Alessio Pellegrinelli

The Clinicopathologic Heterogeneity of Grade 3 Gastroenteropancreatic Neuroendocrine Neoplasms: Morphological Differentiation and Proliferation Identify Different Prognostic Categories

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 (<55% vs. ≥55%). On this basis, median OS was 43.6 months in well-differentiated neoplasms with a Ki-67 index 20-55% (named type A), 24.5 months in poorly differentiated neoplasms with a Ki-67 index 20-55% (type B), and 5.3 months (p < 0.0001) in poorly differentiated neoplasms with a Ki-67 index ≥55% (type C). 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.

Heterogeneity of acquired resistance to anti-EGFR monoclonal antibodies in patients with metastatic colorectal cancer

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.

Ewing sarcoma of the small bowel: a study of seven cases, including one with the uncommonly reported EWSR1-FEV translocation.

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.

IMP3 expression in small-intestine neuroendocrine neoplasms: a new predictor of recurrence

PurposeSmall-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.MethodsFrom 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.ResultsThe 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.).ConclusionsIn 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.

Identification and characterization of a novel SCYL3-NTRK1 rearrangement in a colorectal cancer patient

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.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.

Ki67 proliferative index of the neuroendocrine component drives MANEC prognosis

Mixed adenoneuroendocrine carcinomas (MANECs) are composed of a poorly differentiated neuroendocrine carcinoma (NEC) and a non-neuroendocrine (non-NEC) neoplastic epithelial component, each representing at least 30% of the tumor. At present, prognostic factors for MANECs remain largely unexplored. We investigated the clinical-pathologic features of a large multicenter series of digestive system MANECs. Surgical specimens of 200 MANEC candidates were centrally reviewed; diagnosis was confirmed in 160 cases. While morphology, proliferation (mitotic count (MC), Ki67 index) and immunophenotype (p53, SSTR2a, beta-Catenin, Bcl-2, p16, Rb1, ALDH, mismatch repair proteins and CD117) were investigated separately in both components, genomic (TP53, KRAS, BRAF) alterations were searched for on the entire tumor. Data were correlated with overall survival (OS). MANEC sites were: 92 colorectal, 44 gastroesophageal and 24 pancreatobiliary. Median OS was 13.2 months. After adjustment for primary site, Ki67 index of the NEC component (but not of the non-NEC component) was the most powerful prognostic marker. At multivariable analysis, patients with Ki67 ≥ 55% had an 8-fold risk of death (hazard ratio (HR) 7.83; 95% confidence interval (CI) 4.17-14.7; P < 0.0001) and a median OS of 12.2 months compared to those with Ki67 < 55% (median OS 40.5 months). MC (HR 1.51; 95% CI 1.03-2.20, P = 0.04) was a weaker prognostic index. Colorectal primary site (HR 1.60; 95% CI 1.11-2.32; P = 0.01) was significantly associated with poorer survival. No single immunomarker, in either component, was statistically significant. This retrospective analysis of a large series of digestive system MANECs, showed that the NEC component, particularly its Ki67 index, was the main prognostic driver.

Ki-67 and presence of liver metastases identify different progression-risk classes in pancreatic neuroendocrine neoplasms (pNEN) undergoing resection

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.

Differential histopathologic parameters in colorectal cancer liver metastases resected after triplets plus bevacizumab or cetuximab: a pooled analysis of five prospective trials

BackgroundMany 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.MethodsWe 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.ResultsBoth 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 < 0.001) and an unsignificant advantage from cetuximab vs bevacizumab was evident in RFS (p = 0.116). In the pushing HGP (N = 66), a significant benefit from bevacizumab vs cetuximab (p = 0.017) was observed. No difference was described in the replacement HGP (N = 65, p = 0.615).ConclusionsThe histopathologic response is the only independent determinant of survival in patients resected after triplets plus a biologic. When associated with triplet chemotherapy, bevacizumab induces a higher histopathologic response rate than cetuximab. The assessment of HGPs should be further explored as a predictor of benefit from available targeted agents.

Orbital lesions, an exceedingly rare site of neuroendocrine tumor metastasis

Orbital lesions, an exceedingly rare site of neuroendocrine tumor metastasis Sara Pusceddu1, Massimo Milione2, Silvia Ortolani3, Alessio Pellegrinelli2, Marco Brugia4, Filippo de Braud1, Lorenzo Antonuzzo4,5 1Department of Medical Oncology, Fondazione IRCCS “Istituto Nazionale dei Tumori”, 20133 Milan, Italy. 2Department of Phatology, Fondazione IRCCS “Istituto Nazionale dei Tumori”, 20133 Milan, Italy. 3Department of Medical Oncology, Azienda Ospedaliera Universitaria Integrata, University of Verona, 37134 Verona, Italy. 4Department of Medical Oncology, Azienda Ospedaliera Universitaria Careggi, 50134 Firenze, Italy. 5Medical Genetics, University of Siena, 53100 Siena, Italy. Correspondence to: Dr. Sara Pusceddu, Department of Medical Oncology, Fondazione IRCCS “Istituto Nazionale dei Tumori”, Via G. Venezian 1, 20133 Milan, Italy. E-mail: sara.pusceddu@istitutotumori.mi.it Neuroendocrine tumors are rare neoplasms arising primarily in the gastrointestinal tract and lung. The liver is the most common site of metastases, but these tumors can rarely metastasize to atypical sites. Surgery is the only curative approach while the optimal medical treatment is debated. From this perspective, a multidisciplinary approach for each single case becomes very important. In this report we describe the case of a male affected by a single intraorbital metastasis from a midgut well differentiated neuroendocrine tumor. The patient refused surgical removal and therefore he was at first treated with stereotactic radiotherapy and systemic treatment with a somatostatin analog (SSA). After achieving a stable disease for four months he underwent primary tumor resection. Six years after the initial diagnosis, the patient is currently stable and receiving SSA at standard dose.

Abstract B57: Comprehensive molecular characterization of acquired resistance to anti-EGFR monoclonal antibodies (MoAbs) in patients with metastatic colorectal cancer (mCRC)

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 genes9 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: 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.