Search

Supplementary Data 1. This supplementary data comprises a step-by-step description on how our gene-expression based classification models were generated and validated

Supplementary Figure 1. Highlights the EFS and OS for the cohort of neuroblastoma patients with MYCN-amplified disease (n=114, Fig 1a) and for th subcohort of patients >18 months of age with stage 4, MYCN non-amplified disease (n=102, fig. 1b). F, favorable; UF, unfavorable.

Purpose: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression–based classification and established prognostic markers.Experimental Design: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan–Meier estimates and Cox regression analyses.Results: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients [5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001] and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 [EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20–8.02; OS: HR, 25.54; 95% CI, 8.40–77.66; both P < 0.001]. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation.Conclusions: Combination of gene expression–based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial. Clin Cancer Res; 21(8); 1904–15. ©2014 AACR.See related commentary by Attiyeh and Maris, p. 1782

Supplementary Table 1. Clinical co-variates for the 709 patients who participated in the study. This supplementary table comprises detailed information on clinical co-variates for all 709 neuroblastoma patients who participated in the study.

Supplementary Table 2. External performance validation of the top five classifiers. This supplementary table comprises the external performance metrics of the top five classifiers. Indicated are the values for classification accuracy, sensitivity , specificity and Matthew's correlation coefficient (MCC) in the prediction of 325 patients of the test set who fulfilled the criteria for classifier training (Favorable, n=187; Unfavorable, n=138).

Supplementary Table 4. Transcripts that contribute to the SVM_th10 classifier. This supplementary table highlights the transcripts that constitute the SVM_th10 classifier. Indicated for each feature are the Oligo-ID, the Gene symbols, the Entrez gene IDs, the RefSeq IDs, the Gene IDs and the Transcript IDs.

Legends to Supplementaries. This documents comprises the legends to the supplementary figures and tables

Supplementary Data 2. This supplementary data comprises the r-algorithm scripts required to perform the both model selection and validation as described in the step-by-step protocol

International audience; Aims Renal cell carcinomas (RCCs) represent 2-5% of kidney malignancies in children and adolescents. Appropriate diagnostic and classification are crucial for the correct management of the patients and in order to avoid inappropriate pre-operative chemotherapy, which is usually recommended if a Wilms' tumour is suspected. Methods and results A French-Italian series of 93 renal cell carcinomas collected from 1990 to 2019 in patients aged less than 18 years was reclassified according to the 2016 World Health Organization (WHO) classification and the latest literature. TFE3 and TFEB fluorescence in-situ hybridisation (FISH) analyses and a panel of immunohistochemical stains were applied. The median age at diagnosis was 11 years (range = 9 months-17 years). MiT family (MiTF) translocation RCCs accounted for 52% of the tumours, followed by papillary (20%) and unclassified RCCs (13%). Other subtypes, such as SDHB-deficient and fumarate hydratase-deficient RCCs, represented 1-3% of the cases. We also described a case of ALK-rearranged RCC with a metanephric adenoma-like morphology. Conclusion A precise histological diagnosis is mandatory, as targeted therapy could be applied for some RCC subtypes, i.e. MiTF-translocation and ALK-translocation RCC. Moreover, some RCC subtypes may be associated with a predisposition syndrome that will impact patients' and family's management and genetic counselling. A precise RCC subtype is also mandatory for the clinical management of the patients and inclusion in new prospective clinical trials.

Purpose Genomic tests can identify ER-positive HER2-negative localized breast cancer patients who may not benefit from adjuvant chemotherapy. Such tests seem especially interesting in “intermediate” clinico-pathological risk categories. The psychological impact of the decision uncertainty in these women remains largely unexplored. We assessed the clinical and psychological impact of EndoPredict® (EpClin), a clinico-genomic test, in these patients. Methods This multicenter, single arm prospective study (NCT02773004) enrolled patients for which adjuvant chemotherapy was uncertain, based on predefined criteria. The primary endpoint was the proportion of change between initial adjuvant decision and final administration of chemotherapy. Secondary endpoints included post-test (Day 17) and 1-year patient reported outcomes. Results One third of 200 evaluable patients had a high EpClin score (≥3.32867; 10 years cumulative risk of distance failure ≥10%). The overall change rate of chemotherapy decision was 72/200 (35.8%, 95% CI 29.2–42.4). Chemotherapy was withdrawn in 57 cases (28.4% [22.2–34.8]) and added in 15 (7.5% [3.8–11.2]. 6 changes (8%) were based on patients’ decisions. Anxiety and distress levels increased at Day 17 when adding chemotherapy after the test result (p, Highlights • EndoPredict ® (EpClin) allowed a chemotherapy decision modification in 35% of the patients included in the Adendom trial. • Patient-physician concertation is important: 8% of treatment changes are based on patients’ will. • A single-step decision including the test appears preferable to limit anxiety and distress.

The cyclin-dependent kinase 4 (CDK4)/6 inhibitor Palbociclib is a new standard treatment in hormone-receptor positive breast cancer patients. No predictive biomarkers have been identified and no pharmacodynamics has properly been described so far.Patients with early-breast cancer were randomized 3 : 1 to oral palbociclib 125 mg daily for 14 days until the day before the surgery versus no treatment. Primary objective was antiproliferative response defined as a natural logarithm of Ki67 expression at day 15 below 1. Secondary end points were subgroups analyses and safety. Exploratory analyses included search for predictive biomarkers. Immunostainings (Ki67, RB, pRB, p16, pAKT, pER, pCDK2, CyclinD1), FISH (CCND1) and gene expression (GE) arrays were carried out at baseline and at surgery. In addition, activating PIK3CA and AKT1 mutations were assessed at baseline.74 patients were allocated to palbociclib and 26 to control. Most patients (93%) were hormone-receptor (HR)-positive, whereas 8% were HER2-positive. Palbociclib led to significantly more antiproliferative responses when compared with control (58% versus 12%, P 0.001), and to a significantly higher Ki67 decrease (P 0.001). In the HR-positive/HER2-negative subgroup, this antiproliferative effect was even more marked in the palbociclib arm when compared with control (70% versus 9%, P 0.001). Palbociclib treatment led also to a significantly higher decrease from baseline in phospho-Rb when compared with control (P 0.001). Among treated patients, changes in Ki67 correlated with changes in phospho-Rb (Spearman rank r = 0.41, P 0.0001). GE analyses confirmed a major effect on proliferation and cell cycle genes. Among treated patients, CCNE2 expression was significantly more decreased in antiproliferative responders versus nonresponders (P = 0.006).Short-term preoperative palbociclib decreases Ki67 in early-breast cancer patients. Early decrease of Rb phosphorylation correlates with drug's effect on cell proliferation and could potentially identify patients with primary resistance.NCT02008734.

Determining the status of HER2-neu amplification and overexpression in breast cancer is crucial for prognosis but mostly for treatment purposes. Standard techniques include the determination of IHC in combination with in situ hybridization techniques to confirm a HER2-neu amplification in case of IHC2+ using either a core-needle biopsy or a surgical specimen. qPCR has been also demonstrated to be able to determine HER2 status, mostly in core biopsies or in surgical specimens. Fine-needle aspiration is a reliable, quicker and less invasive technique that is widely used for diagnosis of invasive breast cancer. In this study, we assessed the performance of qPCR in invasive breast carcinomas to determine HER2-neu status by using fine-needle aspiration samples and comparing to standard IHC and FISH. From a total of 154 samples from patients who had nodular breast lesions and attended the 1-day-stop clinic at the Gustave Roussy from March 2013 to October 2014, qPCR was able to determine the HER2 status in a mean of 3.7 days (SD 3.1). The overall concordance with standard HER2-testing was very high: 97% (95% CI 0.94 to 0.99); sensitivity was 96% (0.87–1), specificity 98% (0.95–1) and positive and negative predictive values 88% (0.75–1) and 99% (0.98–1), respectively. In conclusion, our study demonstrates that qPCR performed using fine-needle aspiration samples from a primary tumour is a reliable and fast method to determine HER2/neu status in patients with early breast cancer.

// Christophe Massard 1, 2, * , Marianne Oulhen 2, 3, * , Sylvestre Le Moulec 4 , Nathalie Auger 5 , Stephanie Foulon 6 , Aurelie Abou-Lovergne 1 , Fanny Billiot 2, 3 , Alexander Valent 5 , Virginie Marty 7 , Yohann Loriot 1, 2 , Karim Fizazi 1, 2 , Philippe Vielh 2, 3, 5, # , Francoise Farace 2, 3, # 1 Gustave Roussy, Universite Paris-Saclay, Department of Medicine, F-94805, Villejuif, France 2 INSERM, U981 “Identification of Molecular Predictors and New Targets for Cancer Treatment”, F-94805, Villejuif, France 3 Gustave Roussy, Universite Paris-Saclay, “Circulating Tumor Cells” Translational Platform, AMMICA CNRS UMS3655 – INSERM US23, F-94805, Villejuif, France 4 Hopital d’Instruction des Armees du Val de Grâce, Department of Oncology, F-75005, Paris, France 5 Gustave Roussy, Universite Paris-Saclay, Department of Biopathology, F-94805, Villejuif, France 6 Gustave Roussy, Universite Paris-Saclay, Department of Biostatistics and Epidemiology, F-94805, Villejuif, France 7 Gustave Roussy, Universite Paris-Saclay, “Histo Cytopathology” Translational Platform, AMMICA CNRS UMS3655 – INSERM US23, F-94805, Villejuif, France * These authors contributed equally to the study # These authors contributed equally to the study Correspondence to: Francoise Farace, email: francoise.farace@gustaveroussy.fr Keywords: prostate cancer, biopsy, circulating tumor cells, androgen receptor, TMPRSS2-ERG translocation Received: March 24, 2016 Accepted: June 17, 2016 Published: July 04, 2016 ABSTRACT Molecular characterization of cancer samples is hampered by tumor tissue availability in metastatic castration-resistant prostate cancer (mCRPC) patients. We reported the results of prospective PETRUS study of biomarker assessment in paired primary prostatic tumors, metastatic biopsies and circulating tumor cells (CTCs). Among 54 mCRPC patients enrolled, 38 (70%) had biopsies containing more than 50% tumour cells. 28 (52%) patients were analyzed for both tissue samples and CTCs. FISH for AR -amplification and TMPRSS2-ERG translocation were successful in 54% and 32% in metastatic biopsies and primary tumors, respectively. By comparing CellSearch and filtration (ISET)-enrichment combined to four color immunofluorescent staining, we showed that CellSearch and ISET isolated distinct subpopulations of CTCs: CTCs undergoing epithelial-to-mesenchymal transition, CTC clusters and large CTCs with cytomorphological characteristics but no detectable markers were isolated using ISET. Epithelial CTCs detected by the CellSearch were mostly lost during the ISET-filtration. AR -amplification was detected in CellSearch-captured CTCs, but not in ISET-enriched CTCs which harbor exclusively AR gain of copies. Eighty-eight percent concordance for ERG -rearrangement was observed between metastatic biopsies and CTCs even if additional ERG -alteration patterns were detected in ISET-enriched CTCs indicating a higher heterogeneity in CTCs. Molecular screening of metastatic biopsies is achievable in a multicenter context. Our data indicate that CTCs detected by the CellSearch and the ISET-filtration systems are not only phenotypically but also genetically different. Close attention must be paid to CTC characterization since neither approach tested here fully reflects the tremendous phenotypic and genetic heterogeneity present in CTCs from mCRPC patients.

Simple Summary Cytopathology cannot distinguish benign from malignant follicular lesions in 20–30% of cases. These indeterminate cases includes the so-called follicular neoplasms (FNs) according to The Bethesda System for Reporting Thyroid Cytopathology. Frozen samples from 66 classic follicular adenomas (cFAs) and carcinomas (cFTCs) studied by array-comparative genomic hybridization identified three specific alterations of cFTCs (losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X) confirmed by fluorescent in situ hybridization (FISH) in a second independent series of 60 touch preparations from frozen samples of cFAs and cFTCs. In a third independent set of 27 cases of already stained pre-operative fine-needle aspiration cytology samples diagnosed as FNs and histologically verified, FISH analysis using these three markers identified half of cFTCs. Specificity of our assay for identifying cFTCs is higher than 98% which might be comparable with BRAF600E testing in cases of suspicion of classic papillary thyroid carcinomas. Abstract Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.

Myeloid sarcoma (MS) is a rare extra-medullary tumour of the myeloid lineage, which can be a difficult diagnosis to make.We report the case of a 73-year-old male with a right-sided nasopharyngeal mass revealed on CT scan and MRI.An initial cytological and histological examination suggested a high-grade lymphoma. Nevertheless, the final diagnosis was a MS with an unusual involvement of the nasopharynx that was treated with a conventional induction leukemia therapy. Eight months later, the patient had persistent thrombocytopenia and a bone marrow aspiration showed the dysplasia of a high grade myelodysplastic syndrome and cytogenetics detected t(3;21). The patient was treated with a 5-Azacitidine (Vidaza) protocol until overt progression and disease evolution.In conclusion few cases of MS involving the nasopharynx have been reported. Its diagnosis is often difficult and should be considered especially when a high index of suspicion is present and the immunophenotype of the malignant haematological cells is not clearly in favour of a lymphoma.

The goal of this study was CGH array profiling of breast cancer from Malian women in order to define differences with those from USA. CGH array was performed in 28 samples, 17 with a triple negative phenotype. The profiles were compared to those of 106 tumors from USA. 6 chromosomal regions (6p21, 9q34, 11q13, 12q24, 17q25 and 22q12.1-22q13.1) were identified with a significant higher rate of copy number alterations. These regions contain several genes of interest including BCR. FISH and IHC confirmed that BCR was amplified and overexpressed particularly in triple negative tumors. Finally, 5 regions presented a high level of amplification in two or more samples, including 2 regions located between 9p22.3-9p23 and 9p23-9p24.1. This study confirms that breast cancers from African women present biological differences with those from USA. Larger studies are needed to go further in the identification of therapeutic targets that would be specific to African women.

Background: The status of the HER2 gene is not static and may change between the primary tumor, lymph node metastases and distant metastases. This status change can be a consequence of the natural evolution of the tumor or can be induced by therapy. The HER2 gene status is, in the majority of cases, established at the moment of diagnosis. After chemotherapy, monitoring this marker can be a real challenge because of ploidy changes induced by drugs. We made a HER2 genetic examination by Fluorescent in situ Hybridization (FISH) of invasive breast cancers before and after taxane treatment. After treatment we found supernumerary HER2 gene copies up to 15 copies in the cases non-amplified at diagnosis. Material and methods: All 410 invasive breast carcinoma samples were collected in a single laboratory (Service de Pathologie, Centre Jean Perrin). Tumor samples were collected by biopsy at diagnosis or by surgical excision after taxanes chemotherapy. For all cases we had matched samples, one before and one after therapy. Tumor samples were formalin-fixed paraffin-embedded. IHC staining was done to detect the expression of HER2 protein. Only cases which were IHC 2+ or IHC 1+ (with scattered HER2 heterogeneous staining) were checked by FISH. Results: After taxane-based neoadjuvant chemotherapy (4–8 cycles) thirty patients (HER2 negative at Dg) out of 344 showed supernumerary HER2 gene copies detected in giant cells with large polyploid nuclei. The proportion of polyploid post-therapy cells among regular-size cancer cells (diploid) was from 15 to 30%. The FISH examination of polyploid cells revealed an increase of HER2 copy number (and chromosome 17centromere) up to 15. The same copy number raise was observed with EGFR and ALK FISH probes. The ratio of all 30 cases with two distinct tumor cell populations (near diploid cells and giant cells) was up to 1.65 (not amplified). In one third of cases the average number of HER2 gene copies was higher than 6. Conclusions and recommendations: As a result of taxane administration, polyploid cells are formed and that most (if not all) chromosomes have been replicated simultaneously because of absent chromosomal segregation secondary to microtubule inhibition by taxanes. This HER2 copy increase is distinct from the mechanisms accounting for a more restricted gene amplification during carcinogenesis. The viability of giant polyploid cells is particularly low and most if not all of these cells succumb to mitotic catastrophe, an onco-suppressive mechanism causing cell cycle arrest and cell death. Irrespective of this consideration, it would be a critical error to classify cases as HER2 amplified solely only the bases of HER2 copy number >6. We recommend that the detection of more than 6 HER2 copies after taxane-based chemotherapy should be prompt a validation step involving the exclusion of polyploid cells from the analysis, as well as hybridization with a second and third probe specific for a chromosome other than 17. This validation step appears crucial to avoid unnecessary, costly treatments with HER2-targeted therapies after the failure of taxane-based chemotherapies. The clinico-pathological parameters of tumors with giant cells will be discussed. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-08-04.

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678 ; see the Perspective by Zanetti and Mahadevan ) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells.

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.

Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells.

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor α was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

In the past decade, microarray technology has become a major tool for high-throughput comprehensive analysis of gene expression, genotyping and re-sequencing applications. High-throughput microarrays are used for expression profiling analyses with the aims of gene or pathway discovery, tumor subclassification or relapse risk assessment. The introduction of microarray CGH provides a powerful tool to precisely detect and quantify genomic aberrations and map these directly onto the human genome. This review summarizes the current status of the application of microarray technology to neuroblastoma research.

CPT-11 (irinotecan) is a DNA-topoisomerase I inhibitor with preclinical activity against neuroblastoma (NB) xenografts. The aim was to establish in vivo an NB xenograft resistant to CPT-11 in order to study the resistance mechanisms acquired in a therapeutic setting. IGR-NB8 is an immature NB xenograft with MYCN amplification and 1p deletion, which is sensitive to CPT-11. Athymic mice bearing advanced-stage subcutaneous tumours were treated with CPT-11 (27 mg kg(-1) day(-1) x 5) every 21 days (1 cycle) for a maximum of four cycles. After tumour regrowth, a new in vivo passage was performed and the CPT-11 treatment was repeated. After the third passage, a resistant xenograft was obtained (IGRNB8-R). The tumour growth delay (TGD) was reduced from 115 at passage 1 to 40 at passage 4 and no complete or partial regression was observed. After further exposure to the drug, up to 28 passages, the resistant xenograft was definitively established with a TGD from 17 at passage 28. Resistant tumours reverted to sensitive tumours after 15 passages without treatment. IGR-NB8-R remained sensitive to cyclophosphamide and cisplatin and cross-resistance was observed with the topoisomerase I inhibitor topotecan. No quantitative or qualitative topoisomerase I modifications were observed. The level of expression of multidrug resistance 1 (MDR1), MDR-associated protein 1 (MRP1) and, breast cancer resistance protein, three members of the ATP-binding cassette transporter family was not modified over passages. Our results suggest a novel resistance mechanism, probably not involving the mechanisms usually observed in vitro.

Background The genetic heterogeneity of HER2 gene amplification (GA) in breast cancer has previously been described, but the clinical significance of this phenomenon remains unknown. We studied the genetic categories of a series of consecutive 2+ IHC cases over 5 years, with a focus on cases with HER2 GA detected in minor clone(s). We compared the HER2 status in primary tumors and positive axillary lymph nodes (ALN) when available to test the hypothesis that HER2 amplified cells are more aggressive and metastase quicker than non amplified cells. Material and methods: From January 1st. 2006 to May 30. 2011, 4491 invasive breast carcinomas had HER2 immunohistochemical (IHC) and/or HER2 gene status evaluation on their tumor sample in Institut Gustave Roussy. The distribution according to their IHC was as follows: 0 in 2915 cases (65%), 1+ in 569 cases (12.6%), 2+ in 536 cases (11.8%) and 3+ in 471 cases (10.6%). All 2+ samples were checked by Fluorescence in situ hybridization (FISH). For each case we analysed 100 invasive cells in 10 microscopic fields. Heterogeneous amplification was defined as presence of 5–50% of amplified cells (ratio>2,2) within the tumors otherwise classified as not amplified or borderline (ratio for 100 cells: Results: FISH with HER2/cen17 probes can classify IHC 2+ tumors into 5 genetic categories: normal gene status (two chromosomes (chr) 17 and two HER2 genes): 90 cases; chr 17 “ polysomy “/17q gain: 145 cases; HER2 gain: 122 cases; HER2 amplification (major clone): 135 cases and chr 17 monosomy: 44 cases. We focused our interest to IHC 2+ cases where the major clone showed no HER2 amplification, but minority clone (cut off 5%) showed the HER2 amplification (ratio >2,2 or >6 HER2 copies per cell). We found 48 such cases (10% of 2+ cases). These cases had been reported as not amplified (42/48) or borderline (6/48). To find out if the amplified cells are those which metastase, we checked 10 involved ALN: they all showed the similar genetic heterogeneity as a primary tumor with a global ratio < 2,2. Other cases with involved ALN are in ongoing study by FISH. Conclusions: The genetic heterogeneity of IHC 2+ tumors is a common event and five different genetic categories can be detected. A part of the HER2 negative cases contained one or several HER2 amplified clones. Preliminary results show a similar genetic heterogeneity of metastatic population. Our results on the small series of cases do not confirm the hypothesis that the only amplified clones metastase, but all clones (amplified and not amplified) has a metastatic capacity. A multi-center collaboration is needed to collect the high number of cases with heterogeneous amplification to: 1) Find out a proportion of such patients in HER2 IHC 2+ group (10% in our study). 2) Study a status of the gene in relapse cases. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-05-07.

Purpose: To optimize neuroblastoma treatment stratification, we aimed at developing a novel risk estimation system by integrating gene expression–based classification and established prognostic markers. Experimental Design: Gene expression profiles were generated from 709 neuroblastoma specimens using customized 4 × 44 K microarrays. Classification models were built using 75 tumors with contrasting courses of disease. Validation was performed in an independent test set (n = 634) by Kaplan–Meier estimates and Cox regression analyses. Results: The best-performing classifier predicted patient outcome with an accuracy of 0.95 (sensitivity, 0.93; specificity, 0.97) in the validation cohort. The highest potential clinical value of this predictor was observed for current low-risk patients [5-year event-free survival (EFS), 0.84 ± 0.02 vs. 0.29 ± 0.10; 5-year overall survival (OS), 0.99 ± 0.01 vs. 0.76 ± 0.11; both P < 0.001] and intermediate-risk patients (5-year EFS, 0.88 ± 0.06 vs. 0.41 ± 0.10; 5-year OS, 1.0 vs. 0.70 ± 0.09; both P < 0.001). In multivariate Cox regression models for low-risk/intermediate-risk patients, the classifier outperformed risk assessment of the current German trial NB2004 [EFS: hazard ratio (HR), 5.07; 95% confidence interval (CI), 3.20–8.02; OS: HR, 25.54; 95% CI, 8.40–77.66; both P < 0.001]. On the basis of these findings, we propose to integrate the classifier into a revised risk stratification system for low-risk/intermediate-risk patients. According to this system, we identified novel subgroups with poor outcome (5-year EFS, 0.19 ± 0.08; 5-year OS, 0.59 ± 0.1), for whom we propose intensified treatment, and with beneficial outcome (5-year EFS, 0.87 ± 0.05; 5-year OS, 1.0), who may benefit from treatment de-escalation. Conclusions: Combination of gene expression–based classification and established prognostic markers improves risk estimation of patients with low-risk/intermediate-risk neuroblastoma. We propose to implement our revised treatment stratification system in a prospective clinical trial. Clin Cancer Res; 21(8); 1904–15. ©2014 AACR. See related commentary by Attiyeh and Maris, p. 1782

Background: Identification of MET genetic alteration, mutation, or amplification in oropharyngeal squamous cell carcinoma (OPSCC) could lead to development of MET selective kinase inhibitors. The aim of this study was to assess the frequency and prognostic value of MET gene mutation, amplification, and protein expression in primary OPSCC.Methods: A retrospective chart review was conducted of patients treated for single primary OPSCC between January 2007 and December 2009. Pre-treatment OPSCC tissue samples were analyzed for MET mutations, gene amplification, and overexpression using Sanger sequencing, FISH analysis, and immunohistochemistry respectively. Univariate and multivariate analyses were used to analyze correlations between molecular abnormalities and patient survival.Results: 143 patients were included in this study. Six cases (4%) were identified that had a genetic variation, but previously described mutations such as p. Tyr1235Asp (Y1235D) or p.Tyr1230Cys (Y1230C) were not detected. There were 15 high polysomy cases, and only 3 cases met the criteria for true MET amplification, with >= 10% amplified cells per case. Immunohistochemistry evaluation showed 43% of cases were c-MET negative and in 57% c-MET was observed at the tumor cell level. Multivariate analysis showed no significant association between MET mutation, amplification, or expression and survival.Conclusions: Our study shows a low frequency of MET mutations and amplification in this cohort of OPSCC. There was no significant correlation between MET mutations, amplification, or expression and patient survival. These results suggest that patient selection based on these MET genetic abnormalities may not be a reliable strategy for therapeutic intervention in OPSCC.

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca2+-dependent NAD(P)H oxidase that generates H2O2 by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138Tox). Human and porcine p138Tox have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a CaenorhabditiscDNA and with rbohA, a protein involved in theArabidopsis NADPH oxidase. p138Tox shows also similarity to the p65Mox and to the gp91Phox in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91Phox, p138Tox shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91Phox with the p47Phox cytosolic factor are absent in p138Tox. The expression of porcine p138Tox mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138Tox is a differentiation marker of thyrocytes. The gene of human p138Tox has been localized on chromosome 15q15.

Background Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with “gold standard FISH” for evaluation of HER2 amplification status. Methods This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. Results The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. Conclusion These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

Reverse transcriptase-polymerase chain reactions using foetal brain RNA with reverse and forward primers of the first, second and third NTRK4 region allowed us to obtain three amplified NTRK4 fragments. The specificity of amplified fragments was checked by digestion with restriction endonucleases AvrII, HindIII and PspII for the first, second and third regions, respectively. Each restriction site was specific for each amplified fragment. The fragment of the NTRK4 first region was also sequenced and the sequence determined was identical to the human NTRK4 sequence. The three amplified fragments were cloned in pBS. For the Southern technique, plasmid pBS-NTRK4a (with an insert of 1052 bp) detected a human 9-kb HindIII sequence which was localised unambiguously on chromosome 6. For fluorescence in situ hybridisation, the three plasmids, pBS-NTRK4a, pBS-NTRK4b (insert 924 bp) and pBS-NTRK4c (insert 1114 bp) were pooled and used as a probe. This NTRK4 probe was localised on 6p21. Of 50 metaphases analysed, 49 contained twin spot signals on both sister chromatids.

Background The progesterone receptor (PR) is expressed by ∼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples. Methods Archived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade. Results A total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p

Purpose: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma. Experimental Design: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n = 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n = 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level. Results: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (κ = 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%. Conclusions: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance. Clin Cancer Res; 17(4); 792–804. ©2011 AACR.

Purpose For a comprehensive overview of the genetic alterations of neuroblastoma, their association and clinical significance, we conducted a whole-genome DNA copy number analysis. Patients and Methods A series of 493 neuroblastoma (NB) samples was investigated by array-based comparative genomic hybridization in two consecutive steps (224, then 269 patients). Results Genomic analysis identified several types of profiles. Tumors presenting exclusively whole-chromosome copy number variations were associated with excellent survival. No disease-related death was observed in this group. In contrast, tumors with any type of segmental chromosome alterations characterized patients with a high risk of relapse. Patients with both numerical and segmental abnormalities clearly shared the higher risk of relapse of segmental-only patients. In a multivariate analysis, taking into account the genomic profile, but also previously described individual genetic and clinical markers with prognostic significance, the presence of segmental alterations with (HR, 7.3; 95% CI, 3.7 to 14.5; P < .001) or without MYCN amplification (HR, 4.5; 95% CI, 2.4 to 8.4; P < .001) was the strongest predictor of relapse; the other significant variables were age older than 18 months (HR, 1.8; 95% CI, 1.2 to 2.8; P = .004) and stage 4 (HR, 1.8; 95% CI, 1.2 to 2.7; P = .005). Finally, within tumors showing segmental alterations, stage 4, age, MYCN amplification, 1p and 11q deletions, and 1q gain were independent predictors of decreased overall survival. Conclusion The analysis of the overall genomic pattern, which probably unravels particular genomic instability mechanisms rather than the analysis of individual markers, is essential to predict relapse in NB patients. It adds critical prognostic information to conventional markers and should be included in future treatment stratification.

Stage 4 neuroblastoma (NB) are heterogeneous regarding their clinical presentations and behavior. Indeed infants (stage 4S and non‐stage 4S of age 365days). Our study aimed at: (i) identifying age‐based genomic and gene expression profiles of stage 4 NB supporting this clinical stratification; and (ii) finding a stage 4S NB signature. Differential genome and transcriptome analyses of a learning set of MYCN‐non amplified stage 4 NB tumors at diagnosis (n=29 tumors including 12 stage 4S) were performed using 1Mb BAC microarrays and Agilent 22K probes oligo‐microarrays. mRNA chips data following filtering yielded informative genes before supervised hierarchical clustering to identify relationship among tumor samples. After confirmation by quantitative RT‐PCR, a stage 4S NB's gene cluster was obtained and submitted to a validation set (n=22 tumors). Genomic abnormalities of infant's tumors (whole chromosomes gains or loss) differ radically from that of children (intra‐chromosomal rearrangements) but could not discriminate infants with 4S from those without this presentation. In contrast, differential gene expression by looking at both individual genes and whole biological pathways leads to a molecular stage 4S NB portrait which provides new biological clues about this fascinating entity.

In the last decade, new molecular techniques were introduced into pathology laboratories. Cytology also benefited from the innovations emerging from this new era. Molecular cytopathology (MCP) can be defined as molecular studies applied on all types of cytological specimens, namely gynaecology cytology, exfoliative non- gynaecology cytology and fine needle aspirates. The development of many new ancillary techniques has paralleled the emergence of clinical cytology as a major diagnostic specialty. Clinical applications of these techniques have been growing in the last decade. The widespread acceptance of liquid-based systems in gynaecological cytology emphasises the relation between cells and molecules. The increased use of morphology and molecular biology in human papillomavirus-induced lesions for example, showed the potential to optimise, in one single brushed sample, diagnosis and research. Cytology samples from serous effusions, the pulmonary tree, urine, and aspirations, among others, are now likely to be studied by different molecular techniques for diagnosis, prognosis, or even assessment of therapeutic targets. In this review, the main published results concerning the application of molecular techniques in different fields of cytopathology are highlighted, and their applications discussed.

Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to analyze the status of key molecular markers such as ERG gene rearrangement and AR amplification in tumor tissues (primitive tumor specimens and metastasis biopsies) and CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were detected using two methods, CellSearch (Veridex LLC, Raritan, NJ, USA) and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Epithelial (cytokeratins, EpCAM (Epithelial Cell Adhesion Molecule)) and mesenchymal (vimentin, N-cadherin) markers were tested. Molecular characterization of CTCs was performed on filters by a two step method combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH). Image analysis was performed using an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. ERG gene rearrangement and AR copy number gains were examined both in CTCs enriched on filters and in the cell fractions recovered from the magnests after CellSearch and spread on slides for FISH. Primitive tumor specimens and metastasis biopsies were also collected and analyzed by FISH. Results: Higher levels of CTCs were detected by filtration enrichment combined to four-color immunofluorescent staining compared to CellSearch. Filtration enriched CTCs expressed epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process in most PCa patients. A higher level of gene variability and tumor heterogeneity was observed in filtration enriched CTCs compared to those captured by the CellSearch. ERG gene rearrangement and AR amplification in prostate tissue, metastases, and filtration enriched CTCs were analysed by FISH. ERG gene rearrangement and AR copy number gain status present within the tumor tissue was in concordance with that found in filtration enriched CTCs. However, FISH experiments also revealed a much higher degree of cell-to-cell variability and tumor heterogeneity in filtration enriched CTCs compared to tumor tissues. Conclusion: We show here that prostate CTCs are highly heterogeneous in term of EMT markers and ERG gene rearrangement and AR amplification marker status. The high degree of cell-to-cell variability in ERG and AR gene patterns present in CTCs reveals important tumor heterogeneity and could have important implications for developing personalized anticancer strategies based on the use of CTCs in PCa patients. Citation Format: Christophe Massard, Marianne Oulhen, Alexander Valent, Sylvestre Lemoulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2014-3079

Neuroblastoma, the most common solid extracranial neoplasm in children, shows an appreciable variability in clinical evolution. Amplification of the MYCN oncogene in this tumor is detected in 25 to 30% of cases and is associated with poor clinical outcome. In this study, quantitative polymerase chain reaction and fluorescence in situ hybridization were used to determine MYCN amplification status in 46 neuroblastoma tumors. MYCN amplification was detected in tumors from 11 patients. Fluorescence in situ hybridization revealed the presence of micronuclei containing amplified MYCN sequences in 8 of the 11 tumors. Micronuclei are indicative of spontaneous elimination or loss of amplified sequences by tumor cells. Because the elimination of amplified sequences can be enhanced in vitro by specific drugs such as hydroxyurea, our observations suggest a new therapeutic strategy specifically targeted to cells with amplified genes.

Background: Hormonal therapy is considered one of the most effective treatment options for patients with estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancers (BC); however only a 50% objective response rate is observed as 1st line therapy of ER positive (ERpos) tumors (Bonneterre, 2001). An IHC diagnostic test to identify the activated form of the ER and PR has previous been described (ASCO 2013 abst # 592, 593 & 5602). The nuclear morphological of distribution of the ER and PR can be categorized in two patterns, a presumably not-activated D (diffuse pattern) or activated A (aggregated pattern). This classification is correlated with antiestrogen treatment outcome (ASCO 2013, abst # 592), and correlated with antiprogestin activity in vitro (AACR 2013, abst # 1317). Clinical activity of antiprogestins has been previously described (Robertson 1999, Jonat 2002). It is generally accepted that expression of PR is under the transcriptional control of ER, thus targeting ER can potentially suppresses PR expression. However, in breast cancer tissues although both ER and PR are present in the tumor specimen, there is low level of co-expression of ER/PR at the cellular level (ASCO 2013, abstract # 596). The goal of the study is to elucidate whether or not the activated form of ER and PR were coexpressed. Method: 293 ERpos or PRpos BC were obtained from the Oscar-Lambret Cancer Center, Lille, France. Each sample was analyzed with specific antibodies for ERα, PR A or PR B; a subset (53 for PR A and 49 for PR B cases) was analyzed by immunochemical duel staining on the same paraffin tumor section for ERα and PR A or PR B. Tumors were deemed receptor positive if >10% of the tumors cells (tc) were stained. Samples processed for duel staining came from the same dataset and were selected if they had both ERα and PR A and/or PR B present in more than 10% and less than 80% of stained tc. The distribution of ERα, PR A, PR B tc was described. Determination of the activated form of ERα (AERpos) and PR A (APRpos A), PR B (APRpos B) was performed using the previously described IHC technique at 100X magnification. Results: Standard receptor positivity (% of tumors tested) was as follows; ERα 85%, PR A 61% and PR B 65%, either PR A or B 68%. Duel staining receptor positivity (% of tumors) showed for the PR A subset: 72% ER pos, 74% PR A pos, and 9% ER/PR (% of tumors co-expressing); for the PR B subset: ER 60%, PR B 100%, and 13% ER/PR (% of co-expressing); Subnuclear Morphology: 216 cases were ER and PR positive, and of these, 23% were AERpos, 23% were APRpos A, 24%, APRpos B% and 31% APRpos for either A or B. In the 216 case dataset, 18% of the tumors had both AER and APR A or APR B; However, using the duel staining method no cases had more than 10% of the tc co-expressing i.e. AERpos and APRpos, indicating that the co-expression was principally at the tumor level and not at the cellular level. Conclusion: As known, BC commonly express both ER and PR; however the co-expression of ER and PR in malignant cells is uncommon, and this co-expression of ER and PR could be explained by chance. Although AER and APR are expressed within the same tumor, the activated forms of the ER and PR are not co-expressed at the cellular level. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-13.

Purpose: The diagnostic test of ALK rearrangement in non-small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine needle aspirations. The present study was designed to evaluate -1) whether ALK-rearrangement diagnosis could be performed using circulating tumor cells (CTCs), -2) whether CTCs harboring ALK-rearrangement could be monitored in ALK-positive patients treated by crizotinib. Patients and Methods: CTCs were isolated in 18 ALK-positive and 14 ALK-negative patients by blood filtration using ISET (Isolation by Size Epithelial Tumor cells) and tested by filter adapted-fluorescence in situ hybridization (FA-FISH), a FISH method optimized for filters. Numbers of ALK-rearranged cells and patterns of ALK-rearrangement were determined in CTCs and compared to those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratin, E-cadherin) and mesenchymal (vimentin, N-cadherin) markers expression. ALK-rearranged CTCs were monitored in ALK-positive patients treated by crizotinib. Results: ALK-rearranged CTCs [ranging from 4 to 34 CTCs /1mL] were detected in all ALK-positive patients, while no or only one ALK-rearranged CTCs was detected in blood samples obtained from ALK-negative patients. ALK-rearranged CTCs harboured a unique (3’ 5’) split pattern while heterogeneous patterns (3’ 5’, only 3’) of splits were present in tumors. ALK-rearranged CTCs exclusively expressed a mesenchymal phenotype and contrasted with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in levels of ALK-rearranged CTCs and in CTCs harboring a gain of ALK native copies were detected under crizotinib treatment. Conclusion: We report that ALK-rearrangement can be detected in CTCs of ALK-positive NSCLC patients using ISET and FA-FISH, enabling diagnostic testing for crizotinib treatment. Our results suggest that CTCs harboring a unique ALK-rearrangement and mesenchymal phenotype may arise from the clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC. The molecular characterization of CTCs in patients undergoing crizotinib treatment might provide new insights into mechanism of resistance to ALK tyrosine kinase inhibitors, and possible strategies to overcome this resistance. Citation Format: Emma Pailler, Julien Adam, Amélie Barthelemy, Marianne Oulhen, Nathalie Auger, Alexander Valent, David Planchard, Melissa Taylor, Isabelle Borget, Fabrice Andre, Jean-Charles Soria, Philippe Vielh, Benjamin Besse, Françoise Farace. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1470. doi:10.1158/1538-7445.AM2013-1470

Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to characterize the status of key molecular markers such as ERG gene rearrangement, and AR and c-myc amplifications, according to epithelial and/or mesenchymal marker expression in CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were isolated from the blood of 40 patients at day 0 and day 30 of abiraterone treatment. CTCs were detected using two methods, CellSearch® (Veridex LLC, Raritan, NJ, USA) and ISET (Isolation by Size of Epithelial Tumor cells). Molecular characterization of CTCs was performed on ISET filters by combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH®). To optimize pan-keratin detection, four pancytokeratin antibodies, including AE1/AE3, KL1, C11, and A45-B/B3, were tested on the LnCAP prostate cell line spiked in normal blood cells and on PCa CTCs enriched by ISET. Epithelial (cytokeratins, E-cadherin, EpCAM (Epithelial Cell Adhesion Molecule)), mesenchymal (vimentin, N-cadherin), PTEN, androgen receptor (AR) and Ki67 marker expression was detected by immunofluorescent staining on ISET filters according to a method we previously established. FA-FISH® was optimized for the detection of ERG gene rearrangement and AR and c-myc amplification or gain of copy number. A two step method combining immunofluorescent staining and FA-FISH® was established on an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. Results: Higher CTC levels were detected by ISET compared to CellSearch®. Quantitative analysis of the intensity values for each tested pan-keratins antibodies showed that KL1 and A45-B/B3 clones gave the strongest immunofluorescent signals both on the LnCAP cell line and PCa CTCs. CTCs expressing epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers were detected in most patients suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process. The loss of PTEN expression was also observed using hematopoietic cells present on ISET filters as a positive control of PTEN expression. ERG gene rearrangement, AR and c-myc amplifications were detected. The presence of these markers within CTC subsets expressing epithelial and/or mesenchymal markers is currently studied. Conclusion: Several molecular markers such as ERG gene rearrangement, AR and c-myc amplifications were detected in PCa CTCs. The presence of these markers in CTCs according to their EMT status will be presented. Citation Format: Marianne Oulhen, Christophe Massard, Alexander Valent, Rachel Young, Sylvestre Le Moulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1471. doi:10.1158/1538-7445.AM2013-1471

Background: Antihormonal agents (AH) are some of the most clinically useful anticancer drugs. AH resistance and the availability of alternative treatments have made patient selection increasingly important for AH drug development. We hypothesized that the progesterone receptor (PR) may be constitutively active because of the aberrant biological pathways and that selecting patients with baseline tumor PR activation would predict for antiprogestin activity. Activated PR (APR) can be visualized in nuclei as fluorescent aggregates via fluorescent green protein engineered PR cell lines in vitro and by immunofluorescence (IF) in tissues. The detection of PR foci has been associated with transcription and gene activation in vitro. PR phosphorylation (phos) is induced by a variety of pathways. We have developed a IHC method that allows APR determination in tumor biopsies on a routine basis (SABCS 2012). The predictive nature of the observation of APR by IHC in vitro has been tested with onapristone (ONA) for cell survival and proliferation. Methods: 8 human breast cancer (BT-474, CAMA-1, EVSA-T, HCC-1954, MCF-7, MDA-MB-231, T-47D, ZR-75-1) and 2 human endometrial cancer (Ishikawa, HEC-1-A) cell lines were studied. Cytoblocks for APR analysis were made for each time point and experimental condition, baseline ER/PR expression was determined by IHC and PR phos was analyzed with specific antibodies (pSER162, 190, 294, 400, 554) by Western Blotting and IHC. Culture conditions: normal and stripped FBS, +/- ONA and growth factor/hormone exposure (EGF, FGF2, E2 and P4) in triplicate. Analysis was performed at baseline (BL), 6h, 96h and 7 days. Cell viability was assessed by MTS assay, proliferation was studied by Ki67 analysis. APR foci were determined by IHC and read by a pathologist blinded to the experimental conditions. Results: At BL only two cell lines were PR pos/APR pos (T47D, CAMA-1) and all other cell lines where PR pos/APR neg (BT-474, EVSA-T, HCC-1954, MCF-7) or PR neg/APR neg (Ishikawa, HEC-1-A, MDA-MB-231, ZR-75-1). ONA exerted inhibitory effect only in APR pos cell lines. In stripped FBS at 6h, T47D APR pos status was converted to APR neg by ONA except with E2 stimulation which required longer treatment. The CAMA-1 data are not yet available. No APR neg cell lines were converted into APR pos except in one experiment after protracted exposure to EGF. ONA generally decreased phos; phos decrease measured by WB did not correlate with growth inhibition. Phos status determined by IHC and Ki67 proliferation assessment are currently in process and will be presented with the confirmatory data. Conclusions: Baseline APR positivity is predictive of ONA activity in vitro. ONA converts APR pos cells to APR neg within 6h in most cases, a time frame consistent with PR biology. The growth inhibitory effects of ONA were not correlated to PR phos in the cell lines and conditions tested. The APR IHC methodology may be applicable in the clinical setting. Citation Format: Erard Gilles, Laura Caplier, Alexander Valent, Guillaume Serin, Anne Gompel, Jacques Bosq, Alexander Zukiwski. Antiprogestin drug development: in vitro validation of a potential clinical biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2013-1317

Introduction: ERCC1 allows removal of branched DNA structures at essential steps in DNA repair pathways such as Nucleotide Excision Repair (NER), Fanconia-dependent Interstrand Cross-Link Repair (ICL-R), long-patch base excision repair (BER), and Single Strand Annealing (SSA). ERCC1 expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. The most frequent method to determine ERCC1 status is the evaluation of gene expression (IHC or RT-PCR). Here, we studied the prevalence of ERCC1 copy number variation (CNV) using FISH. Methods: FISH was performed with Poseidon™ dual color probe set, ERCC1 (19q13) and ZNF443 (19p13) on a panel of 17 NSCLC cell lines and 324 FFPE tumor samples from resected NSCLC patients (103 from IGR, Villejuif, France, 221 from MDACC, Houston, USA). Gene amplification (GA) and high polysomy (HP) were defined following actual consensus rules for EGFR assessment. Results: ERCC1 copy number was increased (>2) in a large majority of cell lines (13/17) of which three displayed gene amplification (GA) (H2342, CN=5; H1299, CN>12; HOP62, CN>15). In human samples, overall ERCC1 gene abnormalities were detected in 65 cases (20.1%) including 31 GA (6.9%, the majority with clusters) and 34 HP (13.7%, more frequent in squamous cell carcinomas). In patients with ERCC1 expression status available (immunohistochemistry) we found a non-significant trend for higher ERCC1 expression in HP patients compared to GA and low CNV patients (P=0.06). Despite a 3-fold increase in IC50 of cisplatin in cell lines with ERCC1 gene amplification compared to normal cells (with no CNV) the ERCC1 FISH status seemed unable to predict chemosensitivity in resected NSCLC patients. In patients not treated with chemotherapy, DFS was significantly longer in patients with normal ERCC1 FISH status of the tumor compared to patients with ERCC1 gene amplification (P=0.02). Conclusion: ERCC1 copy number variation is a frequent event in NSCLC and could reflect genomic instability, but the clinical relevance of this observation needs further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3098. doi:1538-7445.AM2012-3098