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Next generation sequencing Immunohistochemistry for HER-2 and MET Dual color silver in situ hybridization (SISH) for HER-2 and MET

Supplementary Figure 1: Patients with acquired MET amplification have shorter PFS as compared to those with MET-negative tumors. PFS duration expressed as month time units is shown for individual patients with acquired MET amplification (blue histograms) and MET-negative (red histograms) tumors (panel A). Kaplan Meier curves show shorter median PFS of MET-amplified (blue) as compared with MET-negative (red) patients; moreover, around 40% and 15% patients MET-negative (but none of MET-amplified subjects) were still progression-free at 10 and even 20 months, respectively (panel B).

Purpose: Even if RAS-BRAF wild-type and HER2/MET–negative metastatic colorectal cancer (mCRC) patients frequently respond to anti-EGFR mAbs, acquired resistance almost invariably occurs. Mechanisms of resistance to EGFR blockade include the emergence of KRAS, NRAS, and EGFR extracellular domain mutations as well as HER2/MET alterations. However, these findings derive from retrospective studies that analyzed one single resistance mechanism at a time; moreover, it is still unclear how molecular heterogeneity affects clonal evolution in patients. In this work, we aimed at extensively characterizing and correlating the molecular characteristics of tissue- and blood-based data in a prospective cohort of patients with mCRC who received anti-EGFR antibodies.Experimental design: Twenty-two RAS-BRAF wild-type, HER2/MET–negative mCRC patients progressing on anti-EGFR therapy after initial response underwent rebiopsy. Next-generation sequencing and silver in situ hybridization (SISH)/IHC analyses were performed both on archival tumors and postprogression samples. Circulating tumor (ctDNA) molecular profiles were obtained in matched tissue–plasma samples.Results: RAS mutations and HER2/MET amplification were the most frequently detected resistance mechanisms in both tissue and blood sample analysis. On the other hand, BRAF and EGFR ectodomain mutations were much rarer. Patients with acquired MET amplification showed worse PFS on anti-EGFRs. We detected both intralesion heterogeneity, as suggested by co-occurrence of different resistance mechanisms in the same sample, and interlesion heterogeneity. The combined analysis of tissue and blood (ctDNA) results highlights the complexity of clonal evolution triggered by EGFR blockade.Conclusions: Our results indicate that it may be extremely challenging to target the complex landscape of molecular heterogeneity associated with emergence of resistance to targeted therapies in patients with mCRC. Clin Cancer Res; 23(10); 2414–22. ©2016 AACR.

Introduction: Poorly differentiated neuroendocrine carcinomas (NECs) are characterized by aggressive clinical course and poor prognosis. No reliable prognostic markers have been validated to date; thus, the definition of a specific NEC prognostic algorithm represents a clinical need. This study aimed to analyze a large NEC case series to validate the specific prognostic factors identified in previous studies on gastro-entero-pancreatic and lung NECs and to assess if further prognostic parameters can be isolated. Methods: A pooled analysis of four NEC retrospective studies was performed to evaluate the prognostic role of Ki-67 cut-off, the overall survival (OS) according to primary cancer site, and further prognostic parameters using multivariable Cox proportional hazards model and machine learning random survival forest (RSF). Results: 422 NECs were analyzed. The most represented tumor site was the colorectum (n = 156, 37%), followed by the lungs (n = 111, 26%), gastroesophageal site (n = 83, 20%; 66 gastric, 79%) and pancreas (n = 42, 10%). The Ki-67 index was the most relevant predictor, followed by morphology (pure or mixed/combined NECs), stage, and site. The predicted RSF response for survival at 1, 2, or 3 years showed decreasing survival with increasing Ki-67, pure NEC morphology, stage III–IV, and colorectal NEC disease. Patients with Ki-67 Conclusion: The most effective parameters to predict OS for NEC patients could be Ki-67, pure or mixed/combined morphology, stage, and site.

© 2019 Wiley Periodicals, Inc. The histological diagnosis of peripheral T-cell lymphoma (PTCL) can represent a challenge, particularly in the case of closely related entities such as angioimmunoblastic T-lymphoma (AITL), PTCL-not otherwise specified (PTCL-NOS), and ALK-negative anaplastic large-cell lymphoma (ALCL). Although gene expression profiling and next generations sequencing have been proven to define specific features recurrently associated with distinct entities, genomic-based stratifications have not yet led to definitive diagnostic criteria and/or entered into the routine clinical practice. Herein, to improve the current molecular classification between AITL and PTCL-NOS, we analyzed the transcriptional profiles from 503 PTCLs stratified according to their molecular configuration and integrated them with genomic data of recurrently mutated genes (RHOA G17V , TET2, IDH2 R172 , and DNMT3A) in 53 cases (39 AITLs and 14 PTCL-NOSs) included in the series. Our analysis unraveled that the mutational status of RHOA G17V , TET2, and DNMT3A poorly correlated, individually, with peculiar transcriptional fingerprints. Conversely, in IDH2 R172 samples a strong transcriptional signature was identified that could act as a surrogate for mutational status. The integrated analysis of clinical, mutational, and molecular data led to a simplified 19-gene signature that retains high accuracy in differentiating the main nodal PTCL entities. The expression levels of those genes were confirmed in an independent cohort profiled by RNA-sequencing.

High-grade Gastroenteropancreatic Neuroendocrine neoplasms (H-NENs) comprehend well-differentiated tumors (NET G3) and poorly differentiated carcinomas (NEC) with proliferative activity indexes as mitotic count (MC) &gt, 20 mitoses/10 HPF and Ki-67 &gt, 20%. At present, no specific therapy for H-NENs exists and the several evidences of microenvironment involvement in their pathogenesis pave the way for tailored therapies. Forty-five consecutive cases, with available information about T-cell, immune, and non-immune markers, from surgical pathology and clinical databases of 2 Italian institutions were immunostained for Arginase, CD33, CD163 and CD66 myeloid markers. The association between features was assessed by Spearman’s correlation coefficient. A unsupervised K-means algorithm was used to identify clusters of patients according to inputs of microenvironment features and the relationship between clusters and clinicopathological features, including cancer-specific survival (CSS), was analyzed. The H-NEN population was composed of 6 (13.3%) NET G3 and 39 (86.7%) NEC. Overall, significant positive associations were found between myeloid (CD33, CD163 and Arginase) and T/immune markers (CD3, CD4, CD8, PD-1 and HLA-I). Myeloid and T-cell markers CD3 and CD8 identified two clusters of patients from unsupervised K-means analysis. Cases grouped in cluster 1 with more myeloid infiltrates, T cell, HLA and expression of inhibitory receptors and ligands in the stroma (PD-1, PD-L1) had significantly better CSS than patients in cluster 2. Multivariable analysis showed that Ki-67 (&gt, 55 vs. &lt, 55, HR 8.60, CI 95% 2.61–28.33, p &lt, 0.0001) and cluster (1 vs. 2, HR 0.43, CI 95% 0.20–0.93, p = 0.03) were significantly associated with survival. High grade gastroenteropancreatic neuroendocrine neoplasms can be further classified into two prognostic sub-populations of tumors driven by different tumor microenvironments and immune features able to generate the framework for evaluating new therapeutic strategies.

Background: Little information is available concerning prognostic factors for bronchopulmonary large cell neuroendocrine carcinomas (BP-LCNECs) and even less is known about combined LCNECs (Co-LCNECs). We investigated whether an integrated morphological, immunohistochemical, and molecular approach could be used for their prognostic evaluation. Methods: Morphological (including combined features), proliferative (mitotic count/Ki-67 index), immunohistochemical (napsin A, p40, TTF-1, CD44, OTP, SSTR2A, SSTR5, mASH1, p53, RB1, and MDM2), and genomic (TP53, RB1, ATM, JAK2, KRAS, and STK11) findings were analyzed in BP-LCNECs from 5 Italian centers, and correlated with overall survival (OS). The Ki-67 index was expressed as the percentage of positive cells in hot spots as indicated in the WHO 2019 Digestive System Tumors and, for Co-LCNECs, the Ki-67 index was evaluated only in the LCNEC component. Results: A total of 111 LCNECs were distinguished into 70 pure LCNECs, 35 Co-LCNECs (27 with adenocarcinoma [ADC] and 8 with squamous cell carcinoma [SqCC]), and 6 LCNECs with only napsin A immunoreactivity. The Ki-67 index cutoff at 55% evaluated in the neuroendocrine component was the most powerful predictor of OS (log-rank p = 0.0001) in all LCNECs; 34 cases had a Ki-67 index p = 0.0001) were also observed between pure and Co-LCNECs. A significant difference in OS was found between pure LCNECs-A and Co-LCNECs-A (p < 0.05) but not between pure LCNECs-B and Co-LCNECs-B. Co-LCNEC-ADC and LCNEC napsin A+ cases had longer OS than pure LCNEC and Co-LCNEC-SqCC cases (log-rank p = 0.0001). On multivariable analysis, tumor location, pure versus combined features, and napsin A, but no single gene mutation, were significantly associated with OS after adjustment for Ki-67 index and study center (p < 0.05). Conclusions: The Ki-67 proliferation index and the morphological characterization of combined features in LCNECs seem to be important tools for predicting clinical outcome in BP-LCNECs.

In colorectal cancer patients, chromosomal rearrangements involving NTRK1 gene (encoding the TRKA protein) are shown in a small subset of patients and are associated with the constitutive activation of the kinase domain of TRKA. In turn, activated TRKA-fusion proteins are associated with proliferation and survival in colorectal cancer tumors. Here we report the identification and functional characterization of a new SCYL3-NTRK1 fusion gene in a 61-year-old colorectal cancer patient. To our knowledge, this fusion protein has never been previously documented in oncological patients. We show that this novel fusion is oncogenic and sensitive to TRKA inhibitors. As suggested by other pieces of evidence, entrectinib - an orally available pan-TRK, ROS1 and ALK inhibitor - may have particular efficacy in patients with NTRK rearrangements. Therefore, screening for rearrangements involving NTRK genes may help identifying a subset of patients able to derive benefit from treatment with entrectinib or other targeted inhibitors.

Small-intestine neuroendocrine neoplasms are heterogeneous neoplasms arising from endocrine cells of the intestinal mucosa. Ki-67 is the main determinant of prognosis in neuroendocrine neoplasms. However, the search for new prognostic makers represents a key point with regard to small-intestine neuroendocrine neoplasms. The oncofetal protein IMP3 plays a role in cell growth and its expression has a prognostic value in lung neoplasms. From January 1998 to August 2015, all the consecutive small-intestine neuroendocrine neoplasms patients suitable for surgery were included: 51 patients (32 males, median age 68 years) had small-intestine neuroendocrine neoplasms classified according to the WHO 2010 classification. In all the cases IMP3 expression was evaluated on primary tumors and, when available, on nodal and distant metastases. The medical records and pathological slides of these patients were used to determine the clinical characteristics, pathological diagnoses, and outcome information. The overall 5-year and 10-year survival rate were 53.9 and 42% respectively. At Cox proportional hazards regression grading was the major factor influencing both OS and progression-free survival at univariate (p = 0.0002 and 0.0051, respectively) and multivariate analysis (p = 0.0004 and 0.0043, respectively). Also IMP3 expression at the nodal metastases resulted a factor significantly associated with progression-free survival at both univariate (p = 0.0066) and multivariate analysis (p = 0.0059, HR 3.58). IMP3 expression did not correlate with the Ki-67 (p = n.s.). In this study, IMP3 at the nodal site resulted to be associated with low progression-free survival in small-intestine neuroendocrine neoplasms, independently of the Ki-67 index. We suggest that the integration of IMP3 and Ki-67 would help better stratify the risk of progression in small-intestine neuroendocrine neoplasms.

Gastroblastoma (GB) is a rare gastric epithelial-mesenchymal neoplasm, first described by Miettinen et al. So far, all reported cases described the tumor in children or young adults, and similarities with other childhood blastomas have been postulated. We report a case of GB in a 43-year-old patient with long follow up and no recurrence up to 100 months after surgery. So far, this is the second case of GB occurring in the adult age >40-year-old. Hence, GB should be considered in the differential diagnosis of microscopically comparable conditions in adults carrying a worse prognosis and different clinical approach.

Background: In gastroenteropancreatic (GEP) high-grade neuroendocrine neoplasms (H-NENs), Ki-67 threshold of 55% defines three prognosis subclasses: neuroendocrine tumor (NET) G3, neuroendocrine carcinoma (NEC) Methods: In GEP-NEN patients, we evaluated: (i) 55% threshold for Ki-67 labeling index for further stratifying NEC and (ii) immunoreactivity and gene mutations by immunohistochemistry and targeted next-generation sequencing (T-NGS). Results: Fifteen NETs G3 and 39 NECs were identified. Ki-67 labeling index was TP53, 32.9%; KRAS, 5.5%; BRAF, 4.1%) were detected in 46.6% NENs, significantly enriched in NEC ≥55% (76.7%) compared to NEC p = 0.03). Median OS was 4.3 years in NET G3, 1.8 years in NEC p <0.0001); it was 2.3 years with NGS wild-type, 0.7 years with ≥1 mutation (p <0.0001), 0.8 years in PD-L1-positive patients, and 1.7 years in PD-L1-negative subjects (p = 0.0004). In multivariate analysis, only the proposed subclassification approach yielded statistically significant differences between groups (NEC p = 0.005; NEC ≥55% vs. NET G3, HR 25.8, 95% CI 3.9–169, p = 0.0007). Conclusions: These findings identify NEC ≥55% as a biologically and prognostically distinct subtype and pave the way for more personalized treatment.

In pancreatic neuroendocrine neoplasms (pNEN), size ≤2 cm and Ki-67 3% suggest indolent behavior, but no factor alone predicts prognosis. We investigated factors predictive of tumor progression in 80 pNENs surgically resected in a single Institution from 1995 to 2015. At multivariable analysis the only two independent variables related to PFS were Ki-67 (HR 2.97; 95%CI 1.26-7.02) and presence of synchronous liver metastases (HR 3.60; 95%CI 1.70-7.61). Using Ki-67 3% and M0 as reference, the HR for tumor progression was 3.21 (95%CI 1.18-8.74) for M0 patients with Ki-67 3-20%, 5.06 (2.29-11.2) for M1 patients with Ki-67 ≤ 20% and 24.3 (6.64-89.2) for those with Ki-67 20%. Tumor size (≤2 vs.2 cm) was not a predictive factor at any analysis. Intra-class correlation of Ki-67 values on pre-surgical biopsies vs. surgical specimens was 0.99 and Ki-67 classes were correctly identified in 97% of biopsies. Ki-67 and presence of liver metastases are the major prognostic factors in pNEN and identify different progression risks regardless of tumor size. Pre-surgical pNEN biopsy for Ki-67 assessment should be included in the evaluation of patients with 1-2 cm tumors to help in the decision on whether to perform surgical resection.

Background Many factors, including histopathologic parameters, seem to influence the prognosis of patients undergoing resection of colorectal cancer liver metastases (CRCLM), although their relative weight is unclear. Histopathologic growth patterns (HGPs) of CRCLM may affect sensitivity to antiangiogenics. We aimed at evaluating differences in histopathologic parameters of response according to the use of bevacizumab or cetuximab as first-line targeted agents, and at exploring the prognostic and predictive role of HGPs. Methods We performed a comprehensive histopathologic characterisation of CRCLM from 159 patients who underwent secondary resection, after receiving triplets FOLFOXIRI (folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan) or COI (capecitabine, oxaliplatin, and irinotecan) plus bevacizumab (N = 103) vs cetuximab (N = 56) in five first-line no-profit clinical trials. Results Both major histopathologic response (tumour regression grade TRG1–2, 32 vs 14%, p = 0.013) and infarct-like necrosis (80 vs 64%, p = 0.035) were significantly higher in the bevacizumab than in the cetuximab group. Achieving major response positively affected relapse-free survival (RFS) (p = 0.012) and overall survival (OS) (p = 0.045), also in multivariable models (RFS, p = 0.008; OS, p = 0.033). In the desmoplastic HGP (N = 28), a higher percentage of major response was reported (57 vs 17% in pushing and 22% in replacement HGP, p

Correction to: Blood Cancer Journal (2016) 6, e491; doi:10.1038/bcj.2016.99; published online 4 November 2016 Following the online publication of this article, it was noticed that the author names and affiliations were missing from the HTML version of this article. The complete names and affiliations are as follows

Secondary resection is a chance of cure for a subgroup of metastatic colorectal cancer (mCRC) patients with unresectable liver-limited disease. Medical treatment has a dual goal: to induce tumour shrinkage and to prevent disease relapse. The aims of the present analysis were to assess the efficacy of FOLFOXIRI plus bevacizumab in this setting, and to investigate whether this regimen could revert the poor prognosis of high-risk patients defined by clinical and molecular factors. We performed a pooled analysis of patients with unresectable and liver-limited mCRC, treated with first-line FOLFOXIRI plus bevacizumab in three prospective clinical trials by Gruppo Oncologico del Nord Ovest. 205 (37.9%) patients with liver-limited disease were selected, out of 541 treated patients. Liver metastases were synchronous, ≥4 and bilobar in 90%, 61%, and 79% of cases, respectively. The largest diameter was5 cm in 42% of cases, and ≥6 segments were involved in 25%. Seventy-four patients (36.1%) underwent R0 or R1 resection of metastases. R2 resections were performed in 17 cases (8.3%). Having6 involved segments (p 0.001) and achieving RECIST response (p = 0.019) were associated with higher chances of resection. R0/R1 resected patients had significantly longer median progression-free survival (PFS) (18.1 versus 10.7 months, HR: 0.48 [0.35-0.66], p 0.001) and overall survival (OS) (44.3 versus 24.4 months, HR: 0.32 [0.22-0.48], p 0.001) compared with other patients, both in the univariate and multivariate analyses (PFS p = 0.025; OS p 0.001). The 5-year PFS and OS rate in R0 resected patients were 12% and 43%, respectively. Neither negative baseline characteristics nor high clinical risk scores or RAS/BRAF mutations were associated with poor post-resection outcomes. In conclusion, FOLFOXIRI plus bevacizumab demonstrates efficacy in the conversion setting with considerable long-term outcome results independent of clinical and molecular prognostic factors (NCT00719797, NCT01163396 and NCT02271464).

Abnormal expression of succinate dehydrogenase, (SDH), in particular of the B subunit (SDHB), is implicated in the pathogenesis of neuroendocrine tumors. This study evaluates the distribution of SDHB in WHO grading G1 and G2 intestinal, well-differentiated neuroendocrine tumors and corresponding lymph node or liver metastases. We collected ileal well-differentiated neuroendocrine tumors specimens from consecutive patients with prior primary resection and distant synchronous or metachronous liver metastases. We obtained 195 specimens from primary tumors (n = 106) and metastases (n = 89). The expression (E) of SDHB and the immunostaining intensity (I) were evaluated semiquantitatively and combined into a single score. SDHB score was evaluated in primitive tumor and metastatic specimens. SDHB was found in all tumor cells. Mean SDHB expression was 72.7 % ± 17.1 % in primitive specimens and 27.9 % ± 24.6 % in metastatic specimens (p

Historically, the differential diagnosis between different nodal peripheral T-cell lymphoma (PTCL) subtypes based on morphological and phenotypic grounds has posed great challenges. In the last few years, our knowledge of the molecular bases of different PTCLs has significantly expanded. However, peripheral T-cell lymphomas not otherwise specified (PTCL-NOSs) are still regarded to as a heterogeneous category encompassing PTCL cases not fitting other, more homogeneous, subtypes. In fact, PTCL-NOS is one of the few lymphoma subtypes where no recurrent driver mutations have been reported so far. In order to better characterized the PTCL-NOS genomic landscape, we decided to investigate 11 PTCL-NOS patients by a whole genome sequencing (WGS) approach (median coverage 27X). Ten out of eleven samples were collected from FFPE blocks and 2 were removed from analysis: one due to low cancer cell fraction (CCF) and the other based on cluster generation issues during sequencing likely caused by a hyper-fragmented DNA. Among the remaining 9 cases, we extracted 59,617 somatic base substitutions (range 2,471-10,756, median 6,358 per patient) and 20,531 small insertion-deletions (indels) (range 84-6,397, median 1,580). We were able to characterize the spectrum of FFPE-induced artefacts, mostly composed of point mutations and indels within LINE-1 (L1) elements, predominantly of the L1PA family. This is a crucial quality control step that could be applied to similar future studies from archive samples. Four samples were heavily involved by FFPE-related artefacts and were excluded for this reason. Using a non-negative matrix factorization (NNMF) algorithm we investigated for the first time the PTCL-NOS mutational signature landscape. We did not find novel processes in this entity, but rather known processes operative in other lymphoid malignancies. Among those: signatures 1 and 5, deriving from the age-related process of spontaneous deamination of methylated cytosines; signatures 2 and 13 deriving from aberrant activity of the APOBEC family of DNA deaminases; signatures 17 and 8, pertaining to two yet poorly characterized processes. The contribution of different processes to the mutational spectrum of each case was profoundly heterogeneous. Combining our data set with 64 previously published whole exome sequencing cases (23 ALCL, 15 AITLs, 9 PTCL-NOSs and 16 EATL-II), we confirmed the lack of recurrent driver mutations among PTCL-NOS. Taking advantage of WGS data, we therefore focused on structural variants (SVs: inversions, translocations, internal tandem duplications and deletions) and copy number alterations (CNAs). We found 372 SVs, with a stunning median of 73 per sample (range 56-86). Even more interesting, at least one complex event was observed in all but one patients, including one whole genome duplication (WGD) and five chromothripsis events in three patients, suggesting a critical role of SVs in shaping the PTCL-NOS genome. We found that known onco-drivers were recurrently disrupted by such events: the most frequent target was CDKN2A, deleted in 4 out of 5 patients, 2 of which carried homozygous deletions. Interestingly, PTEN loss was observed in 2 out of 4 CDKN2A-deleted patients. Given the high prevalence of these deletions, we extended our observation to an independent validation set of ALCLs (n=56), AITL (n=22) and PTCL-NOS (n=59) investigated by FISH (n=36), next generation sequencing (n=25) or SNP6 array series (n=76). Overall, CDKN2A was deleted in 22/59 (37%) PTCL-NOSs cases, and in 17/22 (77%) both alleles were lost. PTEN was deleted in 12/59 (20%) PTCL-NOS cases, all of which also carried a CDKN2A loss. Strikingly, the co-occurrence of CDKN2A and PTEN was found only among PTCL-NOS, and in none of the other entities. With the limitations of the small sample size, the presence of CDKN2A bi-allelic deletions was associated with inferior survival (25% [95% CI: 9-66%] 5-y OS for deleted cases vs 52% [95% CI: 28-96%] for wt/hemizygous cases, p=0.042) among patients treated with an autologous bone marrow transplant front line program for advance stage and high-risk disease (n=19). Our observations point at SVs as a main driver of PTCL-NOS, often involving known cancer genes and their downstream pathways. Furthermore, our data highlighted recurrent gene deletions that may be relevant for differential diagnosis within this category of lymphomas. Disclosures Bolli: Celgene: Honoraria. Chiappella:Roche: Other: lecture fees; Amgen: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Nanostring: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Teva: Other: lecture fees. Corradini:Celgene: Honoraria, Other: Advisory Board & Lecturer; Novartis: Honoraria, Other: Advisory Board & Lecturer; Roche: Honoraria, Other: Advisory Board & Lecturer; Sanofi: Honoraria, Other: Advisory Board & Lecturer; Gilead: Honoraria, Other: Advisory Board & Lecturer; Sandoz: Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board & Lecturer; Takeda: Honoraria, Other: Advisory Board & Lecturer; Amgen: Honoraria, Other: Advisory Board & Lecturer; Janssen: Honoraria, Other: Lecturer.

Peripheral T-cell lymphomas (PTCLs) represent a rare and heterogeneous group of aggressive non-Hodgkin's lymphomas comprising different entities. Anthracycline-based regimens (usually CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone) followed by stem cell transplantation are considered the standard of care in the front-line setting. However, long-term disease control can be achieved only in 30-40% of young patients. Increasing evidences highlight the role of tumor microenvironment in sustaining tumor progression and aggressiveness. Among microenvironment components, a central role is played by cancer associated fibroblast (CAFs). Recent studies demonstrated that this is applicable not only to solid tumors but also to haematological malignancies. Here, we report for the first time the isolation and characterization of PTCL-CAFs and their role in promoting cell proliferation. We kept in culture a skin biopsy of a cutaneous localization of a peripheral T cell lymphoma. Fibroblast-like cells grown out from the biopsy were isolated, cultivated for few passages and then characterized. Flow cytometric analyses revealed that the cell population entirely expressed cell-surface antigens specific for fibroblasts, such as CD140b and CD90, while they did not express CD45 and CD31, thus excluding hematopoietic or endothelial origin. Biochemical analyses confirmed that all cells were positive for vimentin, while αSMA expression highlighted the activated status of the cells isolated. We then assessed the in vitro effect of CAFs on PTCL cell growth by co-colture experiments. For this purpose, we coltured for 8 days the PTCL cell line OCI-Ly12 in fresh medium or in medium previously conditioned by CAFs or normal skin fibroblasts (NFs). PTCL cell growth was not affected by co-colture either with CAFs or with NFs. Moreover, the presence of conditioned medium did not alter the response of OCI-Ly12 cells to CHOP treatments. We then assessed the in vivo effect of PTCL-CAFs. We subcutaneously injected CAFs alone, OCI-Ly12 cells alone and OCI-Ly12 together with CAFs (ratio 1:1) in NOD/SCID mice and monitored tumor growth for 20 days. In contrast to the effect observed in vitro, co-injection of CAFs with cancer cells substantially promoted tumor growth (tumor volume OCI-Ly12 0,87±0,47 cm3; OCI-Ly12+CAFs 2,30±0,26 cm3). As expected, CAFs alone did not develop a measurable mass. Subsequent immunohistochemical staining with CD31 revealed the presence of more vascular structures in tumors originated by OCI-Ly12+CAFs co-injection than by OCI-Ly12 alone, suggesting that CAFs presence sustains tumor growth by promoting angiogenesis. In summary, we show that PTCL-derived CAFs promote cancer cell growth in vivo, while leaving unaffected cell growth in vitro and the response to chemotherapy. This is the first evidence of the role played by the microenvironment in promoting malignant cell proliferation in PTCLs. Citation Format: Martina Magni, Sara Rizzitano, Alessio Pellegrinelli, Paolo Corradini, Cristiana Carniti. Peripheral T cell lymphoma-associated fibroblasts promote tumor growth in an in vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3123.

Background/Aims: Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) are defined as neuroendocrine neoplasms (NENs) with a Ki-67 index >20% according to the 2010 WHO classification. Some reports suggest that this category is heterogeneous. We retrospectively studied a series of 136 patients affected by grade 3 GEP-NECs with the aim to clarify the prognostic role of tumor morphological differentiation, proliferation, defect in mismatch repair proteins (MMRd), CD117 expression, and site of origin. The primary endpoint was the correlation between these parameters and the overall survival (OS). Methods: Univariate and multivariable Cox proportional hazards regression analyses were used to assess the prognostic significance of various clinical and histopathologic features. Results: With a median follow-up of 81 months, the median OS was 12.9 months. At multivariate analysis, morphological differentiation, Ki-67 index, MMRd, stage, and CD117 expression were independent prognostic markers in NECs. Three different prognostic categories of NECs were identified according to the degree of morphologic differentiation (well vs. poorly differentiated) and Ki-67 index (Conclusions: The present study suggests that GEP-NECs represent a heterogeneous group of neoplasms which can be better classified in different prognostic categories using both tumor morphology and Ki-67 index.

In a phase II study, we showed that temozolomide (TMZ) was tolerable and active in heavily pre-treated patients with advanced colorectal cancer (CRC) and MGMT methylation. A schedule of dose-dense TMZ may have enhanced activity due to the higher cumulative dose and induction of MGMT depletion, even in resistant tumors. Thirty-two patients with chemorefractory MGMT-methylated CRC were treated with TMZ at a daily dose of 75 mg/m2 for 21 consecutive days every 4 weeks, for up to six cycles or until the occurrence of progressive disease/unacceptable toxicity. The primary endpoint was treatment activity in terms of objective response rate (ORR). MGMT protein expression was tested by immunohistochemistry (IHC) on two pooled cohorts: patients from the previous study of standard-dose TMZ and those from the current investigation. From November 2013 to December 2014, 32 patients were treated at Fondazione IRCCS Istituto Nazionale dei Tumori. We observed only three episodes of grade 3 asthenia and no significant myelotoxicity. The ORR was 16 % (all partial responses occurring in RAS-BRAF-mutated tumors). Median progression-free survival (PFS) and overall survival (OS) were 2.3 and 6.7 months, respectively. Patients with MGMT-low expression by IHC had a significantly higher ORR (p

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

Aims: The role of tumour metabolic and proliferative indices in predicting non-small-cell lung cancer (NSCLC) patients' prognosis is unclear. We correlated fluorine 18 ((18) F)-fluorodeoxyglucose (FDG)-positron emission tomography (PET) value and Ki67 index to patients' survival, taking into account tumour heterogeneity, disease characteristics and genetic aberrations. Methods and results: A series of 383 NSCLCs was arranged into tissue microarrays and Ki67 staining was analysed by immunohistochemistry. The maximum standardized uptake (SUV(MAX) ) value detected by (18) F-FDG-PET analysis was calculated over a region of interest. Large-cell and squamous cell carcinomas had higher proliferative and metabolic activities than adenocarcinomas, and the two measures were correlated significantly. The hot-spot Ki67 value was correlated with patients' survival and the cut-off to discriminate patients in the survival risk groups was 20%. Ki67 hot-spot values were greater in anaplastic lymphoma kinase (ALK) rearranged tumours. Adenocarcinomas showed the highest intratumour heterogeneity in proliferative activity and the hot-spot Ki67 value predicted only the prognosis of patients in this group. Although tumour metabolic activity was not associated with patients' prognosis, a SUV(MAX) > 2 was related to nodal metastases, tumour size and grade. Conclusions: Our results highlight how tumour heterogeneity influences evaluation of prognostic biomarkers. Our data support Ki67 evaluation to estimate NSCLC patients' prognosis, particularly for adenocarcinoma.

Aims Primary Ewing sarcoma of the ileum has rarely been documented. Little is known about its pathogenesis and clinical implications, and it would be helpful to identify novel molecular markers. EWSR1–FEV translocation is exceedingly rare in Ewing sarcoma, as FEV expression is restricted to prostate, brain and serotonin neuroendocrine cells (NE) and related tumours. Methods and Results Paraffin sections or snap-frozen material were used in this investigation. Tumours were investigated by means of immunohistochemistry, RT–PCR (EWSR1–FLI1, EWSR1–ERG and EWSR1–FEV transcripts), FISH analysis (EWSR1 break-apart and specific EWSR1–FEV translocation) and spectral karyotyping (SKY). Ten ileal neuroendocrine tumours (INET) made up the control group for EWSR1–FEV translocation. Among 445 Ewing sarcomas cases spanning a period of 20 years, seven (1.6%) arose in the ileum. All tumours were immunoreactive for synaptophysin, CD99, FLI1 and vimentin. FISH identified EWSR1 rearrangement in all cases, with EWSR1–FLI1 transcripts being detected in all but one tumour showing the uncommon EWSR1–FEV rearrangement, with SKY, RT–PCR and FISH confirmation. The mean survival of EWSR1–FLI1 patients was 14 months, whereas the EWSR1–FEV patient was alive after 15 years despite several recurrences controlled by surgery alone. No INET showed EWSR1 translocation. Conclusions Most primary Ewing sarcomas of the ileum show the common EWSR1–FLI1 translocation, but EWSR1–FEV could be specific for tumours arising in the ileum and showing better prognosis.

INTRODUCTION: The diagnosis of Peripheral T-cell lymphomas not otherwise specified (PTCL-NOS) is currentlybased on an "exclusion criteria" model, since PTCL-NOS lack pathognomonic features. Nevertheless, based on gene expression data, Iqbal et al(Blood 2014) have recently identified two different PTCL-NOS subgroups with different biological and prognostic features that accounts for approximately 80% of the cases and have different biological and prognostic features: one characterized by TBX21 overexpression and T-CD8+ molecular profile; the other by GATA3 overexpression and T-CD4+ profile. Herein, we used a wide comprehensive gene expression profiling (GEP) data set in order to further investigate the molecular features of different PTCL-NOS molecular entities. MATHERIAL AND METHODS: A data set were created including samples from 8 main published series (GSE6338, GSE14879, GSE19067, GSE19069, GSE58445 and GSE65823 at http://www.ncbi.nlm.nih.gov/geo/; E-TABM-702 and E-TABM-783 at https://www.ebi.ac.uk/arrayexpress) for a total of 541 patients. R/Bioconductor was used to generate and analyze the gene expression data. We applied the CIBERSORT algorithm (Gentles et al, Nat Gen 2015), which connects specific global expression profiles to the relative prevalence of tissue components (tumour and microenvironment cells). RESULTS: We first re-classified each sample included in the investigated data set based on previously published signatures (Iqbal et al. Blood 2014, Agnelli et al. Blood 2012). This approach led to a final data set of 144 PTCL-NOS (28%), 127 AITL (23%), 69 ALCL Alk neg (12%), 56 ALCL Alk pos (10%), 59 NK (11%) %), together with 86 healthy T-cell tissues. To our knowledge, this is the largest GEP data set ever described in PTCL so far. In the 144 PTCL-NOS cases, two main molecular clusters were extracted based on published signature, replicating previous findings: the first was characterized by both GATA3 expression (GATA3+) and T-CD4+ cell origin; the second by TBX21 expression (TBX21+) and T-CD8+ cell origin. Approximately 30% of all PTCL-NOS were characterized by neither GATA3 (GATA-) nor TBX21 expression (TBX21-), and for this reason they were classified as "double negative" PTCL-NOS. Based on data obtained by the CIBERSORT algorithm we found that the contribution of cellular microenvironment components was extremely heterogeneous and variable through the entire PTCL-NOS series. A significant T-CD4+ and T-CD8+ cell enrichment was reported among GATA3+ and TBX21+ groups, respectively. Interestingly, a fraction of GATA3+ PTCL-NOS (n=11, 7.6%) was characterized by a significant γδ T-cell component. Conversely, PTCL-NOS GATA3+ patients without γδ T-cell component signature were characterized by a low non-T-cell microenvironment component. This may reflect the major tumour infiltration due to higher proliferation rate as suggested by the strong GATA3 correlation with MIB1 and MYC expression (p Based on the clinical data available for 105/144 PTCL-NOS, poor overall survival (OS) was associated with GATA3 expression (p=0.03) [3-y OS 26.4% (range 20-32.8%) vs 47.3% (range 40-54.5%)]. However, such a poor outcome was less evident when we limited the clinical evaluation to patients younger than 60 years (40/105). Among low GATA3 expressors, TBX21 expression was associated with better OS compared to other "double negative" PTCL-NOS (p=0.07) [3-y OS 66.7% (range 51-82.4%) vs 36.4% (range 22-51%)]. Higher CIBERSORT-predicted T-CD8+ contribution was associated with better OS among patients younger than 60 years (p=0.03) [3-y OS 70% (range 55.6-84.5%) vs 30% (range 21.7-38.3%)]. No other variables or clusters were associated with significant impact on OS. CONCLUSION: Our study based on an innovative computational approach and a large and comprehensive gene expression data set, confirmed the great molecular heterogeneity of PTCL-NOS, suggesting that the current molecular classification of PTCL-NOS may be further improved in the future. Disclosures No relevant conflicts of interest to declare.

Background: Peripheral T-cell lymphomas (PTCL) have an aggressive clinical course with a poor 5-year overall survival with conventional therapy. Autologous stem cell transplantation (autoSCT) and allogeneic stem cell transplantation (alloSCT) can improve long-term disease control in first and second remission, respectively. Prognostic factors are not able to discriminate patients with chemorefractory disease. Preliminary evidence from gene expression profiling (GEP) and immunoistochemical study have suggested that the majority of PTCL-not otherwise specified (PTCL-NOS) can be subdivided in two different groups based on GATA3 and Tbet (transcription regulators of T-helper 2 and T-helper 1 lymphocytes) expression with a poorer prognosis in the former group. Because GEP studies are not feasible in routine clinical practice, we investigated whether expression changes of those proteins could be evaluated by immunohistochemistry (IHC) and used to predict prognosis. Material and Methods: We collected paraffin tissues from 63 consecutive patients (pts) with a diagnosis of PTCL [subtypes: PTCL-NOS (n=32), ALK-negative anaplastic large cell lymphomas (n=15, ALCL ALK-negative), angioimmunoblastic (n=16, AITL)] treated at different Italian Institutions. Sections were analyzed for Ki-67, GATA3, Tbet and CCR4 expression by IHC. Results were expressed as percentages of positive tumor cells. Histology was centrally reviewed. Median age was 58 years (range, 18-76 years); 20 of 63 (32%) pts were characterized by intermediate-high/high IPI; 47 of 63 (74%) pts were candidated to transplantation whereas 16 (25%) were not due to age ≥65 years (n=14) or limited stage/IPI 0 (n=2). Fifty-nine pts (93%) received anthracycline-based induction chemotherapy. Twenty of 47 (43%) pts underwent autoSCT in first (n=11) or subsequent remissions; 23 of 47 (49%) underwent alloSCT in first remission (n=3) or at relapse (n=20). The median follow-up of alive pts was 30 months (range, 8-250). Results: Expression of GATA3 was highly variable, but was significantly higher on average in PTCL-NOS [34% positive tumor cells (range, 1-99%)] than in other subtypes [11% (range,1-97%) in ALK-negative ALCL (p=0.027); 12% (range, 1-82) in AITL (p=0.0025)]; The mean value of Ki67 expression was higher in ALK-negative ALCL [87% (range, 70-95%)] as compared to other subtypes [46% (range,15-90%) in PTCL-NOS (p In PTCL-NOS (n=32), ROC curve analysis identified a cut-off of 27% for GATA3 with 16 pts showing a GATA3 expression higher than the cut-off value. While no significant difference in baseline clinical characteristics was observed among the two groups, pts with GATA3 ≥27% showed a significantly lower CR rate following induction [19% versus 62% (p=0.02)]. Cases with high GATA3 expression were significantly associated with a reduced 5-year PFS and OS as compared to those with low expression [PFS: 6% (95%CI:1%-24%) versus 49% (95%CI:22%-71%), (p=0,004); OS: 39% (95%CI:13%-65%) versus 72% (95%CI:35%-90%) (p=0,04) respectively]. In multivariate analysis including age, IPI score ≥2 and sex, high GATA3 expression retained a strong prognostic value in terms of PFS (p Conclusions: Our analysis identifies GATA3 ICH expression as a strong prognostic and predictive biomarker among PTCL-NOS. Pts with high GATA3 expression values were characterized by chemorefractory disease and poorer outcome even with transplantation strategies. Validation of this biomarker in a larger series of patients is ongoing. Disclosures No relevant conflicts of interest to declare.

Background: Acquired resistance to anti-EGFR MoAbs (cetuximab and panitumumab) represents a challenge in the treatment of mCRC, but its molecular mechanisms are not completely understood. Even in presence of RAS-BRAF-PI3KCA ”quadruple wt” and HER-2/MET negative status for protein expression and gene amplification, pts who primarily respond to anti-EGFR MoAbs will eventually develop secondary resistance. Prior retrospective and small series uniquely investigated a single biomarker, showing in some cases the emergence of KRAS mutations,HER-2 or MET amplifications. Our study aimed at comprehensively describing all known molecular alterations possibly associated with acquired resistance. Methods: Pts with mCRC were prospectively treated with cetuximab- or panitumumab-based therapy until progressive disease. All archival tumors were defined as RAS-BRAF-PI3KCA”quadruple wt” by Sanger sequencing, as well as HER-2/MET negative by both immunohistochemistry (IHC) and in-situ hybridization (ISH). At the time of disease progression, tumor re-biopsy was performed on the most accessible site of metastasis, as institutional procedure for a wide phase 1 screening program. On both archival tissue and re-biopsy, next generation sequencing of 50 genes' hotspot regions included in the Hotspot Cancer Panel v2 (Life Technologies) was performed by using the Ion Torrent Personal Genome Machine platform (Life Technologies). Moreover,HER-2/MET status were repeated on tumor re-biopsy by IHC and ISH. Results: Seventeen pts were recruited. All had prior objective response to anti-EGFRs. Next-generation sequencing confirmed RAS-BRAF-PI3KCA wt status on archival tumors. The results of our analyses on tumor re-biopsies are shown in the table: IDNGS: Acquired mutations (% mutant alleles)cellularity% mutant alleles normalized for cellularityHER2 ISHMET ISH#1KRAS Q61H (37%)80%46%--#2-Amplified-#3BRAF V600E (13%)90%14%--#4NRAS Q61R (13%)40%32%--#5--Amplified#6KRAS Q61K (4%)70%6% Acquired RAS or BRAF mutations were found in 4 (23%) and 1 (6%) cases, respectively. Acquired HER-2 or MET amplification were found in 4 (23%) and 2 (12%) cases, respectively. As shown for patient #6, some degree of intra-tumor heterogeneity may exist due to concomitant presence of low represented RAS-mutated and HER-2 amplified sub-clones. In some cases (35%), a detectable acquired mechanism of resistance remains unknown. Conclusions: Based on our results, currently known molecular alterations associated with acquired resistance were mainly mutually exclusive. In a relevant subset of cases additional molecular profiling is warranted. Citation Format: Filippo Pietrantonio, Rosa Berenato, Federica Perrone, Annunziata Gloghini, Elena Tamborini, Benedetta Picciani, Adele Busico, Giulio Settanni, Chiara Costanza Volpi, Ambra Vittoria Gualeni, Alessio Pellegrinelli, Massimo Milione, Marta Caporale, Monica Niger, Maria Di Bartolomeo, Filippo de Braud. Comprehensive molecular characterization of acquired resistance to anti-EGFR monoclonal antibodies (MoAbs) in patients with metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B57.

Background: We showed that standard dose TMZ (150 mg/mq/day day1-5q28) is tolerable and active in 32 heavily pre-treated patients with advanced CRC and MGMT promoter methylation (Pietrantonio et al., Ann Oncol 2014). In a subsequent study, we included 32 pts treated with dose-dense TMZ (75 mg/mq/day day 1-21q28, for up to 6 cycles or until progression/unacceptable toxicity). Additional predictive markers are needed for improved selection of patients for TMZ therapy in metastatic CRC. Retained MGMT expression by IHC was proposed as marker for negative selection of pts with brain tumors. We conducted a retrospective analysis to assess MGMT expression in our 2 studies and to correlate this with the outcomes. Methods: All 64 patients included in the 2 studies were defined as MGMT methylation-positive according to methylation-specific PCR. A total of 40 patients had tissue available for IHC. Expression of nuclear MGMT protein was defined scored semiquantitatively according to extension and intensity. Using ROC analysis, IHC score Results: Herein, we report for the first time the results of the dose-dense TMZ study. We obtained 4 confirmed partial responses and 2 stable disease, accounting for a response rate of 12% and a clinical benefit of 18%. Median PFS was 1.8 months. OS data are not mature. Thus, the results are comparable to those obtained in the previous study. MGMT-low expression was found in 15 (38%) samples and MGMT-high in 25 (62%). RAS-BRAF mutations were found in 28 (70%) pts and were not correlated with MGMT expression (p = 1). Response rate was significantly higher in patients with MGMT-low (53%) vs. those with MGMT-high (p Conclusions: In this translational study on MGMT methylated CRC, absence of MGMT expression was significantly correlated with tumor response and benefit from TMZ treatment. Citation Format: Filippo De Braud, Filippo Pietrantonio, Maria Di Bartolomeo, Katia Fiorella Dotti, Claudia Maggi, Roberto Iacovelli, Marta Caporale, Rosa Berenato, Federica Perrone, Elena Tamborini, Alessio Pellegrinelli, Stefano Federici, Fabrizio Festinese, Giuseppe Pelosi, Ilaria Bossi, Paola Valentina Consonni, Susanna Maggi, Massimo Milione. MGMT immunohistochemistry (IHC) improves patients’ selection for temozolomide (TMZ) treatment in advanced chemorefractory MGMT-methylated colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-119. doi:10.1158/1538-7445.AM2015-LB-119

529 Background: Pathologic response of HCRM to neoadjuvant tx is associated to better outcome. In retrospective analyses Bev-based tx improved tumor regression grade (TRG), while cetuximab (Cmab) increased RECIST response and R0 resections in prospective studies. The aim of this analysis was to assess the effect of Bev vs Cmab-based tx in terms of HCRM pathological response and outcome. Methods: Fifty-eight pts with HCRM were resected at National cancer Institute of Milan from 2007 to 2012 following Bev (n=30) or Cmab (n=28) based neoadjuvant tx. Pathological response was classified from TRG 1 for pathological complete response (pCR), to TRG5 for no response; TRG 1-3, for histological response; TRG 4-5 for scarce/no response (Rubbia-Brandt L, Ann Oncol 2007). Results: KRAS missing in 9 pts (3 Bev/6 Cmab), mutated in 18 pts (60%) in Bev vs 4 (14%) in Cmab group.Median number of resected metastases: 4.6 vs 3.9; mean TRG: 2.9 (2.2-4) in Bev vs 3.8 (3-5) in Cmab group (p=0.04 at Kruskal-Wallis test). Pathological response rate was higher for Bev vs Cmab (70% vs 39%; p=0.037 at Chi-square test). Bev also increased pCRs (20% vs 0%, p=0.024 at Fisher’s test). With generalized estimating equation for all resected nodules, mean TRG was lower in Bev vs Cmab group: 2.6 (2-4) vs 3.7 (3-4), p

Purpose: Even if RAS-BRAF wild-type and HER2/MET–negative metastatic colorectal cancer (mCRC) patients frequently respond to anti-EGFR mAbs, acquired resistance almost invariably occurs. Mechanisms of resistance to EGFR blockade include the emergence of KRAS, NRAS, and EGFR extracellular domain mutations as well as HER2/MET alterations. However, these findings derive from retrospective studies that analyzed one single resistance mechanism at a time; moreover, it is still unclear how molecular heterogeneity affects clonal evolution in patients. In this work, we aimed at extensively characterizing and correlating the molecular characteristics of tissue- and blood-based data in a prospective cohort of patients with mCRC who received anti-EGFR antibodies. Experimental design: Twenty-two RAS-BRAF wild-type, HER2/MET–negative mCRC patients progressing on anti-EGFR therapy after initial response underwent rebiopsy. Next-generation sequencing and silver in situ hybridization (SISH)/IHC analyses were performed both on archival tumors and postprogression samples. Circulating tumor (ctDNA) molecular profiles were obtained in matched tissue–plasma samples. Results: RAS mutations and HER2/MET amplification were the most frequently detected resistance mechanisms in both tissue and blood sample analysis. On the other hand, BRAF and EGFR ectodomain mutations were much rarer. Patients with acquired MET amplification showed worse PFS on anti-EGFRs. We detected both intralesion heterogeneity, as suggested by co-occurrence of different resistance mechanisms in the same sample, and interlesion heterogeneity. The combined analysis of tissue and blood (ctDNA) results highlights the complexity of clonal evolution triggered by EGFR blockade. Conclusions: Our results indicate that it may be extremely challenging to target the complex landscape of molecular heterogeneity associated with emergence of resistance to targeted therapies in patients with mCRC. Clin Cancer Res; 23(10); 2414–22. ©2016 AACR.

Background Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Patients and methods Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). Results KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not—being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Conclusions Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.