Luca Di Tommaso

AXL-associated tumor inflammation as a poor prognostic signature in chemotherapy-treated triple-negative breast cancer patients

A subgroup of triple-negative breast cancer (TNBC) shows epithelial-to-mesenchymal transition (EMT) features, which are sustained by the interaction between cancer cells and tumor-associated macrophages (TAMs). In this study, the clinical relevance of 30 EMT-related kinases and the potential cross-talk with TAMs were investigated in a cohort of 203 TNBC patients treated with adjuvant chemotherapy. The prognostic value of the evaluated markers was validated in two independent cohorts of TNBC patients treated with adjuvant chemotherapy (N=95; N=137). In vitro, we investigated the potential synergism between cancer cells and TAMs. We found that the EMT-related kinase AXL showed the highest correlation with the frequency of CD163-positive macrophages (rS=0.503; P<0.0001). Relapsing TNBC patients presented high expression of AXL (P<0.0001) and CD163 (P<0.018), but only AXL retained independent prognostic significance in multivariate analysis (relapse-free survival, P=0.002; overall survival P=0.001). In vitro analysis demonstrated that AXL-expressing TNBC cells were able to polarize human macrophages towards an M2-like phenotype, and modulate a specific pattern of pro-tumor cytokines and chemokines. Selective AXL inhibition impaired the activity of M2-like macrophages, reducing cancer cell invasiveness, and restoring the sensitivity of breast cancer cells to chemotherapeutic drugs. These data suggest that the EMT-related kinase AXL overexpressed in cancer cells has prognostic significance, and contributes to the functional skewing of macrophage functions in TNBC. AXL inhibition may represent a novel strategy to target cancer cells, as well as tumor-promoting TAMs in TNBC.

Increased Infiltration of Natural Killer and T Cells in Colorectal Liver Metastases Improves Patient Overall Survival

IntroductionCancer heterogeneity and degree of intra-tumoral immune cells represent variables affecting overall survival (OS). The present study investigated the impact of natural killer (NK) and T cells infiltrating colorectal liver metastases (CLM) in patients undergoing hepatectomy after neoadjuvant chemotherapy.MethodsThe frequencies of intra-tumoral, marginal, and peritumoral CD3+ T and NKp46+ NK cells were determined for 121 patients. OS was assessed in relation to prognostic factors.ResultsAt univariate analysis, several variables, including T and N of the primary tumor, metachronous CLM, radiological response, and higher density of intra-tumoral CD3+ T cell (>1%/mm2) and of NKp46+ NK cells (>1 cell/mm2), were associated with OS. Only increased frequencies of intra-tumoral CD3+ T cells (pxa0=xa00.005) and NKp46+ NK cells (pxa0=xa00.004) correlated with OS at multivariate analysis. The logistic regression revealed that metachronous CLM (ORxa0=xa02.781; pxa0=xa00.002), the use of an epidermal growth factor receptor inhibitor (ORxa0=xa03.891; pxa0=xa00.001), and radiological response (ORxa0=xa03.219; pxa0=xa00.001) were associated with higher infiltration of these cells.ConclusionsHigh frequencies of NK and T cells in response to chemotherapy predict OS in CLM patients. These findings provide important insights that can help physicians to choose the best treatment option and adopt more predictive follow-up strategies for patients with CLM.

A Phosphokinome-based screen uncovers new drug synergies for cancer driven by liver-specific gain of non-oncogenic RTKs.

Genetic mutations leading to oncogenic variants of receptor tyrosine kinases (RTKs) are frequent events during tumorigenesis; however, the cellular vulnerability to nononcogenic RTK fluctuations has not been characterized. Here, we demonstrated genetically that in the liver subtle increases in wild‐type Met RTK levels are sufficient for spontaneous tumors in mice (Alb‐R26Met), conceptually illustrating how the shift from physiological to pathological conditions results from slight perturbations in signaling dosage. By analyzing 96 different genes in a panel of tumor samples, we demonstrated that liver tumorigenesis modeled by Alb‐R26Met mice corresponds to a subset of hepatocellular carcinoma (HCC) patients, thus establishing the clinical relevance of this HCC mouse model. We elucidated the regulatory networks underlying tumorigenesis by combining a phosphokinome screen with bioinformatics analysis. We then used the signaling diversity results obtained from Alb‐R26Met HCC versus control livers to design an “educated guess” drug screen, which led to the identification of new, deleterious synthetic lethal interactions. In particular, we report synergistic effects of mitogen‐activated protein kinase kinase, ribosomal S6 kinase, and cyclin‐dependent kinase 1/2 in combination with Bcl‐XL inhibition on a panel of liver cancer cells. Focusing on mitogen‐activated protein kinase kinase and Bcl‐XL targeting, we mechanistically demonstrated concomitant down‐regulation of phosphorylated extracellular signal–regulated kinase and myeloid cell leukemia 1 levels. Of note, a phosphorylated extracellular signal–regulated kinase+/BCL‐XL+/myeloid cell leukemia 1+ signature, deregulated in Alb‐R26Met tumors, characterizes a subgroup of HCC patients with poor prognosis. Conclusion: Our genetic studies highlight the heightened vulnerability of liver cells to subtle changes in nononcogenic RTK levels, allowing them to acquire a molecular profile that facilitates the full tumorigenic program; furthermore, our outcomes uncover new synthetic lethal interactions as potential therapies for a cluster of HCC patients. (Hepatology 2017;66:1644–1661).

A Large Set of miRNAs Is Dysregulated from the Earliest Steps of Human Hepatocellular Carcinoma Development

Hepatocellular carcinoma (HCC) typically results from a stepwise process characterized by the development of premalignant lesions, such as low- or high-grade dysplastic nodules (LGDNs and HGDNs, respectively), in a cirrhotic setting. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional regulation of gene expression that can act as oncogenes or tumor suppressors. Whether and which miRNAs are involved in the early stages of HCC development remains elusive. Here, small-RNA sequencing was applied to profile miRNA expression in 55 samples (cirrhotic nodules; CNs), LGDNs, HGDNs, early HCCs, and small progressed HCCs, obtained from 17 patients bearing HCCs of different etiologies. An miRNA expression signature of 62 miRNAs distinguishing small progressed HCCs from matched CNs was identified. Interestingly, 52 of these miRNAs discriminated CNs from LGDNs/HGDNs, regardless of etiology, and remained modified along the tumorigenic process. Functional analysis of the predicted mRNA targets of deregulated miRNAs identified common modifications between the early and late stages of HCC development likely involved in the stepwise process of HCC development. Our results demonstrate that miRNA deregulation happens very early in HCC in humans, implying their crucial role in the tumorigenic process. The identification of miRNAs discriminating CNs from neoplastic nodules may have relevant translational implications in early diagnosis.

Filamin A expression predicts early recurrence of hepatocellular carcinoma after hepatectomy

Recurrence of hepatocellular carcinoma (HCC) after hepatectomy is very high. A predictive marker of early recurrence (ER) capable of personalizing follow‐up and developing a new target therapy would be beneficial. The overexpression of Filamin‐A (FLNA), a cytoskeleton protein with scaffolding properties, has recently been associated with progression in tumours. The aim of this study was to test the expression of FLNA in a cohort of patients operated for HCC.

Phenotypic and molecular changes in nodule-in-nodule hepatocellular carcinoma with pathogenetic implications

Nodule‐in‐nodule (N/N) hepatocellular carcinoma (HCC) is a convincing proof of multistep hepatocarcinogenesis. In this lesion, an inner HCC develops within an outer, more differentiated, tumour, which can be rapidly taken over by the former so that N/N HCC is rarely detected.

CXCR4/CXCL12 Signaling and Protumor Macrophages in Primary Tumors and Sentinel Lymph Nodes Are Involved in Luminal B Breast Cancer Progression

Luminal B breast cancers (BC) have a more aggressive behavior associated with a higher rate of tumor relapse and worse prognosis compared to luminal A tumors. In this study, we evaluated the involvement of specific epithelial-to-mesenchymal transition- (EMT-) and immune-related pathways in the dissemination of luminal B BC cells. The expression of 42 EMT- and immune-related genes was evaluated in matched sentinel lymph nodes (SLNs) analyzed by the one-step nucleic acid amplification assay (OSNA) and primary tumors of 40 luminal B BC patients by gene array and immunohistochemistry. The results were validated in an independent group of 150 luminal B tumors by immunohistochemistry and immunofluorescence and using gene expression data from 315 luminal B BC patients included in the Metabric dataset. We found that the expression of CXCR4 (p = 3.28E − 02) and CD163 (p = 6.92E − 03) was significantly upregulated in SLNs of recurrent luminal B BC patients. Luminal B primary tumors overexpressing CXCR4 were characterized by an increased expression of vimentin and a high content of CD163-positive macrophages. Bioinformatics analysis confirmed the correlation of CXCR4 with CXCL12, VIM, and CD163 expression and LN involvement. Our results suggest that the upregulation of the CXCR4/CXCL12 pathway and the presence of protumor macrophages in the primary tumor and SLNs sustain the aggressiveness of an important subgroup of luminal B BC.

CYP1A2 is a predictor of HCC recurrence in HCV-related chronic liver disease: A retrospective multicentric validation study

BACKGROUNDnAlthough hepatic resection is a potentially curative treatment for hepatocellular carcinoma (HCC), post-operative prognosis remains unsatisfactory due to the high incidence of recurrence. Several clinicopathological markers have been associated with HCC recurrence, but none has been validated. Extratumoral expression of cytochrome P4501A2 (CYP1A2) was recently proposed as predictor of HCC recurrence.nnnAIMSnTo validate extratumoral CYP1A2 as predictor of HCC recurrence and to determine its applicability to pretreatment liver biopsy.nnnMETHODSnSurgically resected HCC (n.180) with clinicopathological data and follow up were retrospectively studied (HCV n.54; HBV n.91; NAFLD/NASH n.35). CYP1A2 expression was evaluated using an immunohistochemical assay and semiquantitative analysis.nnnRESULTSnEtiology-stratified analysis showed that low CYP1A2 expression was independently associated with recurrence-free survival in HCV patients (HR 2.814, 95% CI 1.300-6.093, p=0.009); this association was lost in the whole cohort. Pretreatment liver biopsy and paired surgical specimens showed concordant CYP1A2 expression in the vast majority of cases (87%), with NPV of 100%, PPV of 81.25%, and a Cohen kappa of 0.72 (substantial agreement).nnnCONCLUSIONnWe validated the extratumoral expression of CYP1A2 as a biomarker of HCC recurrence in HCV patients. CYP1A2 analysis in pretreatment liver biopsy can be of help to stratify HCC patients for personalized treatment.