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Supplementary Data from Marked Activity of Irinotecan and Rapamycin Combination toward Colon Cancer Cells In vivo and In vitro Is Mediated through Cooperative Modulation of the Mammalian Target of Rapamycin/Hypoxia-Inducible Factor-1α Axis

Purpose: Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1α (HIF-1α), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1α are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1α. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models.Experimental Design: Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro.Results: In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1α axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1α rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways.Conclusion: These results identify HIF-1α as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

KRAS is the most frequently mutated oncogene in non-small cell lung cancers (NSCLC), with a frequency of around 30%, and encoding a GTPAse that cycles between active form (GTP-bound) to inactive form (GDP-bound). The

Simple Summary Non-small cell lung cancer (NSCLC) is the most common cancer in the world. For common EGFR mutations, treatment is based on different inhibitors. Despite the excellent disease control with inhibitors, acquired resistance inevitably occurs and remains a biological challenge. This leads to the discovery of novel biomarkers and some possible drug targets. Resistance mechanisms could be involved as gene mutations, amplifications or fusions, which could be detected by different molecular techniques on different types of biological samples. Histological transformation is another mechanism of resistance with some biological predictive factors that needs tumor biopsy. The place of liquid biopsy also depends on the generation/line of inhibitors and could be a good candidate for molecular monitoring. This article is based on the literature and proposes actual and future directions in clinical and translational research. Abstract Non-small cell lung cancer (NSCLC) is the most common cancer in the world. Activating epidermal growth factor receptor (EGFR) gene mutations are a positive predictive factor for EGFR tyrosine kinase inhibitors (TKIs). For common EGFR mutations (Del19, L858R), the standard first-line treatment is actually third-generation TKI, osimertinib. In the case of first-line treatment by first (erlotinib, gefitinib)- or second-generation (afatinib) TKIs, osimertinib is approved in second-line treatment for patients with T790M EGFR mutation. Despite the excellent disease control results with EGFR TKIs, acquired resistance inevitably occurs and remains a biological challenge. This leads to the discovery of novel biomarkers and possible drug targets, which vary among the generation/line of EGFR TKIs. Besides EGFR second/third mutations, alternative mechanisms could be involved, such as gene amplification or gene fusion, which could be detected by different molecular techniques on different types of biological samples. Histological transformation is another mechanism of resistance with some biological predictive factors that needs tumor biopsy. The place of liquid biopsy also depends on the generation/line of EGFR TKIs and should be a good candidate for molecular monitoring. This article is based on the literature and proposes actual and future directions in clinical and translational research.

KRAS is the most frequently mutated oncogene in non-small cell lung cancers (NSCLC), with a frequency of around 30%, and encoding a GTPAse that cycles between active form (GTP-bound) to inactive form (GDP-bound). The KRAS mutations favor the active form with inhibition of GTPAse activity. KRAS mutations are often with poor response of EGFR targeted therapies. KRAS mutations are good predictive factor for immunotherapy. The lack of success with direct targeting of KRAS proteins, downstream inhibition of KRAS effector pathways, and other strategies contributed to a focus on developing mutation-specific KRAS inhibitors. KRAS p.G12C mutation is one of the most frequent KRAS mutation in NSCLC, especially in current and former smokers (over 40%), which occurs among approximately 12–14% of NSCLC tumors. The mutated cysteine resides next to a pocket (P2) of the switch II region, and P2 is present only in the inactive GDP-bound KRAS. Small molecules such as sotorasib are now the first targeted drugs for KRAS G12C mutation, preventing conversion of the mutant protein to GTP-bound active state. Little is known about primary or acquired resistance. Acquired resistance does occur and may be due to genetic alterations in the nucleotide exchange function or adaptative mechanisms in either downstream pathways or in newly expressed KRAS G12C mutation.

The prognostic value of exon 19 and 21 EGFR mutations in stage IV non-small cell lung cancer (NSCLC) is well established. We aimed to evaluate the prognostic value of the mutations in surgically resected NSCLC. We retrospectively reviewed data from 1798 surgically resected NSCLC adenocarcinomas between 2007 and 2017 in three departments of thoracic surgery (Nancy/Strasbourg, France, and Torino, Italy) for whom mutational status was known. Overall survival (OS) was evaluated using log-rank and Cox proportional hazard models. EGFR exon 19 deletion was observed in 108 patients (55.1%) and exon 21 L858R mutations were observed in 88 patients (44.9%). In stage I, the median OS was not significantly different between exons 19 and 21 (p = 0.54), while, in stage II, the median OS reached 65 months [95% confidence interval (CI) 41.67–88.33] for exon 19 mutations and decreased to 48 months for exon 21 mutations (95% CI 44.21–51.79; p = 0.027). In multivariate analysis, exon 19 deletion remained a favorable prognostic factor [hazard ratio (HR) 0.314, 95% CI 0.098–0.997; p = 0.05]. In stage III, the median OS reached 66 months (95% CI 44.67–87.32) for exon 19 mutations and decreased to 32 months for exon 21 mutations (95% CI 29.86–34.14; p = 0.03). In multivariate analysis, exon 19 deletion remained a significantly favorable prognostic factor (HR 0.165, 95% CI 0.027–0.999; p = 0.05). The prognostic value of EGFR exon 19 and 21 mutations appears to be different according to disease stage in surgically resected NSCLC.

Non-small cell lung cancer (NSCLC) is the most common cancer in the world. Activating epidermal growth factor receptor (EGFR) gene mutations are a positive predictive factor for EGFR tyrosine kinase inhibitors (TKIs). For common EGFR mutations (Del19, L858R), the standard first-line treatment is actually third-generation TKI, osimertinib. In the case of first-line treatment by first (erlotinib, gefitinib)- or second-generation (afatinib) TKIs, osimertinib is approved in second-line treatment for patients with T790M EGFR mutation. Despite the excellent disease control results with EGFR TKIs, acquired resistance inevitably occurs and remains a biological challenge. This leads to the discovery of novel biomarkers and possible drug targets, which vary among the generation/line of EGFR TKIs. Besides EGFR second/third mutations, alternative mechanisms could be involved, such as gene amplification or gene fusion, which could be detected by different molecular techniques on different types of biological samples. Histological transformation is another mechanism of resistance with some biological predictive factors that needs tumor biopsy. The place of liquid biopsy also depends on the generation/line of EGFR TKIs and should be a good candidate for molecular monitoring. This article is based on the literature and proposes actual and future directions in clinical and translational research.

OBJECTIVES Epidermal growth factor receptor (mEGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogen homologue (mKRAS) mutations are the two main oncogenic drivers in resected non-small-cell lung cancer (NSCLC). We aimed to evaluate the correlation between histopathological prognostic factors and these mutations in resected NSCLC. METHODS We retrospectively reviewed data from 841 patients who underwent a surgical resection with a curative intent for NSCLC between 2007 and 2012. RESULTS KRAS mutations were observed in 255 patients (32%) and mEGFR in 103 patients (12%). A correlation was observed between mKRAS patients and lymph node involvement [Cramer's V: 0.451, P < 0.001, OR: 7.5 (95% CI: 5.3-10.7), P < 0.001]. Otherwise, a correlation was observed between mKRAS and the risk of harbouring 2 N2 stations [Cramer's V: 0.235, P = 0.02, OR: 3.04 (95% CI: 1.5-6.3), P = 0.004]. High lymph node ratio and angioinvasion were also significantly more frequent in mKRAS [Cramer's V: 0.373, P < 0.001, OR: 6.37 (95% CI: 3.9-10.5), P < 0.001; and Cramer's V: 0.269, P < 0.001, OR: 3.25 (95% CI: 2.4-4.4), P < 0.001, respectively]. Skip N2 and microscopic N2 were significantly more frequent in mEGFR [Cramer's V: 0.459, P < 0.001, OR: 18 (95% CI: 5.6-57.8), P < 0.001; and (Cramer's V: 0.45, P < 0.001 OR: 21.14 (95% CI: 9.2-48.3), P < 0.001, respectively]. CONCLUSIONS We observed a correlation between mKRAS and negative histopathological prognostic factors and between mEGFR and positive prognostic factors. One can wonder whether histopathological prognostic factors are only clinical reflections of molecular alterations.

Background: We aimed to evaluate whether EGFR mutations (mEGFR) and KRAS amino acid substitutions can predict first site of recurrence or metastasis after non-small-cell lung cancer (NSCLC) surgery. Methods: Data were reviewed from 481 patients who underwent thoracic surgery for NSCLC between 2007 and 2012. Results: Patients with KRAS G12C developed significantly more bone metastases compared with the remainder of the cohort (59% vs 16%, P

Summary Background Advances in molecular genetics of sarcoma have enabled the identification of type-specific aberrations. We aimed to assess the clinical effect of systematic implementation of molecular assays to improve sarcoma misdiagnosis. Methods In this multicentre, observational study, we recruited patients from 32 centres of the French Sarcoma Group/Reference Network in Pathology of Sarcomas. Eligibility criteria included: biopsy or surgical resection; suspicion of: dermatofibrosarcoma protuberans (cohort 1), dedifferentiated liposarcoma (cohort 2), Ewing's sarcoma family of tumours (cohort 3), synovial sarcoma (cohort 4), alveolar rhabdomyosarcoma (cohort 5), and myxoid or round cell liposarcoma (cohort 6); review by one sarcoma-expert pathologist; availability of frozen material (except for cohort 1 of patients with dermatofibrosarcoma protuberans because anti-CD34 immunohistochemistry is performed on paraffin-embedded tissue); and patient information. For each case, the pathologist made one primary diagnosis followed by up to two differential diagnoses, based on histological characteristics only. Each diagnosis was classified as certain, probable, or possible. For each case to determine the molecular classification, we did fluorescence in-situ hybridisation on paraffin-embedded samples. We also did comparative genomic hybridisation and quantitative PCR (cohort 2) or reverse transcriptase PCR (cohorts 3–6) on frozen and paraffin-embedded samples. We made a final diagnosis based on the molecular results. The clinical effect of diagnosis correction was assessed by a board of experts. Finding Between June 22, 2009, and Oct 30, 2012, 395 patients were enrolled in the study, of which 384 were eligible for inclusion. The diagnosis was eventually modified by molecular genetics for 53 patients: eight (16%) of 50 patients with dermatofibrosarcoma (cohort 1), seven (23%) of 30 patients with dedifferentiated liposarcoma (cohort 2), 13 (12%) of 112 with Ewing's sarcoma family of tumours (cohort 3), 16 (16%) of 97 patients with synovial sarcoma (cohort 4), seven (15%) of 46 patients with alveolar rhabdomyosarcoma (cohort 5), and two (4%) of 49 patients with myxoid or round cell liposarcoma (cohort 6), with an effect on primary management or prognosis assessment in 45 cases. Interpretation Molecular genetic testing should be mandatory for diagnostic accuracy of sarcoma and appropriate clinical management, even when histological diagnosis is made by pathologist experts in this field. Funding French National Cancer Institute and Nice University Hospital.

Resume Les progres dans le demantelement moleculaire des CBNPC ont conduit a des avancees significatives dans le traitement des tumeurs solides. Les alterations moleculaires qui peuvent etre ciblables par des therapeutiques ciblees correspondent soit a des mutations de genes (Epidermal Growth Factor Receptor [EGFR], KRAS, HER2, BRAF), soit a des rearrangements de genes (Anaplastic Lymphoma Kinase [ALK], ROS1, RET), impliquant qu'outils et methodes soient appropries pour les detecter. Cet article decrit les problematiques liees aux differents types de prelevements realises en vue d'une analyse moleculaire, les criteres de qualite/selection des echantillons tumoraux, les differentes techniques de biologie moleculaires utilisees pour la recherche des mutations, avec en perspective l'utilisation du sequencage de nouvelle generation (NGS), ainsi que l'interpretation des resultats.

Emerging data highlight different clinical behaviors according to KRAS amino acid substitutions (AASs) in patients with non-small-cell lung cancer (NSCLC). We aimed to evaluate whether different KRAS AASs were associated with different responses to chemotherapy.We retrospectively reviewed data from 1190 patients with KRAS mutations who underwent first-line platinum-based chemotherapy for stage IV NSCLC. The response to different chemotherapy regimens was evaluated using the Response Evaluation Criteria In Solid Tumors criteria (v 1.1). Overall survival and time to progression (TTP) were secondary endpoints.Taxane was associated with the best response in the entire cohort (odds ratio, 2.52; 95% confidence interval [CI], 1.82-3.48; P .001), especially in G12V patients (odds ratio, 2.15; 95% CI, 1.05-4.41; P = .036). Taxane was associated with improved TTP in the entire cohort (hazard ratio [HR], 0.31; 95% CI, 0.26-0.38; P .001), especially in G13D patients (HR, 0.47; 95% CI, 0.22-1.01; P = .054). Pemetrexed was associated with the worst TTP in the entire cohort, particularly in G12V patients, who had the worst response rates (HR, 0.55; 95% CI, 0.30-0.99; P = .049). No impact on overall survival was observed according to different chemotherapy regimens and AASs.KRAS-specific AAS appears to induce different responses to chemotherapy regimens after first-line platinum-based chemotherapy in advanced NSCLC.

Epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non–small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%–16% versus 40%; 95% CI, 39%–42%; P

Sarcomas represent a complex and heterogeneous group of rare malignant tumors and their correct diagnosis is often difficult. Recent molecular biological techniques have been of great diagnostic use and there is a need to assess the cost of these procedures in routine clinical practice. Using prospective and observational data from eight molecular biology laboratories in France, we used "microcosting" method to assess the cost of molecular biological techniques in the diagnosis of five types of sarcoma. The mean cost of fluorescence in situ hybridization (FISH) was 318 € (273-393) per sample; mean reverse transcription polymerase chain reaction (RT-PCR) cost ranged from 300 € (229-481) per formalin-fixed, paraffin-embedded specimen to 258 € (213-339) per frozen specimen; mean quantitative polymerase chain reaction (Q-PCR) cost was 184 € (112-229) and mean CGH-array cost was 332 € (329-335). The cost of these recently implemented techniques varied according to the type of sarcoma; the method of tissue collection and local organizational factors including the level of local expertise and investment. The cost of molecular diagnostic techniques needs to be balanced against their respective performance.

Objectives The impact of skip N2 metastases (i.e. N2 lymph node metastases without N1) on survival in surgically resected non-small lung cancer remains an intriguing and rarely investigated topic. The goal of our study was to elucidate (i) skip N2 influence on overall survival (OS) and time to recurrence (TTR) in patients with resected lung adenocarcinoma and (ii) its link with epidermal growth factor receptor ( EGFR ) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS ) mutations. Methods A retrospective analysis of 279 consecutive patients with lung pN2 adenocarcinoma, operated in two institutions between 2003 and 2013, was conducted. OS and TTR were calculated using the Kaplan-Meier method. Crude and multivariable-adjusted comparisons by skip N2 for OS and TTR were performed using the Cox method with shared frailty (accounting for the within-centre correlation). Associations between skip N2 metastasis, clinicopathological characteristics and EGFR and KRAS mutations were investigated using the Fisher exact test and Cramer's V -test. Results The mean age at the time of surgery was 63 years (±12), and the median follow-up time was 36 months (min 3; max 101). Skip N2 was observed in 54 patients (19%). EGFR mutations were observed in 38 patients (14%); KRAS mutations were seen in 86 patients (31%). Patients with skip N2 metastasis were predominantly non-smokers ( P = 0.001), underwent segmentectomy or limited resections ( P = 0.004) and were not submitted to adjuvant therapy ( P = 0.022). Moreover, there was a correlation between EGFR mutations and skip N2 (Cramer's V : 0.25, P < 0.001). Indeed, EGFR mutations were significantly more frequent in skip N2 tumours (33%) compared with non-skip tumours (10%), P < 0.001. No correlation between skip N2 and KRAS mutations was observed (Cramer's V : 0.05, P = 0.46). The multivariable-adjusted model showed a significant skip N2 protective effect on OS (hazard ratio, HR 0.503; P = 0.014; 95% confidence interval, CI: 0.291-0.8704) but not on TTR (HR 0.788; P = 0.446; 95% CI: 0.427-1.454). Conclusions In our series, lung adenocarcinoma skip N2 metastasis demonstrated a favourable prognosis. The presence of EGFR mutations could have significance in the better survival and in the specific anatomic pathway of lymphatic metastases exhibited by skip N2 tumours.

// Stephane Renaud 1, 2, 3 , Francesco Guerrera 1, 4 , Joseph Seitlinger 1 , Lorena Costardi 4 , Mickael Schaeffer 5 , Benoit Romain 3, 6 , Claudio Mossetti 4 , Anne Claire-Voegeli 7 , Pier Luigi Filosso 4 , Michele Legrain 7 , Enrico Ruffini 4 , Pierre-Emmanuel Falcoz 1 , Alberto Oliaro 4 , Gilbert Massard 1 1 Department of Thoracic Surgery, Strasbourg University Hospital, Strasbourg, France 2 Department of Thoracic Surgery, Nancy University Hospital, Nancy, France 3 Research Unit EA3430, Tumoral Progression and Micro-Environment, Epidemiological and Translational Approaches, Strasbourg University, Strasbourg, France 4 Department of Thoracic Surgery, Azienda Ospedaliera Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 5 Department of Biostatistics, Strasbourg University Hospital, Strasbourg, France 6 Department of General and Digestive Surgery, Strasbourg University Hospital, Strasbourg, France 7 Department of Molecular Biology, Strasbourg University Hospital, Strasbourg, France Correspondence to: Stephane Renaud, email: sterenaud0@gmail.com Keywords: lung metastasectomy, colorectal cancer, KRAS, surgery, codon Received: August 25, 2016 Accepted: November 21, 2016 Published: November 29, 2016 ABSTRACT Introduction: The utilization of molecular markers as routinely used biomarkers is steadily increasing. We aimed to evaluate the potential different prognostic values of KRAS exon 2 codons 12 and 13 after lung metastasectomy in colorectal cancer (CRC). Results: KRAS codon 12 mutations were observed in 116 patients (77%), whereas codon 13 mutations were observed in 34 patients (23%). KRAS codon 13 mutations were associated with both longer time to pulmonary recurrence (TTPR) (median TTPR: 78 months (95% CI: 50.61–82.56) vs 56 months (95% CI: 68.71–127.51), P = 0.008) and improved overall survival (OS) (median OS: 82 months vs 54 months (95% CI: 48.93–59.07), P = 0.009). Multivariate analysis confirmed that codon 13 mutations were associated with better outcomes (TTPR: HR: 0.40 (95% CI: 0.17–0.93), P = 0.033); OS: HR: 0.39 (95% CI: 0.14–1.07), P = 0.07). Otherwise, no significant difference in OS ( P = 0.78) or TTPR ( P = 0.72) based on the type of amino-acid substitutions was observed among KRAS codon 12 mutations. Materials and Methods: We retrospectively reviewed data from 525 patients who underwent a lung metastasectomy for CRC in two departments of thoracic surgery from 1998 to 2015 and focused on 150 patients that had KRAS exon 2 codon 12/13 mutations. Conclusions: KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC.

OBJECTIVES The role of perioperative chemotherapy (POC) and targeted therapies in lung metastasectomy for colorectal cancer (CRC) is still subject to debate. We aimed to evaluate whether POC and targeted therapies were associated with different outcomes according to the mutational status. METHODS We reviewed data from 223 patients who underwent pulmonary metastasectomy for CRC from 1998 to 2015 and for whom the V-Ki-ras2 Kirsten sarcoma viral oncogene homologue (KRAS) and V-raf Murine sarcoma viral oncogene homologue B1 (BRAF) mutational statuses were known. RESULTS A total of 167 patients (74%) underwent POC: 62 (37%) received neoadjuvant therapy, 59 (35%) were in the adjuvant setting and 46 (28%) were in both the neoadjuvant and adjuvant settings. POC did not significantly influence either the loco-regional recurrence free survival (LRRFS) (P = 0.21) or the overall survival (OS) (P = 0.29). Furthermore, in cases of adjuvant chemotherapy, outcomes were not significantly different in cases of neoadjuvant chemotherapy or both neoadjuvant and adjuvant treatment (P = 0.26 for OS, P = 0.14 for LRRFS). For patients with KRAS mutation, perioperative bevacizumab was associated with a significant improvement in both LRRFS [70 months (41.58–98.42) vs 24 months (1.15–46.86), P = 0.001] and OS [101 vs 55 months (49.77–60.23), P = 0.004]. However, this benefit was only significant in cases of KRAS exon 2 codon 12 mutations [median OS: 101 months (83.97–118.02) vs 60 months (53–66.99), P

To the Editor: Somatic epidermal growth factor receptor ( EGFR ) mutations are now routinely integrated in the molecular diagnosis of nonsmall cell lung cancers (NSCLC) [1, 2]. Thus, germ-line EGFR mutations are rarely mentioned or looked for in the context of patients with a family history of cancer, as their association with NSCLC familial cancer risk is not well established. Moreover, the predictive value of these mutations for response to EGFR tyrosine kinase inhibitors (TKIs) is not well known [3]. We report here two different heterozygous germ-line EGFR variants identified in two Caucasian NSCLC patients, who demonstrated different responses to EGFR-TKI. A 63-year-old Caucasian, male former smoker with no family history of cancer (fig. 1a), was admitted for dyspnoea in August 2011, leading to discovery of a lower left lobe lung tumour and pleural effusion. Trans-thoracic biopsy diagnosed an invasive, acinar-predominant adenocarcinoma, classified cT2a N3 M1a (stage IV). Molecular analyses of the tumour by direct sequencing identified two concomitant heterozygous EGFR exon 21 mutations, L858R and V843I, confirmed in two independent experiments (fig. 1b). The V843I variant, but not L858R, was also detected in DNA obtained from a blood sample, with written informed consent, confirming a germ-line mutation (fig. 1c). Following treatment with cisplatin and pemetrexed, the patient relapsed in December 2012 with vertebral metastasis. An EGFR-TKI (erlotinib) was initiated, resulting in a stable disease for 9 months. Figure 1. a–c) Case 1, with epidermal growth factor receptor ( EGFR ) exon 21 mutations (V843I and L858R). a) Pedigree chart. The black case corresponds to the index patient. b) DNA sequencing electrophoretograms for DNA obtained from lung tumour tissue identifying EGFR exon 21 mutations; both mutations are present. c) DNA sequencing electrophoretograms for DNA obtained from blood, identifying one EGFR exon 21 mutation, the V834I variant, is present and confirming …

Aggressive fibromatosis (AF) or desmoid tumor is a rare condition, characterized by deep tissue invasion by a monoclonal fibroblastic neoplasm, developed from musculoaponeurotic structures. Surgery is the treatment of choice, but negative margins can hardly been achieved in large tumors, and can lead to major functional disability. AF medical therapy includes nonsteroids anti-inflammatory drugs, tamoxifen, with inconsistent results. Several reports of imatinib efficacy in AF appear in the literature. Here, we describe for the first time a V530IKITexon 10 mutant that was associated to a dramatic imatinib response in an extraabdominal aggressive fibromatosis. The previously discovered V530I substitution was characterized in the core binding factor AML, but had never been reported in any other condition, so far. In this paper, we discuss theKITexon 10 mutations or polymorphisms that have been described in a variety of KIT-related conditions, including acute myelogenous leukemia, mastocytosis, and aggressive fibromatosis.

Purpose: Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1α (HIF-1α), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1α are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1α. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models. Experimental Design: Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro. Results: In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1α axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1α rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways. Conclusion: These results identify HIF-1α as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

Background: Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. The acute response to stress causes the release of catecholamines from the adrenal medulla accompanied by chromogranin A (CGA). To date, no study has evaluated the prognostic value of CGA in critically ill intensive care unit patients. Methods: We conducted a prospective study of intensive care unit patients by measuring serum procalcitonin (PCT), C-reactive protein (CRP), and CGA at the time of admission. Univariate and multivariate analyses were performed to evaluate the ability of these biomarkers to predict mortality. Results: In 120 consecutive patients, we found positive correlations between CGA and the following: CRP (r2 = 0.216; P = 0.02), PCT (r2 = 0.396; P < 0.001), Simplified Acute Physiologic Score II (SAPS II) (r2 = 0.438; P < 0.001), and the Logistic Organ Dysfunction System (LODS) score (r2 = 0.374; P < 0.001). Nonsurvivors had significantly higher CGA and PCT concentrations than survivors [median (interquartile range): 293.0 μg/L (163.5–699.5 μg/L) vs 86.0 μg/L (53.8–175.3 μg/L) for CGA, and 6.78 μg/L (2.39–22.92 μg/L) vs 0.54 μg/L (0.16–6.28 μg/L) for PCT; P < 0.001 for both comparisons]. In a multivariable linear regression analysis, creatinine (P < 0.001), age (P < 0.001), and SAPS II (P = 0.002) were the only significant independent variables predicting CGA concentration (r2 = 0.352). A multivariate Cox regression analysis identified 3 independent factors predicting death: log-normalized CGA concentration [hazard ratio (HR), 7.248; 95% confidence interval (CI), 3.004–17.487], SAPS II (HR, 1.046; 95% CI, 1.026–1.067), and cardiogenic shock (HR, 3.920; 95% CI, 1.731–8.880). Conclusions: CGA is a strong and independent indicator of prognosis in critically ill nonsurgical patients.

Introduction Recent clinical success of epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) have raised hopes that targeting other deregulated growth factor signaling, such as the hepatocyte growth factor/MET pathway, will lead to new therapeutic options for NSCLC. Furthermore, NSCLC present secondary EGFR-TKIs resistance related to exons 20 and 19 EGFR mutations or more recently to MET amplification. The aim of this study was to determine MET copy number related to EGFR copy number and K-Ras mutations in a targeted TKI naive NSCLC cohort. Methods We investigated 106 frozen tumors from surgically resected NSCLC patients. Genes copy number of MET and EGFR were assessed by quantitative relative real-time polymerase chain reaction and K-Ras mutations by sequencing. Results MET is amplified in 22 cases (21%) and deleted in nine cases (8.5%). EGFR is amplified in 31 cases (29%). K-Ras is mutated in 11 cases (10.5%). As observed for EGFR amplification, MET amplification is never associated with K-Ras mutation. MET amplification could be associated with EGFR amplification. MET amplification is not related to clinical and pathologic features. MET amplification and EGFR amplification showed a trend toward poor prognosis in adenocarcinomas. Conclusion In EGFR-TKIs naive NSCLC patients, MET amplification is a frequent event, which could be associated with EGFR amplification, but not with K-Ras mutation. MET amplification may identify a subset of NSCLC for new targeted therapy. It will also be important to evaluate MET copy number to properly interpret future clinical trials.

Background: Our study aimed to evaluate response rate (RR) to brain metastasis radiotherapy (RT), depending on the genomic status of non-small-cell lung cancer. Material & methods: We retrospectively reviewed 1971 non-small-cell lung cancer files of patients with EGFR and KRAS testing and focused on 157 patients who had undergone RT for brain metastasis. Results: A total of 16 patients (10.2%) harbored EGFR mutations (mEGFR) and 45 patients (28.7%) KRAS (mKRAS). In univariate analysis, RR was significantly higher for mEGFR compared with wild-type EGFR/KRAS (odds ratio [OR]: 4.96; p = 0.05) or mKRAS (OR: 1.81; p = 0.03). In multivariate analysis, KRAS G12V or G12C status was associated with both poor RR (OR: 0.1; p < 0.0001) and overall survival (OR: 3.41; p < 0.0001). Conclusion: mEGFR are associated with higher RR to brain RT than wild-type EGFR/RAS or mKRAS.

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

Contexte La recherche des mutations du gene EGFR est effectuee en routine sur les prelevements de cancers bronchiques non a petites cellules (CBNPC) non epidermoides. La cellularite tumorale de l’echantillon analyse (rapport en pourcentage correspondant au nombre total de cellules tumorales/nombre total de cellules, tous types confondus) est une donnee importante pour interpreter les resultats de biologie moleculaire en fonction de la sensibilite de la technique utilisee. Un pourcentage de cellules tumorales > 20 % est requis pour la recherche de mutation par sequencage direct, et > 10 % pour l’analyse de fragment. La tendance serait de ne pas tenter d’effectuer d’analyses moleculaires sur les echantillons n’atteignant pas ces seuils, et de demander la realisation d’un autre prelevement. Nous rapportons trois cas pour lesquels l’echantillon renfermait moins de 10 % de cellules tumorales, ce qui n’a pas empeche la mise en evidence d’une mutation d’ EGFR en raison d’une amplification genique d’ EGFR . Cas cliniques Un des cas correspondait a une biopsie d’opacite pulmonaire mettant en evidence un adenocarcinome (cellularite tumorale de 7 %), les deux autres a des culots d’inclusion en paraffine de liquides d’epanchement pleural metastatique d’adenocarcinomes pulmonaires (cellularite tumorale de 5 et 7 %). Une mutation de l’exon 19 d’ EGFR etait detectee dans les trois cas, en analyse de fragment et en sequencage direct, avec une intensite de la mutation evaluee a 90 % de cellules mutees dans l’echantillon par rapport au temoin positif. Une relecture anatomo-pathologique confirmait la faible cellularite tumorale et l’analyse moleculaire de nouveaux echantillons des memes prelevements confirmait les resultats initiaux, ce qui ecartait l’hypothese d’un faux-positif. Devant cette discordance, l’analyse du nombre de copies du gene EGFR etait realisee sur coupes en paraffine par technique SISH (Silver In Situ Hybridization, kit Ventana). Elle mettait en evidence une amplification du gene EGFR en clusters dans les trois cas. Discussion La presence de plus de 2 copies du gene EGFR peut influencer la sensibilite de detection des alleles mutes : augmentation de la sensibilite de detection quand l’amplification touche l’allele mute (comme dans nos trois cas), ou au contraire diminution de la sensibilite en cas d’amplification touchant l’allele sauvage. D’apres les donnees de la litterature, environ la moitie des CBNPC mutes EGFR presente une amplification genique associee, amplification touchant preferentiellement l’allele mute. Conclusion Nous montrons a travers trois cas de « petits prelevements » d’adenocarcinomes pulmonaires (une biopsie pulmonaire et deux culots d’inclusion de liquide d’epanchement pleural metastatique) qu’en cas d’amplification genique touchant l’allele mute du gene EGFR dans les cellules tumorales, une mutation peut etre mise en evidence malgre un faible pourcentage de cellules tumorales. Ces elements invitent a ne pas exclure d’emblee des prelevements dont la cellularite tumorale est inferieure au seuil de sensibilite usuel de la technique moleculaire utilisee.

International audience; Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

Crohn's disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn's disease the frequency of circulating CD4(+)CD25(high) T cells that possess regulatory T-cell functions and CD4(+)CD25(low) T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies in a cohort of 66 patients with Crohn's disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4(+)CD25(high) T-cell frequency was significantly lowered in naïve Crohn's disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4(+)CD25(low) T-cell frequency was increased in Crohn's disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4(+)CD25(high) and CD4(+)CD25(low) T-cell frequencies are altered in naïve Crohn's disease resulting in an imbalance between both populations and a relative contraction of the CD4(+)CD25(high) T-cell population.

Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

The graft versus Host Disease (GvHD) is a major complication of allogenic hematopoietic stem cell transplantation (allo-HSCT) in term of frequency and/or mortality. The complex physiopathology is roughly characterized by 3 phases where several inflammation factors and apoptotic pathways are involved. Among them, the chemokines CCL5 and CXCL12 are mainly playing a role in the homing of hematopoietic stem cells (HSC) to bone marrow and the migration of lymphocytes to the target organs. This migration is mediated by adhesion markers like ICAM-1 (Inter Cellular Adhesion Molecule 1) or by growth factor like VEGF (Vascular Endothelial Growth Factor). The final necrotic changes in target organs due to inflammatory mechanisms involves Interleukin 2, 6 and 8 (IL2, IL6, IL8) and the activation of Nuclear Factor kappa B (NFkB). Different Single Nucleotide Polymorphisms (SNP) in several chemokines and cytokines were shown to modify the level of gene expression. The aim of this study was to identify, in the recipient before allo- HSCT, some predictive factors of occurrence of GvHD, among the molecules involved in the different steps of GvHD. After informed consent, 73 patients (26 children and 47 adults) were included. SNPs -C28G and -G403A in the CCL5 promoter and G801A in the 3′UTR region of CXCL12 were studied and expression of CCL5 was analysed by RT-QPCR. To verify their impact on corresponding gene expression, protein measurements of these chemokines (CCL5 and CXCL12) but also of their inflammatory partners IL2, IL6, IL8, VEGF, ICAM-1 and endoglin were done in 39 samples. Among the 73 patients, 41 have developed a GvHD (Grade II-IV) and 32 did not developed GvHD or developed only grade I GvHD. As shown in table 1, the global frequency of SNPs underlined an unbalanced distribution in this cohort. CCL5 CXCL12 -G403A -C28G G801A G/G G/A A/A C/C C/G G/G G/G G/A A/A 43 (62.3%) 20 (29%) 6 (8.7%) 69 (94.5%) 4 (5.5%) 0 36 (49.3%) 34 (46.6%) 3 (4.4%) Table1: Repartition of the different SNP of CCL5 and CXCL12 However, we did not observed a link between the SNP of CCL5, its expression (p=0.812) or its protein concentration (p=0.993) and the occurrence of a GvHD. Serum concentration of VEGF, CXCL12 and Endoglin were different between adults and children. In fact, in adult, the VEGF and Endoglin serum concentrations were significantly higher than in pediatric samples, with, respectively, 30.66 pg/ml [6.92;100.53] vs 9.97 pg/ml [0;31.29] and 18831.49 pg/ml [17787.7;25190.86] vs 11104.52 pg/ml [7742.54;16231.12], in opposition to CXCL12, which is higher in pediatric patients (54,76 pg/ml [43,74 ; 70,82] vs 35,1 pg/ml [22,63 ; 52,12]), but the levels of these proteins were not associated with the occurrence of GvHD. However, when considering only the adult group, significant differences were observed ( table 2), between the group of patients who developed a GvHD (II-IV) and the group of patients with no or low grade GvHD. Adult with GvHD Adult with GvHD (Grade II-IV) Patients with no or GvHD Grade I ICAM-1 113,571.6 pg/mL [75,549.5 – 164,814.1] 32,055.3 pg/mL [26,083.5 – 121,165.7] VEGF 18.53 pg/mL [1.99 – 62.18] 106.46 pg/mL [51.3 – 214.4] Table 2: Statistical difference (p In conclusion, two markers ICAM-1 and VEGF show a significant prognostic value for GvHD severity in adult, when quantify in recipient patient before allografting. Further studies on a wider cohort would help to confirm these results and to precise the role of CCL5 and CXCL12 SNPs.

10507 Background: The EGFR exon 19 deletion and exon 21 L858R mutation are associated with response to EGFR-TKI. However, the correlation with response to EGFR-TKI remains unclear for rare exon 18 and 20 EGFR mutations. We investigated the clinical features of NSCLC patients with exon 18 and exon 20 EGFR mutations, analyzed response to EGFR-TKI and patient's survival. Methods: Between 2005 and 2011, all rare exon 18 and 20 EGFR mutations were collected from 10117 cases of NSCLC in 15 of 28 French NCI molecular genetic laboratories. Results: During the study period, EGFR mutations were identified in 1048 (10.35%) out of the 10117 performed tests and 108 rare mutations (10.3%) were identified in 103 patients, including 25 never identified mutations. Among them, 48 mutations were localized at exon 18 and 60 at exon 20, with 3 out of the 4 identified T790M exon 20 mutations associated with L858R mutation. Only 5 other rare mutations were associated with EGFR 19 or 21 mutations. 38 patients received EGFR-TKI (erlotinib, n=32; gefitinib, n=6). Among the 36 evaluable patients, best response on TKI was progression in 18 patients (50%), stabilization in 11 (30.5%) and partial response in 7 (19.4%). Tumor control rate observed in exon 18 mutations was 61% (8/13) and 50% (13/26) in exon 20 mutations. For the EGFR-TKI treated patients, the median PFS was 6 months (2-53). Median OS was 15 months (1-92), with no difference between the EGFR-TKI treated patients or not. Conclusions: Prevalence of rare EGFR mutations was 1.06% of all tested NSCLC and 10.35% of all EGFR mutations. Tumor control on TKI was observed in half of the patients. Reports of NSCLC harboring rare mutations are important to help the decision-making process in this subset of patients.