome. Because of the lack of validated drug targets, chemotherapy remains an important therapeutic tool in these cancers.Methods:We report the survival of two consecutive series of 267 locally advanced breast cancers (LABC) treated with two different neoadjuvant regimens, either a dose-dense and dose-intense cyclophosphamide–anthracycline (AC) association (historically called SIM) or a conventional sequential association of cyclophosphamide and anthracycline, followed by taxanes (EC-T). We compared pathological responses and survival rates of these two groups and studied their association with tumours features.Results:Although the two regimens showed equivalent pathological complete response (pCR) in the whole population (16 and 12%), the SIM regimen yielded a non-statistically higher pCR rate than EC-T (48% vs 24%, P=0.087) in TN tumours. In the SIM protocol, DFS was statistically higher for TN than for non-TN patients (P=0.019), although we showed that the TN status was associated with an increased initial risk of recurrence in both regimens. This effect gradually decreased and after 2 years, TN was associated with a significantly decreased likelihood of relapse in SIM-treated LABC (hazard ratio (HR)=0.25 (95% CI: 0.07–0.86), P=0.028).Conclusions:AC dose intensification treatment is associated with a very favourable long-term survival rate in TN breast cancers. These observations call for a prospective assessment of such dose-intense AC-based regimens in locally advanced TN tumours.

Cowden disease and Lhermitte Duclos disease, markers of breast carcinoma: Report of two patients

Large data base registries have shown that up to 20% of women with breast cancer have one affected relative and estimate that 5% of breast cancers are attributable to major genes such as BRCA1 or BRCA2 [1]. The management of familial breast cancers requires a specific approach, aiming at early diagnosis, familial surveillance, and specific treatments adjusted to their specific natural history. BRCA1 and BRCA2 linked families have been identified as the commonest genetic condition predisposing to breast cancer but their treatment remain controvertial. Other inherited conditions have been recently identified as risk factors for breast carcinoma. Among them, genodermatosis are of specific interest. These inherited syndromes characterized by hamartomas of ectodermal, endodermal or mesodermal origin affect multiple organs. Most of them have an autosomal dominant pattern of inheritance. The increased incidence of carcinomas reported in these patients gives rise to specific interest. Cowdens disease (CD) and Lhermitte Duclos disease (LDD) are of particular interest, not only for their multisystem abnormalities, but also for their neoplastic risk. CD was initially described in 1962 by Llyod and Denys [2]. Skin, gingival, or digestive papules are considered pathognomonic but thyroid, breast, and the central nervous system can also be involved and need close monitoring [3]. As neurological abnormalities produce non specific symptoms, they appear underdiagnosed. Hydrocephaly, craniomegaly, epilepsy, intracranial hypertension, cerebellar syndrome or LD syndrome have been reported. LDD is a congenital benign cerebellar hamartoma described as a purkinjenoma or a gangliocytoma by Lhermitte and Duclos [4]. The occurrence of fullblown CD with thyroid, digestive or breast lesions in patients with LDD can not be considered as fortuitous. A strong linkage between these two phacomatosis have been commonly accepted. This close association should be recognized and has led to further genetic analysis, resulting in the identification of the Cowdens gene. We report two patients with breast cancer and Cowden or Lhermitte Duclos disease. We will discuss these two congenital disorders, their features, the risk of cancer and recent advances in genetic findings.

Circulating tumor cell detection and transcriptomic profiles in early breast cancer patients

In the early 2000s, studies using high-throughput transcriptomic analyses revealed different aspects of breast cancer biology. Several outcome-based predictors and biologybased classifiers have been proposed: invasiveness gene signature (IGS), intrinsic molecular subtype, wound-response signature, etc. Recently, circulating tumor cell (CTC) cytological detection has been associated with metastasis-free and overall survival in the REMAGUS02 neoadjuvant trial [1]. In this article, we report, for the first time, the transcriptomic analysis of 60 nonmetastatic breast cancers according to CTC detection. The REMAGUS02 trial included breast cancer patients with locally advanced or large breast cancer. CTC detection was carried out at baseline and at the end of chemotherapy using the CellSearch system (Veridex, Raritan, NJ). Total RNA extracted from the pretreatment cancer biopsy was hybridized on the GeneChip Human Genome U1331 2.0 Array (Affymetrix, Santa Clara, CA). Conditions of the sampling procedures and RNA quality control are detailed elsewhere [2]. The genechip robust multi-array average procedure [3] was used to normalize the gene expression data. A hierarchical clustering was then carried out using the 10 000 probe sets that showed the highest values in the interquartile range (IQR). We applied the intrinsic gene set on the complete dataset of 60 samples to define the molecular subtypes and the IGS. We used the defined and validated centroids of 306 genes to discriminate between previously identified molecular breast cancer subtypes. We matched the probe list UniGene ID (Build#204) to the GeneChip Human Genome U133A 2.0 Array, resulting in a list of 294 unique probe sets. Each sample was assigned to the nearest subtype/centroid as determined by the highest Spearman rank order correlation between the gene expression values of the 294 probe sets and the 5 subtype centroids. The IGS score was determined by calculating the Pearson correlation between the probe set expression values of each sample and the 110 reference expression levels of the same genes defining the IGS signature. Single probe set analyses were carried out using the Wilcoxon rank sum test. Finally, because half of the probe sets showed the highest IQR values, a significance analysis of microarray (SAM) was proposed to detect differentially associated genes (DEG) between CTCpositive and -negative patients. At first, unsupervised clustering revealed three major tumor clusters; they unsurprisingly corresponded to triplenegative, human epithelial growth factor receptor 2(HER2)positive/estrogen receptor (ER)-negative, and ER-positive breast cancer immunohistological phenotypes. CTC detection was not statistically different among these three clusters (Figure 1). Breast cancers were then classified according to selected biology-based signatures: the intrinsic subtype classifier and IGS, which is stemness related. CTC detection was not statistically different among subgroups: basal, n = 6/18 (33%); HER2, n = 3/10 (33%); luminal A, n = 2/15 (13%); luminal B, n = 2/10 (20%); normal like, n = 2/7 (29%); low-risk IGS, n = 9/27 (33%); and high-risk IGS, n = 6/33 (18%). Single probe set analyses were then carried out on candidate genes that are directly involved in CTC detection by the CellSearch system (cytokeratin 8, 18, 19, and EpCAM) or that are surrogate markers for breast cancer stem cells (CD24, CD44, ALDH1A1): messenger RNA levels were not correlated with CTC positivity. In addition, at the high false discovery rate of 30%, only 18 DEG were found using a SAM procedure. This is the first study, to date, to correlate CTC detection in nonmetastatic breast cancer with the gene expression profile of the primary tumor. We did not confirm previously published in vitro experiments that suggested that normal-like

High-dose epirubicin and cyclophosphamide every two weeks as first-line chemotherapy for relapsing metastatic breast cancer patients

BACKGROUND Metastatic breast cancer remains incurable with conventional chemotherapy. For any specific chemotherapy, higher dose intensity may be achieved with either increased doses per cycle, or shortened intervals between courses, or both. We demonstrate here the feasibility and encouraging results of a high-dose combination regimen administered every two weeks. PATIENTS AND METHODS Women with metastatic breast cancer were treated every 14 days for 6 courses with 75 mg/m2 epirubicin and 1200 mg/m2 cyclophosphamide, followed by conventionally-delivered (q 3-4 weeks) chemotherapy. The treatment was to be resumed regardless of the neutrophil count, except in instances of febrile neutropenia. Prophylactic oral antibiotherapy was given, while hematopoietic growth factors and stem cell support were not employed. RESULTS Eighty-six patients were treated between May 1986 and June 1995. Their median age was 43 years (26-69). Grade 3-4 neutrophil toxicity was observed after 86% of the courses, resulting in febrile neutropenia in 5%-18% of the patients, and the rehospitalization of 5%-10%. The median given/planned dose intensity was 97% (79-106). The objective response rate in 84 evaluable patients was 54% (95% confidence interval (95% CI): 43-65), with a complete response rate of 11%, and a 14% rate of outright progression. Median progression-free survival was 16 months and median overall survival 32 months. Multivariate analysis retained previous adjuvant chemotherapy as a negative survival prognostic factor. CONCLUSIONS This dose-intensive anthracycline-based regimen is feasible with manageable morbidity despite pronounced myelotoxicity, and yields encouraging survival rates.