Sébastien Salas

Gene expression profiling of colon cancer by DNA microarrays and correlation with histoclinical parameters

Different diagnostic and prognostic groups of colorectal carcinoma (CRC) have been defined. However, accurate diagnosis and prediction of survival are sometimes difficult. Gene expression profiling might improve these classifications and bring new insights into underlying molecular mechanisms. We profiled 50 cancerous and noncancerous colon tissues using DNA microarrrays consisting of ∼8000 spotted human cDNA. Global hierarchical clustering was to some extent able to distinguish clinically relevant subgroups, normal versus cancer tissues and metastatic versus nonmetastatic tumours. Supervised analyses improved these segregations by identifying sets of genes that discriminated between normal and tumour tissues, tumours associated or not with lymph node invasion or genetic instability, and tumours from the right or left colon. A similar approach identified a gene set that divided patients with significantly different 5-year survival (100% in one group and 40% in the other group; P=0.005). Discriminator genes were associated with various cellular processes. An immunohistochemical study on 382 tumour and normal samples deposited onto a tissue microarray subsequently validated the upregulation of NM23 in CRC and a downregulation in poor prognosis tumours. These results suggest that microarrays may provide means to improve the classification of CRC, provide new potential targets against carcinogenesis and new diagnostic and/or prognostic markers and therapeutic targets.

Validated prediction of clinical outcome in sarcomas and multiple types of cancer on the basis of a gene expression signature related to genome complexity

Sarcomas are heterogeneous and aggressive mesenchymal tumors. Histological grading has so far been the best predictor for metastasis-free survival, but it has several limitations, such as moderate reproducibility and poor prognostic value for some histological types. To improve patient grading, we performed genomic and expression profiling in a training set of 183 sarcomas and established a prognostic gene expression signature, complexity index in sarcomas (CINSARC), composed of 67 genes related to mitosis and chromosome management. In a multivariate analysis, CINSARC predicts metastasis outcome in the training set and in an independent 127 sarcomas validation set. It is superior to the Fédération Francaise des Centres de Lutte Contre le Cancer grading system in determining metastatic outcome for sarcoma patients. Furthermore, it also predicts outcome for gastrointestinal stromal tumors (GISTs), breast carcinomas and lymphomas. Application of the signature will permit more selective use of adjuvant therapies for people with sarcomas, leading to decreased iatrogenic morbidity and improved outcomes for such individuals.

Prognostic Factors Influencing Progression-Free Survival Determined From a Series of Sporadic Desmoid Tumors: A Wait-and-See Policy According to Tumor Presentation

PURPOSE Desmoid tumors are mesenchymal fibroblastic/myofibroblastic proliferations with locoregional aggressiveness and high ability to recur after initial treatment. We present the results of the largest series of sporadic desmoid tumors ever published to determine the prognostic factors of these rare tumors. PATIENTS AND METHODS Four hundred twenty-six patients with a desmoid tumor at diagnosis were included, and the following parameters were studied: age, sex, delay between first symptoms and diagnosis, tumor size, tumor site, previous history of surgery or trauma in the area of the primary tumor, surgical margins, and context of abdominal wall desmoids in women of child-bearing age during or shortly after pregnancy. We performed univariate and multivariate analysis for progression-free survival (PFS). RESULTS In univariate analysis, age, tumor size, tumor site, and surgical margins (R2 v R0/R1) had a significant impact on PFS. PFS curves were not significantly different for microscopic assessment of surgical resection quality (R0 v R1). In multivariate analysis, age, tumor size, and tumor site had independent values. Three prognostic groups for PFS were defined on the basis of the number of independent unfavorable prognostic factors (0 or 1, 2, and 3). CONCLUSION This study clearly demonstrates that there are different prognostic subgroups of desmoid tumors that could benefit from different therapeutic strategies, including a wait-and-see policy.

A prognostic nomogram for prediction of recurrence in desmoid fibromatosis.

Objective: To construct a postoperative nomogram to estimate the risk of local recurrence for patients with desmoid tumors. Background: The standard management of desmoid tumors is resection, but many recur locally. Other options include observation or novel chemotherapeutics, but little guidance exists on selecting treatment. Methods: Patients undergoing resection during 1982–2011 for primary or locally recurrent desmoids were identified from a single-institution prospective database. Cox regression analysis was used to assess risk factors and to create a recurrence nomogram, which was validated using an international, multi-institutional data set. Results: Desmoids were treated surgically in 495 patients (median follow-up of 60 months). Of 439 patients undergoing complete gross resection, 100 (23%) had recurrence. Five-year local recurrence–free survival was 69%. Eight patients died of disease, all after R2 resection. Adjuvant radiation was not associated with improved local recurrence–free survival. In multivariate analysis, factors associated with recurrence were extremity location, young age, and large tumor size, but not margin. Abdominal wall tumors had the best outcome (5-year local recurrence–free survival rate of 91%). Age, site, and size were used to construct a nomogram with concordance index of 0.703 in internal validation and 0.659 in external validation. Integration of additional variables (R1 margin, sex, depth, and primary vs recurrent presentation) did not importantly improve concordance (internal concordance index of 0.707). Conclusions: A postoperative nomogram including only size, site, and age predicts local recurrence and can aid in counseling patients. Systemic therapies may be appropriate for young patients with large, extremity desmoids, but surgery alone is curative for most abdominal wall lesions.

Molecular characterization by array comparative genomic hybridization and DNA sequencing of 194 desmoid tumors.

Desmoid tumors are fibroblastic/myofibroblastic proliferations. Previous studies reported that CTNNB1 mutations were detected in 84% and that mutations of the APC gene were found in several cases of sporadic desmoid tumors lacking CTNNB1 mutations. Forty tumors were analyzed by comparative genomic hybridization (CGH). Karyotype and fluorescence in situ hybridization revealed a nonrandom occurrence of trisomy 8 associated with an increased risk of recurrence. We report the first molecular characterization including a large series of patients. We performed array CGH on frozen samples of 194 tumors, and we screened for APC mutations in patients without CNNTB1 mutation. A high frequency of genomically normal tumors was observed. Four relevant and recurrent alterations (loss of 6q, loss of 5q, gain of 20q, and gain of Chromosome 8) were found in 40 out of 46 tumors with chromosomal changes. Gain of Chromosomes 8 and 20 was not associated with an increased risk of recurrence. Cases with loss of 5q had a minimal common region in 5q22.5 including the APC locus. Alterations of APC, including loss of the entire locus, and CTNNB1 mutation could explain the tumorigenesis in 89% of sporadic desmoids tumors and desmoids tumors occurring in the context of Gardners syndrome. A better understanding of the pathogenetic pathways in the initiation and progression of desmoid tumors requires studies of 8q and 20q gains, as well as of 6q and 5q losses, and study of the Wnt/β‐catenin pathway.

Safety and efficacy of regorafenib in patients with advanced soft tissue sarcoma (REGOSARC): a randomised, double-blind, placebo-controlled, phase 2 trial

BACKGROUND Regorafenib is a multikinase inhibitor with proven activity in refractory gastrointestinal stromal tumours and chemotherapy-refractory advanced colorectal cancers. We assessed this agents efficacy and safety in patients with metastatic soft tissue sarcomas previously treated with anthracycline. METHODS In this randomised, double-blind, phase 2 trial undertaken in France and Austria, we enrolled patients aged 18 years and older with advanced soft tissue sarcomas who had received previous doxorubicin or other anthracycline treatment. These patients were randomly assigned (1:1) into one of the following four cohorts: liposarcoma, leiomyosarcoma, synovial sarcoma, and other sarcomas. Participants were treated with oral regorafenib (160 mg per day 3 weeks on and 1 week off) or matched placebo. Patients receiving placebo were offered optional crossover in case of centrally confirmed disease progression. The random allocation schedule was computer-generated with permuted blocks of four patients, with two stratification factors: country (France or Austria) and previous exposure to pazopanib (yes or no). Eligibility criteria included patients with histologically proven advanced and inoperable soft tissue sarcomas with intolerance or failure to doxorubicin or other anthracycline-based chemotherapy and at least one unidimensionally or bidimensionally measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). The primary endpoint was RECIST-based progression-free survival after central radiological review in the intention-to-treat population. Patients, physicians, and radiologists of the panel were masked to treatment allocation. This study is still open for recruitment for an additional stratum (patients previously treated with pazopanib) and registered with ClinicalTrials.gov, NCT01900743. FINDINGS From Aug 5, 2013, to Nov 26, 2014, 182 patients were randomly assigned to one of four cohorts and included in the final analysis. At the cutoff date (Jan 7, 2016), the number of required events was reached for the four cohorts. In the liposarcoma cohort, progression-free survival was 1·1 months (95% CI 0·9-2·3) with regorafenib versus 1·7 months (0·9-1·8) with placebo (HR 0·89 [95% CI 0·48-1·64] p=0·70). In the leiomyosarcoma cohort, progression-free survival was 3·7 months (95% CI 2·5-5·0) with regorafenib versus 1·8 (1·0-2·8) months with placebo (HR 0·46 [95% CI 0·46-0·80] p=0·0045). In the synovial sarcoma cohort, progression-free survival was 5·6 months (95% CI 1·4-11·6) with regorafenib versus 1·0 (0·8-1·4) with placebo (HR 0·10 [95% CI 0·03-0·35] p<0·0001). In the other sarcoma cohort, progression-free survival was 2·9 months (95% CI 1·0-7·8) with regorafenib versus 1·0 (0·9-1·9) with placebo (HR 0·46 [95% CI 0·25-0·81] p=0·0061). Before crossover, the most common clinically significant grade 3 or higher adverse events were arterial hypertension (17 [19%] events in the 89 patients in the regorafenib group vs two [2%] events in the 92 patients in the placebo group), hand and foot skin reaction (14 [15%] vs no events) and asthenia (12 [13%] vs six [6%]). One treatment-related death occurred in the regorafenib group due to liver failure. INTERPRETATION Regorafenib has an important clinical antitumour effect in non-adipocytic soft tissue sarcomas, improving progression-free survival. Regorafenib should be further evaluated in this setting, and its therapeutic role has to be defined in the context of the growing therapeutic armamentarium, already including one approved multikinase inhibitor, pazopanib. FUNDING Bayer HealthCare.

Toxic death case in a patient undergoing gemcitabine-based chemotherapy in relation with cytidine deaminase downregulation.

Gemcitabine is an antimetabolite drug used in the treatment of various solid tumours, including lung, pancreatic or gynaecological cancers. Innovative combinational strategies (e.g. gemcitabine+capecitabine or gemcitabine+oxaliplatin) make gemcitabine an extensively prescribed drug now. Gemcitabine is characterized by a narrow therapeutic index, and its liver elimination depends upon a key enzymatic step, driven by cytidine deaminase (CDA). CDA is prone to gene polymorphism, including the 208A>G mutation, which can result in marked enzymatic deficiency with subsequent impact on drug exposure levels and related toxicities. We have developed a simple and inexpensive method to determine phenotypically CDA status in cancer patients, as an attempt to detect those at risk upon gemcitabine intake. Conjointly to genotypic investigations, this method was used to phenotype, in a retrospective setting, a female patient displaying extremely severe, and eventually lethal, toxicities after administration of a standard gemcitabine/carboplatin protocol. Phenotypic investigation showed a marked CDA deficiency (−75%) in this patient when compared with a reference, nontoxic population. Genetic studies undertaken next to screen mutations, possibly at the origin of this deficiency, showed heterozygosity for the 79A>C single-point mutation, whereas surprisingly the canonical CDA 208A>G polymorphism was not found. Taken together, this case report demonstrates, for the first time, that CDA downregulation can lead to toxic-death in patients exposed to gemcitabine. Besides, we showed here that our cost-effective and simple phenotypic approach should enable, in the future, the detection of deficient patients at risk upon gemcitabine administration.

Nutritional factors as predictors of response to radio-chemotherapy and survival in unresectable squamous head and neck carcinoma

BACKGROUND AND PURPOSE This study sought to evaluate nutritional prognostic factors before treatment in patients with unresectable head and neck cancer treated by concomitant radio-chemotherapy. METHODS AND MATERIALS Seventy-two consecutive patients were treated. We studied the potential effects of CRP, Alb, preAlb, orosomucoid, weight, weight history, BMI, PINI, OPR and NRI on response to treatment, Event-Free Survival (EFS) and Overall Survival (OS). Effects of potential risk factors on OS and on EFS were analyzed by computing Kaplan-Meier estimates, and curves were compared using the log-rank test. RESULTS All biological nutritional factors were statistically correlated with the response to radio-chemotherapy. In multivariate analysis, only CRP (p=0.004) remained statistically significant. A statistical correlation was found between Alb and EFS in multivariate analysis (p=0.04). The factors influencing OS in univariate analysis were Alb (p=0.008), CRP (p=0.004), orosomucoid (p=0.01) and NRI (p=0.01), response to radio-chemotherapy (p<0.001) and staging (p=0.04). In multivariate analysis, only the response to radio-chemotherapy (p<0.001) remained significant. CONCLUSIONS This study illustrates the prognostic value of nutritional status. CRP and Alb may be useful in the assessment of advanced head and neck cancer patients at diagnosis and for stratifying patients taking part in randomized trials.

Ezrin and alpha-smooth muscle actin are immunohistochemical prognostic markers in conventional osteosarcomas

Ezrin is a cytoskeleton linker protein that is actively involved in the metastatic process of cancer cells. We have searched for a prognostic value of ezrin and some of its partners: α-smooth muscle actin and CD44H in 37 patients with an osteosarcoma. Automate immunohistochemistry (IHC) with anti-ezrin, α-smooth muscle actin and CD44H antibodies was performed in 66 specimens: 37 biopsies before chemotherapy, 16 resected tumours of “poor” responders and 13 metastases. The messenger RNA (mRNA) levels of ezrin of 13 frozen biopsies and 4 metastases were evaluated by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). All results were correlated to the following clinical data. Ezrin expression by IHC was found in 62% of 37 biopsies in the different histological subtypes. A good correlation was found between positive or negative samples by IHC and mRNA levels. Ezrin expression was recorded in 84.5% of metastastic samples. The mean expression of ezrin was higher in metastases than biopsies (p = 0.024). In multivariate analysis, ezrin was an independent prognostic marker for event-free survival and overall survival (OS) with p < 0.001 and p = 0.003, respectively, and α-smooth muscle actin for OS only (p = 0.024). Our findings suggest that ezrin and α-smooth muscle actin are predictive IHC prognostic markers for patients with an osteosarcoma.

Molecular characterization of the response to chemotherapy in conventional osteosarcomas: Predictive value of HSD17B10 and IFITM2

The therapy regimen of high‐grade osteosarcoma includes chemotherapy followed by surgical resection and postoperative chemotherapy. The degree of necrosis following definitive surgery remains the only reliable prognostic factor and is used to guide the choice of postoperative chemotherapy. The aim of this study was to find molecular markers able to classify patients with an osteosarcoma as good or poor responders to chemotherapy before beginning treatment. Gene expression screening of 20 nonmetastatic high‐grade osteosarcoma patients was performed using cDNA microarray. Expression of selected relevant genes was validated using QRT‐PCR. Immunohistochemistry on tissue microarrays sections of 73 biopsies was performed to investigate protein expression. Fluorescent in situ hybridization was performed for RPL8 gene. We have found that HSD17B10 gene expression was up‐regulated in poor responders and that immunohistochemistry expression of HSD17B10 on biopsy before treatment was correlatedto response to chemotherapy. Other results include correlationof IFITM2, IFITM3, and RPL8 gene expression to chemotherapy response. A statistical correlation was found between polysomy 8 or gain of RPL8 and good response to chemotherapy. These data suggest that HSD17B10, RPL8, IFITM2, and IFITM3 genes are involved in the response to the chemotherapy and that HSD17B10 may be a therapeutic target. RPL8 and IFITM2 may be useful in the assessment at diagnosis and for stratifying patients taking part in randomized trials.