Massimo Milione

Microenvironment modulated metastatic CD133+/CXCR4+/EpCAM- lung cancer initiating cells sustain tumor dissemination and correlate with poor prognosis

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.

Efficacy of FOLFOXIRI plus bevacizumab in liver-limited metastatic colorectal cancer: A pooled analysis of clinical studies by Gruppo Oncologico del Nord Ovest

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 was >5xa0cm 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%). Having <6 involved segments (pxa0<xa00.001)xa0and achieving RECIST response (pxa0=xa00.019) 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], pxa0<xa00.001) and overall survival (OS) (44.3 versus 24.4 months, HR: 0.32 [0.22-0.48], pxa0<xa00.001) compared with other patients, both in the univariate and multivariate analyses (PFS pxa0=xa00.025; OS pxa0<xa00.001). The 5-year PFS and OS rate in R0 resected patients were 12% and 43%, respectively. Neither negative baseline characteristicsxa0nor high clinical risk scoresxa0or RAS/BRAF mutations were associated with poor post-resection outcomes. In conclusion, FOLFOXIRI plus bevacizumab demonstrates efficacy in the conversion settingxa0with considerable long-term outcome resultsxa0independentxa0of clinical and molecular prognostic factors (NCT00719797, NCT01163396 and NCT02271464).

Dose-Dense Temozolomide in Patients with MGMT-Silenced Chemorefractory Colorectal Cancer

BackgroundIn 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.MethodsThirty-two patients with chemorefractory MGMT-methylated CRC were treated with TMZ at a daily dose of 75xa0mg/m2 for 21 consecutive days every 4xa0weeks, for up to sixxa0cycles 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.ResultsFrom 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 16xa0% (all partial responses occurring in RAS-BRAF-mutated tumors). Median progression-free survival (PFS) and overall survival (OS) were 2.3 and 6.7xa0months, respectively. Patients with MGMT-low expression by IHC had a significantly higher ORR (pu2009<u20090.0001) and PFS (pu2009=u20090.001) compared to those with MGMT-high expression, while no difference was observed in OS.ConclusionsOur data confirm the encouraging activity of TMZ in chemorefractory CRC patients selected for MGMT silencing, even in the RAS-BRAF-mutated population. The role of MGMT IHC as a biomarker for improving patient selection warrants further prospective confirmation.

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.

The histopathological classification, diagnosis and differential diagnosis of mucinous appendiceal neoplasms, appendiceal adenocarcinomas and pseudomyxoma peritonei

The vermiform appendix is the primary site of several distinctive benign and malignant neoplasms. Some can produce the clinical syndrome of pseudomyxoma peritonei (PMP). A consensus on their terminology was reached by an international panel of pathologists and clinicians working under the auspices of the Peritoneal Surface Oncology Group International (PSOGI), and this review discusses the application of the PSOGI classification to routine reporting. We discuss diagnosis and differential diagnosis together with implications for patient management, covering low‐grade appendiceal mucinous neoplasms, high‐grade appendiceal mucinous neoplasms, serrated polyps, adenomas and adenocarcinomas. We do not cover goblet cell tumours or neuroendocrine neoplasms in this paper.

Succinate Dehydrogenase B Subunit Immunohistochemical Expression Predicts Aggressiveness in Well Differentiated Neuroendocrine Tumors of the Ileum

Immunohistochemical loss of the succinate dehydrogenase subunit B (SDHB) has recently been reported as a surrogate biomarker of malignancy in sporadic and familial pheocromocytomas and paragangliomas through the activation of hypoxia pathways. However, data on the prevalence and the clinical implications of SDHB immunoreactivity in ileal neuroendocrine tumors are still lacking. Thirty-one consecutive, advanced primary midgut neuroendocrine tumors and related lymph node or liver metastases from 24 males and seven females were immunohistochemically assessed for SDHB. All patients were G1 tumors (Ki-67 labeling index ≤2%). SDHB immunohistochemistry results were expressed as immunostaining intensity and scored as low or strong according to the internal control represented by normal intestinal cells. Strong positivity for SDHB, with granular cytoplasmatic reactivity, was found in 77% of primary tumors (T), whilst low SDHB expression was detected in 90% of metastases (M). The combined analysis (T+M) confirmed the loss of SDHB expression in 82% of metastases compared to 18% of primary tumors. SDHB expression was inversely correlated with Ki-67 labeling index, which accounted for 1.54% in metastastic sites and 0.7% in primary tumors. A correlation between SDHB expression loss, increased Ki-67 labeling index and biological aggressiveness was shown in advanced midgut neuroendocrine tumors, suggesting a role of tumor suppressor gene.

Protein drug target activation homogeneity in the face of intra-tumor heterogeneity: implications for precision medicine

Introduction: Recent studies indicated tumors may be comprised of heterogeneous molecular subtypes and incongruent molecular portraits may emerge if different areas of the tumor are sampled. This study explored the impact of intra-tumoral heterogeneity in terms of activation/phosphorylation of FDA approved drug targets and downstream kinase substrates. Material and methods: Two independent sets of liver metastases from colorectal cancer were used to evaluate protein kinase-driven signaling networks within different areas using laser capture microdissection and reverse phase protein array. Results: Unsupervised hierarchical clustering analysis indicated that the signaling architecture and activation of the MAPK and AKT-mTOR pathways were consistently maintained within different regions of the same biopsy. Intra-patient variability of the MAPK and AKT-mTOR pathway were <1.06 fold change, while inter-patients variability reached fold change values of 5.01. Conclusions: Protein pathway activation mapping of enriched tumor cells obtained from different regions of the same tumor indicated consistency and robustness independent of the region sampled. This suggests a dominant protein pathway network may be activated in a high percentage of the tumor cell population. Given the genomic intra-tumoral variability, our data suggest that protein/phosphoprotein signaling measurements should be integrated with genomic analysis for precision medicine based analysis.

Giant condyloma acuminatum of the anorectum: successful radical surgery with anal reconstruction.

Buschke-Löwenstein tumor, or giant condyloma acuminatum, is a relatively uncommon lesion of the anus with aggressive local invasive behavior which may present as a large warty tumor of the genital region with expansive and destructive growth. Many sporadic reports have been published suggesting various therapeutic strategies. We report a case of Buschke-Löwenstein tumor treated with conservative surgery followed by reconstructive procedures without a loop colostomy

Abdominosacral resection for locally recurring rectal cancer.

AIM To investigate feasibility and outcome of abdominal-sacral resection for treatment of locally recurrent rectal adenocarcinoma. METHODS A population of patients who underwent an abdominal-sacral resection for posterior recurrent adenocarcinoma of the rectum at the National Cancer Institute of Milano, between 2005 and 2013, is considered. Retrospectively collected data includes patient characteristics, treatment and pathology details regarding the primary and the recurrent rectal tumor surgical resection. A clinical and instrumental follow-up was performed. Surgical and oncological outcome were investigated. Furthermore an analytical review of literature was conducted in order to compare our case series with other reported experiences. RESULTS At the time of abdomino-sacral resection, the mean age of patients was 55 (range, 38-64). The median operating time was 380 min (range, 270-480). Sacral resection was performed at S2/S3 level in 3 patients, S3/S4 in 3 patients and S4/S5 in 4 patients. The median operating time was 380 ± 58 min. Mean intraoperative blood loss was 1750 mL (range, 200-680). The median hospital stay was 22 d. Overall morbidity was 80%, mainly type II complication according to the Clavien-Dindo classification. Microscopically negative margins (R0) is obtained in all patients. Overall 5-year survival after first surgical procedure is 60%, with a median survival from the first surgery of 88 ± 56 mo. The most common site of re-recurrence was intrapelvic. CONCLUSION Sacral resection represents a feasible approach to posterior rectal cancer recurrence without evidence of distant spreading. An accurate staging is essential for planning the best therapy.

Abstract 3465: Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBackground: The evaluation of intratumor heterogeneity has become a major topic in the oncological field and it has major implications in the delivery of individualized therapy. While a number of genomic studies have reported significant intratumoral heterogeneity the analysis of the activation of protein kinase-driven signaling networks, which constitute the targets of most of the targeted compounds, has been sparse, or did not focus specifically on the tumor cells. This study evaluated the activation/phosphorylation of the protein signaling networks and a number of major precision medicine-oriented drug target to assess whether the intratumoral heterogeneity could impact patient stratification for targeted treatment in the context of metastatic disease.nnMethods: 3 independent study sets were used for this analysis: 1) metastatic colorectal cancer (mCRC) liver biopsies were collected from 6 patients; for each biopsy, 2 different regions were microdissected, 2) 18 needle biopsies were collected from 2 different regions of the same lesion of 9 mCRC liver metastases, 3) 7 serial liver biopsies were collected from 3 mCRC before and after treatment. Specimens were frozen upon collection, stored at -80°C and subjected to laser capture microdissection to isolate pure tumor epithelium. Protein pathway activation mapping was performed using reverse phase protein microarray. To assess changes in the activation level of the members of the MAPK and the AKT-mTOR pathway, unsupervised hierarchical clustering analysis and fold change between the different areas of the tumors were evaluated.nnResults: Hierarchical clustering analysis of the activation of 47 key signaling protein drug targets revealed that the signaling activation of any given protein or pathway was largely the same within a patients tumor, no matter where the biopsy was obtained, but large interpatient variation was observed. When we evaluated activation of the MAPK and AKT-mTOR pathways, we found that the relative levels of pathway activation were found to be similar in different regions of the tumor, while each patient looked distinct.nnConclusions: This first of its kind protein drug target activation analysis of microdissected tumor cells from human metastatic tumors revealed that the activated signaling architecture is largely maintained within the same tumor while each patients tumor has a distinct patient-specific signaling portrait. The data suggest that interpatient protein drug target activation heterogeneity is much larger than intratumoral heterogeneity and point to the need to determine precision medicine based profiling on a patient-by-patient basis.nnCitation Format: Erika M. Parasido, Alessandra Silvestri, Claudio Belluco, Vincenzo Canzonieri, Maria Grazia Diodoro, Massimo Milione, Lance A. Liotta, Emanuel F. Petricoin, Mariaelena Pierobon. Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2014-3465