Tumor metabolism assessed by FDG-PET/CT and tumor proliferation assessed by genomic grade index to predict response to neoadjuvant chemotherapy in triple negative breast cancer.

PURPOSE: Survival is increased when pathological complete response (pCR) is reached after neoadjuvant chemotherapy (NAC), especially in triple-negative breast cancer (TNBC) patients. Positron emission tomography/computed tomography (PET/CT) with 18F-fluorodeoxyglucose (FDG) and the genomic grade index (GGI), each separately, showed good potential to predict pCR. Our study was designed to evaluate the predictive value for the therapeutic response of a combination of parameters based on FDG-PET, histoclinical features and molecular markers of proliferation. METHODS: Molecular parameters were measured on pre-treatment biopsy. Tumor metabolic activity was measured using two PET/CT scans, one before and one after 2 cycles of NAC. The pCR was determined on specimen after NAC. Event-free survival (EFS) was estimated using the Kaplan Meier method. RESULTS: Of 55 TNBC patients, 19 (35%) reached pCR after NAC. Tumor grade and Ki67 were not associated with pCR whereas GGI (P = 0.04) and its component KPNA2 (P = 0.04) showed a predictive value. The change of FDG uptake between PET1 and PET2 (ΔSUVmax) was highly associated with pCR (P = 0.0001) but the absolute value of baseline SUVmax was not (P = 0.11). However, the AUC of pCR prediction increased from 0.63 to 0.76 when baseline SUVmax was combined with the GGI (P = 0.016). The only two parameters associated with EFS were ΔSUVmax (P = 0.048) and pathological response (P = 0.014). CONCLUSIONS: The early tumor metabolic change during NAC is a powerful parameter to predict pCR and outcome in TNBC patients. The GGI, determined on pretreatment biopsy, is also predictive of pCR and the combination GGI and baseline SUVmax improves the prediction.

BIRC5 (survivin): a pejorative prognostic marker in stage II/III breast cancer with no response to neoadjuvant chemotherapy.

PURPOSE: Neoadjuvant systemic therapy (NAC) is currently used in the treatment of stage II/III breast cancer. Pathological complete response as a surrogate endpoint for clinical outcomes is not completely validated for all subgroups of breast cancers. Therefore, there is a need for reliable predictive tests of the most effective treatment. METHODS: We used a combination of predictive clinical, pathological, and gene expression-based markers of response to NAC in a prospective phase II multicentre randomized clinical trial in breast cancer patients, with a long follow-up (8 years). This study concerned the subpopulation of 188 patients with similar levels of pathological response rates to sequential epirubicin/cyclophosphamide and docetaxel to determine predictive marker of pCR and DFS. We used a set of 45 genes selected from high throughput analysis and a standardized RT-qPCR. We analyzed the predictive markers of pathological complete response (pCR) and DFS in the overall population and DFS the subpopulation of 159 patients with no pCR. RESULTS: In the overall population, combining both clinical and genomic variables, large tumor size, low TFF1, and MYBL2 overexpression were significantly associated with pCR. T4 Stage, lymphovascular invasion, negative PR status, histological type, and high values of CCNB1 were associated with DFS. In the no pCR population, only lymphovascular invasion and high values of BIRC5 were associated with DFS. CONCLUSIONS: We confirm the importance of ER-related and proliferation genes in the prediction of pCR in NAC-treated breast cancer patients. Furthermore, we identified BIRC5 (survivin) as a main pejorative prognostic factor in patients with breast cancers with no pCR. These results also open perspective for predictive markers of new targeted therapies.

Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network.

BACKGROUND: There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. PATIENTS AND METHODS: EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. RESULTS: Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12-24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08-0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6-21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). CONCLUSIONS: Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment.

Challenging single- and multi-probesets gene expression signatures of pathological complete response to neoadjuvant chemotherapy in breast cancer: experience of the REMAGUS 02 phase II trial.

This study was designed to identify predictive signatures of pathological complete response (pCR) in breast cancer treated by taxane-based regimen, using clinicopathological variables and transcriptomic data (Affymetrix Hgu133 Plus 2.0 devices). The REMAGUS 02 trial (n = 153,training set) and the publicly available M.D. Anderson data set (n = 133, validation set) were used. A re-sampling method was applied. All predictive models were defined using logistic regression and their classification performances were tested through Area Under the Curve (AUC) estimation. A stable set of 42 probesets (31 genes) differentiate pCR or no pCR samples. Single-or 2-probesets signatures, mainly related to ER pathway, were equally predictive of pCR with AUC greater then 0.80. Models including probesets associated with ESR1, MAPT, CA12 or PIGH presented good classification performances. When clinical variables were entered into the model, only CA12 and PIGH, remained informative (p = 0.05 and p = 0.005) showing that a combination of a few genes provided robust and reliable prediction of pCR.

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.

[Cell signalling and cancer: characterisation of therapeutic targets]. / Signalisation cellulaire et cancer: caractérisation de cibles thérapeutiques.

Cellular communication is required for the life of pluricellular organisms. The informations exchanged between cells belong to six major types of order to be executed, opposite each other: proliferate or differentiate; remain attached or migrate; survive or die. The cancer cell is genetically unstable, able to explore all the functions encoded by the genome and to consider every proliferative or migratory advantage for selecting it and transmit it to its descent. All the signalling pathways involved in proliferation or differentiation, in adhesion and migration, in survival and death may be altered by oncogenic alterations. These alterations are precisely those which can be targeted for therapy: from this observation was forged the concept of targeted therapy. We present here some examples of therapeutic targeting at the level of a major proliferation pathway by showing how it was possible to identify and characterise relevant targets, invent original new therapeutic tools and decipher the mechanisms of resistance which occur and hinder the success of targeted therapies. This example is the proliferation signalling pathway which starts from the activation of tyrosine kinase receptors by cognate growth factors and ends by the activation of transcription factors which trigger the transcription of the genes required for DNA replication, after undergoing through numerous intermediate molecules constituting the MAP kinase pathway: RAS, RAF, MEK and ERK.

Three different KRAS statuses in three synchronous colorectal cancers.

Treatments with monoclonal antibodies targeting the epidermal growth factor receptors (EGFR) have improved the prognosis of metastatic colorectal cancer (CRC). Mutated KRAS status is predictive of resistance to anti-EGFR agents and allows the selection of KRAS wild-type patients who may benefit from these targeted therapies. We report an original case of metastatic CRC including three synchronous primary tumors with three different KRAS statuses. We discuss the possible therapeutic impact of this clinical case and the role of multiple samplings for KRAS status determination.

Modeling of response to endocrine therapy in a panel of human luminal breast cancer xenografts.

Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient's tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

[Molecular prognostic and predictive markers of breast cancer treatment]. / Marqueurs pronostiques et prédictifs de réponse aux traitements des cancers du sein.

Breast cancer heterogeneity has been deciphered during the last decade thanks to the use of high-throughput tools. A major clinical concern is the determination of the metastatic risk of the tumours (prognostic factor), but also the optimal choice of the treatment for a given patient and tumour (predictive factor). A significant advance has been obtained from the description of a novel molecular classification of breast cancers. This allowed a refinement in the determination of tumour groups displaying different prognoses. Presently, numerous gene signatures have been published and some of them are on the market, at least in the United States, but prospective validation studies are still ongoing. After a major enthusiasm, numerous questions have been raised, concerning especially the stability of these signatures. An improved knowledge of these limits is also an important factor for an optimal use of these data. It can be concluded that it should be possible to improve patients' care by integrating molecular and clinicopathologic data in a common approach on the one hand, and to further develop new biomarkers predictive of therapeutic efficacy on the other hand.

Establishment and characterisation of a new breast cancer xenograft obtained from a woman carrying a germline BRCA2 mutation.

BACKGROUND: The BRCA2 gene is responsible for a high number of hereditary breast and ovarian cancers, and studies of the BRCA2 biological functions are limited by the lack of models that resemble the patient's tumour features. The aim of this study was to establish and characterise a new human breast carcinoma xenograft obtained from a woman carrying a germline BRCA2 mutation. METHODS: A transplantable xenograft was obtained by grafting a breast cancer sample into nude mice. The biological and genetic profiles of the xenograft were compared with that of the patient's tumour using histology, immunohistochemistry (IHC), BRCA2 sequencing, comparative genomic hybridisation (CGH), and qRT-PCR. Tumour response to standard chemotherapies was evaluated. RESULTS: Histological profile identified the tumour as a basal-like triple-negative breast cancer. Targeted BRCA2 DNA sequencing of the xenograft showed the presence of the mutation previously identified in the carrier. Comparative genomic hybridisation array profiles of the primary tumour and the xenograft revealed a high number of similar genetic alterations. The therapeutic assessment of the xenograft showed sensitivity to anthracyclin-based chemotherapy and resistance to docetaxel. The xenograft was also highly sensitive to radiotherapy and cisplatin-based treatments. CONCLUSIONS: This study describes a new human breast cancer xenograft obtained from a BRCA2-mutated patient. This xenograft provides a new model for the pre-clinical drug development and for the exploration of the drug response biological basis.

[Biomarkers of endocrine therapy-resistance in breast cancers]. / Marqueurs biologiques de résistance à l'hormonothérapie dans les cancers du sein.

During the past decade, molecular signatures allowed a better classification of breast carcinoma and a better evaluation of their prognosis. However, we still need predictive factors of treatment and /or prognosis factors specific of each patient. Regarding hormonal therapy, expression of hormone receptors is essential, but not sufficient to accurately predict response to treatment for all patients. To date, numerous data have identified different pathways of resistance to hormonal treatment, associated with heterogeneity in response to long term treatment. The challenge remains to identify and to anticipate these changes for each patient.

Clinical value of circulating endothelial cells and circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and chemotherapy.

BACKGROUND: We investigated whether circulating tumor cells (CTCs) and circulating endothelial cells (CECs) predict clinical outcome of first-line chemotherapy combined with bevacizumab in metastatic breast cancer patients. PATIENTS AND METHODS: In a French substudy of the MO19391 trial, CTC and CEC counts (CellSearch system) at baseline and changes after two cycles of treatment were correlated with time to progression (TtP). RESULTS: CTC and CEC levels were not correlated in the 67 patients included. At baseline, CTC positivity was a significant prognostic marker for TtP at a threshold of 3 CTC/7.5 ml (P < 0.05) but not at 5 CTC/7.5 ml (P = 0.09). Baseline CEC levels (median 17 CEC/4 ml, range 1-769) were associated with age > or =45 years (P = 0.01), elevated lactate dehydrogenase (P < 0.01) and not with TtP at any threshold. Changes of CTC count during treatment were not a surrogate of TtP, with any of the model tested (threshold based or relative decrease in percent). However, increase in CEC count was associated with improved TtP, at the threshold of 20 CEC/4 ml (P < 0.01). CONCLUSION: Bevacizumab combined with first-line chemotherapy may modify the predictive value of CTC during treatment possibly due to impaired tumor cells intravasation through vessels endothelium. Variations in CEC levels appear to be a promising early surrogate marker of TtP under antiangiogenic treatment.

Single circulating tumor cell detection and overall survival in nonmetastatic breast cancer.

BACKGROUND: Circulation of cancer cells in the blood is a mandatory step for metastasis, but circulating tumor cells (CTC) have a low metastatic efficiency in preclinical animal models. In this prospective study, we reported the clinical outcome of nonmetastatic breast cancer patients according to CTC detection. PATIENTS AND METHODS: In 115 nonmetastatic patients diagnosed with large operable or locally advanced breast cancer, we prospectively detected CTC using the CellSearch system before and after neoadjuvant chemotherapy in a phase II trial (REMAGUS02). RESULTS: At baseline, 23% of patients were CTC positive, but only 10% had >1 CTC/7.5 ml of blood. After a median follow-up of 36 months, CTC detection before chemotherapy was an independent prognostic factor for both distant metastasis-free survival [DMFS; P = 0.01, relative risk (RR) = 5.0, 95% confidence interval (CI) 1.4-17] and overall survival (OS; P = 0.007, RR = 9, 95% CI 1.8-45). CTC detection after chemotherapy was of less significance (P = 0.07 and 0.09, respectively). Moreover, CTC detection showed interesting characteristics as an individual predictive test for metastatic relapses (sensibility 55%, specificity 81%, and global accuracy 77%). CONCLUSIONS: Detection of > or =1 CTC/7.5 ml before neoadjuvant chemotherapy can accurately predict OS. Our findings may change the clinical management of nonmetastatic breast cancer and indicate that the metastatic efficiency of CTC could be higher than previously reported.

[Preoperative concomitant radiochemotherapy in bulky carcinoma of the cervix: Institut Curie experience]. / Chimioradiothérapie concomitante préopératoire dans les carcinomes du col utérin de stades IB2 à IIB : expérience de l'institut Curie.

PURPOSE: To evaluate the treatment results of patients (pts) with Figo stage IB2, IIA, IIB cervical carcinoma (CC) treated with preoperative radiochemotherapy, followed by extended radical hysterectomy. PATIENTS AND METHODS: Retrospective study of 148 women treated at the Institut Curie for operable Figo Stage IB2 to IIB, biopsy proved CC. Among them, 70 pts, median age 46 years, were treated using the same regimen associating primary radiocisplatinum based chemotherapy, intracavitary LDR brachytherapy, followed by extended radical hysterectomy. Kaplan-Meier estimates were used to draw survival curves. Comparisons of survival distribution were assessed by the log-rank test. RESULTS: Complete histological local-regional response was obtained in 56% of the pts (n=39). Residual macroscopic or microscopic disease in the cervix was observed in 28 pts (40%). All but one had in situ microscopic residual CC. Lateral residual disease in the parametria was also present in nine pts, all with residual CC. Pelvic lymph nodes were free from microscopic disease in 56 pts (80%). Eight of 55 (11%) radiological N0 patients had microscopic nodal involvement, as compared to 6/15 (40%) radiological N1 (p=0.03). Seventeen pts (25%) had residual cervix disease but negative nodes. After median follow-up of 40 months (range, 8-141), 38/70 patients (54.1%) are still alive and free of disease, six (8.6%) alive with disease, and 11 (15.8%) patients were lost for follow-up but free of disease. CONCLUSION: The treatment of locally advanced CC needs a new multidisciplinary diagnostic and treatment approach using new therapeutic arms to improve the survival and treatment tolerance among women presenting this disease.

Potentiation of antitumour activity of docetaxel by combination with trastuzumab in a human prostate cancer xenograft model and underlying mechanisms.

Antitumour activity of docetaxel (Taxotere) in hormone-dependent (HD) and hormone-independent (HID) prostate cancer PAC120 xenograft model was previously reported, and its level was associated with HER2 protein expression. In the present study, we evaluate the antitumour effects of docetaxel combined with trastuzumab (Herceptin), an anti-HER2 antibody. Although trastuzumab alone had no effect on tumour growth, it potentiated the antitumour activity of docetaxel in HD tumours and more strongly in HID variants. Using the HID28 variant, we show that docetaxel treatment of tumour-bearing mice induces an increased HER2 mRNA expression of the tyrosine kinase receptor of 25-fold 24 h after docetaxel treatment, while HER2 protein and p-AKT decreased. This was followed by an increase of HER2 protein 3 days (two-fold) after docetaxel treatment and by a strong HER2 release in the serum of treated mice; expression of phospho-ERK, p27, BCL2 and HSP70 concomitantly increased. Similar molecular alterations were induced by docetaxel plus trastuzumab combination, except for that there was a transient and complete disappearance of AR and HSP90 proteins 24 h after treatment. We show that in addition to its known effects on tubulin and mitotic spindles, docetaxel induces complex signalisation pathway mechanisms in surviving cells, including HER2, which can be pharmacologically targeted. This study suggests that the docetaxel/trastuzumab combination may prove an effective therapeutic approach for HER2-expressing hormone-refractory prostate cancer.

Estrogen receptors evolution in neoadjuvant aromatase inhibitor (AI) therapy for breast cancer in elderly women: stability of hormonal receptor expression during treatment.

UNLABELLED: In France, 20% of breast cancers occur in women over the age of 70 and 10% in women over the age of 80. As these women are not included in screening programs, breast cancer is often diagnosed later, at the stage of a large tumor. PURPOSE: To analyze clinical response, possibilities of conservative treatment and course of hormonal receptors in patients receiving neoadjuvant aromatase inhibitor (AI) therapy for at least 6 months. PATIENTS AND METHODS: There were 75 patients, with a mean age of 75 +/- 8 years (range, 58-91 years) received AI for 6 months after the diagnosis of invasive breast cancer with positive hormonal receptors. Clinical and radiologic tumor reduction, the number of conservative treatments and the course of estrogens receptor-labeled cells were determined for each patient. RESULTS: All but 1 of these patients obtained clinical reduction of their tumor. Of these, 86% patients received conservative treatment. In the majority of patients, estrogen receptor (ER) level did not vary between the initial assay and analysis of the operative specimen. DISCUSSION AND CONCLUSION: Aromatase inhibitors are effective as neoadjuvant therapy in ER positive elderly patients with large tumors, as is tamoxifen. Changes in hormone receptor expression during treatment do not predict clinical response. In our experience, neoadjuvant AI therapy should be administered for at least 6 months to optimize clinical response before deciding upon surgery. Discrepancy observed in the literature could be explained by the duration of the treatment.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.