A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation.

BACKGROUND: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. METHODS: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT-PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. RESULTS: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell-cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. CONCLUSION: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

Combined HER2 analysis of biopsies and surgical specimens to optimize detection of trastuzumab-eligible patients in eso-gastric adenocarcinoma: a GERCOR study.

BACKGROUND: HER2 is overexpressed in 10 to 20% of gastro-esophageal adenocarcinoma (GE-ADK), and is a target for trastuzumab in metastatic patients. We conducted a study to compare HER2 expression between diagnostic biopsies (DBs) and surgical specimens (SSs) of GE-ADK, and to determine the influence of non-trastuzumab containing neoadjuvant chemotherapy (NAC) on this expression. PATIENTS AND METHODS: Pathological specimens from biopsies of 228 patients operated on between 2004 and 2011 were collected. Two cohorts treated (n = 141) or not (n = 87) with a NAC were constituted. Two blind independent pathological HER2 analyses on DB and on SS were carried out using immunohistochemistry (IHC) and colorimetric in situ hybridization (CISH). HER-2 overexpression (HER2+) was defined by a score 3+ in IHC, or 2+ with a positive CISH test, according to the specific HER2 scoring guidelines for GE-ADK. RESULTS: Paired HER2 status could be determined for 218 out of the 228 patients (95.6%). HER2+ rates were 13.3% on DB (29/218) and 14.7% on SS (32/218). HER2+ tumors were mainly cardial or esophageal adenocarcinomas, with a well-differentiated, intestinal histological type. HER2 status differed between DB and SS in 6% of cases. When DB analyses were added to SS analyses, the relative increase in HER2+ cases was 13.5% (17.1% for patients with NAC and 23.5% for patients with histological response to NAC, versus 7.1% for patients without NAC, P = 0.4, NS). Differences between DB and SS HER2 expression could be explained by intratumoral heterogeneity and by a HER2 expression decrease in SS after NAC in responding patients possibly due to a higher chemosensitivity of HER2-positive clones. CONCLUSION: The determination of HER2 status on DB provides results that complete those obtained with SS. Combining the analysis of DB and of SS enables to optimize the selection of trastuzumab-eligible patients in case of metastatic relapse, and particularly in previously NAC-responding patients.

FADD protein release mirrors the development and aggressiveness of human non-small cell lung cancer.

BACKGROUND: The need to unfold the underlying mechanisms of lung cancer aggressiveness, the deadliest cancer in the world, is of prime importance. Because Fas-associated death domain protein (FADD) is the key adaptor molecule transmitting the apoptotic signal delivered by death receptors, we studied the presence and correlation of intra- and extracellular FADD protein with development and aggressiveness of non-small cell lung cancer (NSCLC). METHODS: Fifty NSCLC patients were enrolled in this prospective study. Intracellular FADD was detected in patients' tissue by immunohistochemistry. Tumours and distant non-tumoural lung biopsies were cultured through trans-well membrane in order to analyse extracellular FADD. Correlation between different clinical/histological parameters with level/localisation of FADD protein has been investigated. RESULTS: Fas-associated death domain protein could be specifically downregulated in tumoural cells and FADD loss correlated with the presence of extracellular FADD. Indeed, human NSCLC released FADD protein, and tumoural samples released significantly more FADD than non-tumoural (NT) tissue (P=0.000003). The release of FADD by both tumoural and NT tissue increased significantly with the cancer stage, and was correlated with both early and late steps of the metastasis process. CONCLUSION: The release of FADD by human NSCLC could be a new marker of poor prognosis as it correlates positively with both tumour progression and aggressiveness.

Immunohistochemistry to identify EGFR mutations or ALK rearrangements in patients with lung adenocarcinoma.

BACKGROUND: Immunohistochemistry has been proposed as a specific and sensitive method to identify EGFR mutations or ALK rearrangements in lung tumours. PATIENTS AND METHODS: We assessed EGFR and KRAS by direct sequencing in 154 patients with lung adenocarcinoma. ALK rearrangements were assayed by FISH and RT-PCR. Immunohistochemistry was carried out and evaluated closely following published methods using recommended monoclonal rabbit or mouse antibodies. RESULTS: Thirteen of 36 exon 19 EGFR-mutated tumours (36%)-including 12 of 22 with p.Glu746_Ala750del (55%)-were positive with the 6B6 antibody that was raised against p.Glu746_Ala750del. One hundred eleven of 114 EGFR exon 19 wild-type tumours (97%) were negative with 6B6. Four of 21 exon 21 EGFR-mutated tumours (19%)-including 4 of 17 with p.Leu858Arg (24%)-were positive with the 43B2 antibody that was raised against p.Leu858Arg. One hundred twenty-two of 124 (98%) EGFR exon 21 wild-type tumours were negative with 43B2. Two of four ALK rearrangements-including two of three with ELM4-ALK fusion transcripts-were identified with the 5A4 antibody. Eleven of 13 tumours without ALK rearrangement (85%) were negative with 5A4. CONCLUSIONS: Immunohistochemistry is a specific means for identification of EGFR mutations and ALK rearrangements. It suffers, however, from poor sensitivity.

Impact of p53 expression and microsatellite instability on stage III colon cancer disease-free survival in patients treated by 5-fluorouracil and leucovorin with or without oxaliplatin.

BACKGROUND: The aim was to determine the values of p53 tumour expression and microsatellite instability (MSI) phenotype to predict benefit from adjuvant chemotherapy of colon cancer by 5-fluorouracil and leucovorin (FL) alone or with oxaliplatin (FOLFOX). PATIENTS AND METHODS: This retrospective study included 233 unselected patients with stage III colon cancer treated by FL (n = 124) or FOLFOX (n = 109). The impact of p53 expression and MSI on disease-free survival (DFS) was defined using univariate and multivariate analyses. A Cox proportional hazards model was specifically designed to evaluate the interaction between chemotherapy and these genetic alterations. RESULTS: In univariate analyses, addition of oxaliplatin significantly improved DFS provided that tumour overexpressed p53 [hazard ratio (HR) 0.39; 95% confidence interval (CI) 0.19-0.82; P = 0.01] or displayed MSI phenotype (HR 0.17; 95% CI 0.04-0.68; P = 0.01). In multivariate analyses, p53 was confirmed as an independent factor predictive of benefit from FOLFOX (P = 0.03), while the interaction of MSI with chemotherapy could not be determined in the absence of relapse in the MSI group treated with FOLFOX. CONCLUSION: Our observations indicate that MSI status and p53 expression may influence the impact of oxaliplatin on adjuvant treatment of stage III colon cancer patients.

Establishment and Characterization of a Stromal Cell Line Derived From a Patient With Thoracic Endometriosis.

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10-7 mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and a-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

The NER proteins are differentially expressed in ever smokers and in never smokers with lung adenocarcinoma.

BACKGROUND: The expression levels of excision repair cross-complementation group 1 (ERCC1), replication protein A (RPA) and xeroderma pigmentosum group F (XPF) nucleotide excision repair proteins may be important in the response to platin-based therapy in lung cancer patients. It is not known whether ERCC1, RPA and XPF expression levels differ between ever smokers (ES) and never smokers (NS). PATIENTS AND METHODS: ERCC1, RPA and XPF expression levels were immunohistochemically evaluated in 125 patients with resected lung adenocarcinoma (AC) and carefully reviewed smoking status. RESULTS: ERCC1 was correlated with XPF (P = 0.001), but not with RPA (P = 0.11). In the univariate analysis, ERCC1 and XPF levels were higher in NS compared with ES (P = 0.004 and P = 0.003, respectively). In the multivariate analysis, the smoking status was predictive of the ERCC1 level [odds ratio (OR) 2.5, 95% confidence interval (CI) 1.03-6.2] after adjustment for variables linked to the smoking status, including age and the presence of bronchioloalveolar (BAC) features. The smoking status was also predictive of both RPA (OR 6.7, 95% CI 1.5-33.3) and XPF levels (OR 12.5, 95% CI 2.9-50) after adjusting for age, sex and BAC features. CONCLUSION: In patients with resected lung AC, ERCC1, RPA and XPF expression levels are higher in NS compared with ES.

[Benign schwannoma with high uptake of 18 fluorodeoxglucose (PET-Scan)]. / Un cas de schwannome bénin avec hypermétabolisme sur la tomoscintigraphie TEP au 18fluorodésoxyglucose.

INTRODUCTION: In patients with a previous history of malignancy, the occurrence of a mediastinal mass with significant uptake of 18 Fluorodeoxyglucose on a PET-scan may lead to biopsy or resection. CASE REPORT: We report the case of a posterior mediastinal mass, with significant uptake of 18 Fluorodeoxyglucose on PET- scan, in a patient with a previous history of testicular seminoma. The lesion was actually a benign schwannoma. CONCLUSIONS: In the case of a mediastinal mass with conventional imaging being in favour of a neurogenic tumour a PET scan cannot confirm benignity or malignancy.

Establishment and Characterization of a Stromal Cell Line Derived From a Patient With Thoracic Endometriosis.

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10-7 mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and α-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.

[Intrapulmonary bronchogenic cysts]. / Kystes bronchogéniques intrapulmonaires.

INTRODUCTION: Bronchogenic cysts are congenital malformations that are usually located in the mediastinum. Intrapulmonary location is rare. OBSERVATION: Four cases of intrapulmonary bronchogenic cysts are reported in order to discuss their clinical and radiological presentation and their treatment. CONCLUSION: Intrapulmonary bronchogenic cysts diagnostic is often missed. This condition must however be known so as to foresee a resection in order to prevent a potential complication.

[Cystic mediastinal schwannoma appended to the vagus nerve]. / Schwannomes kystiques du médiastin développés au dépend du nerf vague.

INTRODUCTION: Schwannomas are a form of rare tumor, arising from neural tissue and representing 2 % of mediastinal tumors. They are usually located in the posterior mediastinum, most often in the paravertebral gutters and typically appended to intercostal nerves. CASE REPORTS: We report two cases of unusual mediastinal schwannomas, appended to the vagus nerve. The schwannoma was located in the subcarinal region in the first case and in the right para-tracheal region in the second case. The lesions were thought to be bronchogenic cysts preoperatively in both cases because of a cystic appearance on preoperative CT scan and endobronchial ultrasonography. A surgical approach was adopted to remove the tumors. Video-assisted thoracoscopy was used in one case and robotic-assisted surgery in the second case, without any complication, allowing for complete resection and to establish a certain pathological diagnosis. CONCLUSION: Despite this location and cystic presentation being unusual, schwannoma should be considered as a possible cause of cystic lesions in the mediastinum. Minimally invasive surgery allows for complete resection and definitive pathological diagnosis.

[Endobronchial ultrasonography (EBUS) for the internist]. / Écho-endoscopie bronchique (EBUS) pour l'interniste.

Endobronchial ultrasonography (EBUS) is a recent mini-invasive technique allowing transbronchial needle aspiration (TBNA) of mediastinal lymph nodes as well as peribronchial lesions. EBUS was initially developed for lung cancer mediastinal staging. Over the years, indications for EBUS have been progressively extended to the scope of inflammatory disorders, mediastinal lymphomas, and infectious diseases. Particularly in immunosuppressed patients, including HIV-infected patients, EBUS allows the diagnosis of several diseases that involve the mediastinum, avoiding invasive surgical explorations such as mediastinoscopy or thoracoscopy. This review aims at discussing the technical aspects, and specifies indications, results, and limits of EBUS for the internist.

Impact of the extent of extraprostatic extension defined by Epstein's method in patients with negative surgical margins and negative lymph node invasion.

BACKGROUND: To assess the impact of the degree of extraprostatic extension (EPE) on biochemical recurrence (BCR) and utility of the original Epstein's criteria to define EPE in a cohort of pT3aN0 without positive surgical margin (PSM). METHODS: A two-center retrospective analysis was performed on data from 490 pT3aN0 patients who underwent radical prostatectomy between 2000 and 2012. Patients with neoadjuvant and/or adjuvant therapy, detectable PSA and PSM were excluded. Our pathologists used Epstein's criteria to report the degree of EPE. When pathology reports did not reflect the terms 'focal' or 'established' (non-focal), slides were analyzed by a single genitourinary pathologist for final evaluation. The end point was defined by BCR. RESULTS: Selection criteria yielded 247 patients. Mean follow-up was 56.3±4.6 months; mean age at surgery was 62.5 years. Sixty-one (24.7%) patients experienced BCR during follow-up. Patients with focal extension had a 5-year recurrence-free survival of 89% versus 80% for those with non-focal extension (P=0.0018). In multivariate analysis, both pathologic Gleason score (hazard ratio 2.5; 95% confidence interval 1.4-4.5; P=0.002) and the extent of EPE (hazard ratio 1.8; 95% confidence interval 1.1-3.5; P=0.029) were significant predictors of BCR. CONCLUSIONS: The extent of EPE is an independent predictor of BCR in pT3aN0 prostate cancer without PSM. This study reinforces the utility of the subjective Epstein approach already adopted by most pathologists for quantification of the extent of EPE.

Clinical performance of transperineal template guided mapping biopsy for therapeutic decision making in low risk prostate cancer. / Rendimiento clínico de biopsia de mapeo guiada por plantilla transperineal para la toma de decisiones terapéuticas en el cáncer de próstata de bajo riesgo.

OBJECTIVES: To evaluate the role of Transperineal Template guided Mapping Biopsy (TTMB) in determining the management strategy in patients with low risk prostate cancer (PCa). METHODS: We retroscpectively evaluated 169 patients who underwent TTMB at our institution from February 2008 to June 2011. Ninety eight of them harbored indolent PCa defined as: Prostate Specific Antigen<10ng/ml, Gleason score 6 or less, clinical stage T2a or less, unilateral disease and a maximum of one third positive cores at first biopsy and<50% of the core involved. TTMB results were analyzed for Gleason score upgrading and upstaging as compared to initial TransRectal UltraSound (TRUS) biopsies and its influence on the change in the treatment decisions. RESULTS: TTMB detected cancer in 64 (65%) patients. The upgrade, upstage and both were noted in 33% (n=21), 12% (n=8) and 7% (n=5) respectively of the detected cancers. The disease characteristics was similar to initial TRUS in 30 (48%) patients and TTMB was negative in 34 (35%) patients. Prostate volume was significantly smaller in patients with upgrade and/or upstage noted at TTMB (45.4 vs 37.9; P=.03). TTMB results influenced 73.5% of upgraded and/or upstaged patients to receive radical treatment while 81% of the patients with unmodified stage and/or grade continued active surveillance or focal therapy. CONCLUSIONS: In patients with low risk PCa diagnosed by TRUS, subsequent TTMB demonstrated cancer upgrade and/or upstage in about one-third of the patients and resulted in eventual change in treatment decision.

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes.

CD245 is a human surface antigen expressed on peripheral blood lymphocytes, initially delineated by two monoclonal antibodies DY12 and DY35. Until now, CD245 molecular and functional characteristics remained largely unknown. We combined immunological and proteomic approaches and identified CD245 as the unconventional myosin 18A, a highly conserved motor enzyme reported as a receptor for the surfactant protein A (SP-A), that plays a critical role in cytoskeleton organization and Golgi budding. We report that the recruitment of CD245 strongly enhanced NK cell cytotoxicity. Further, we show that the enhancement of the NK lymphocytes killing ability toward CD137-ligand expressing target cells could result from the induction of CD137 expression following CD245 engagement. The SP-A receptor could therefore represent a novel and promising target in cancer immunotherapy.

Human papillomavirus genotype as a major determinant of the course of cervical cancer.

PURPOSE: To determine whether the prognosis of invasive cancers of the uterine cervix is related to the type of human papillomavirus (HPV) associated with the tumor. PATIENTS AND METHODS: Two hundred ninety-seven patients with invasive cervical cancer were prospectively registered from 1986 to 1994. HPV typing was performed on DNA extracted from frozen tumor specimens by means of Southern blot hybridization (SBH) and polymerase chain reaction (PCR) techniques. The median follow-up was 38 months. RESULTS: HPV sequences were detected in 246 patients (83%): 150 patients had HPV16, 31 patients had HPV18, and 14 patients had one of the intermediate-oncogenic-risk HPV types (HPV31, 33, 35, 52, 58). In 51 patients, HPV type remained undetermined, and in 51 patients, no viral sequences were found. No significant associations were observed between virologic data and tumor stage or node status. The 5-year disease-free survival (DFS) rate was 100% for patients with intermediate-risk HPV-associated tumors, 58% for patients with HPV16-positive tumors, and 38% for patients with HPV18-positive tumors (P = .02). In multivariate analysis, patients with HPV18-associated tumors had a relative risk (RR) of death 2.4 times greater (95% confidence interval [CI], 1.29-4.59) than that for patients with HPV16, and 4.4 times greater (95% CI, 3.48-5.32) than that for patients with a tumor associated with a viral type different from HPV16/18. CONCLUSION: The prognosis for invasive cancers of the uterine cervix is dependent on the oncogenic potential of the associated HPV type. HPV typing may provide a prognostic indicator for individual patients and is of potential use in defining specific therapies against HPV-harboring tumor cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.