FACILITATE: A real-world, multicenter, prospective study investigating the utility of a rapid, fully automated real-time PCR assay versus local reference methods for detecting epidermal growth factor receptor variants in NSCLC.

Accurate testing for epidermal growth factor receptor (EGFR) variants is essential for informing treatment decisions in non-small cell lung cancer (NSCLC). Automated diagnostic workflows may allow more streamlined initiation of targeted treatments, where appropriate, while comprehensive variant analysis is ongoing. FACILITATE, a real-world, prospective, multicenter, European study, evaluated performance and analytical turnaround time of the Idylla™ EGFR Mutation Test compared with local reference methods. Sixteen sites obtained formalin-fixed paraffin-embedded biopsy samples with ≥ 10% neoplastic cells from patients with NSCLC. Consecutive 5 µm sections from patient samples were tested for clinically relevant NSCLC-associated EGFR variants using the Idylla™ EGFR Mutation Test and local reference methods; performance (concordance) and analytical turnaround time were compared. Between January 2019 and November 2020, 1,474 parallel analyses were conducted. Overall percentage agreement was 97.7% [n = 1,418; 95% confidence interval (CI): 96.8-98.3], positive agreement, 87.4% (n = 182; 95% CI: 81.8-91.4) and negative agreement, 99.2% (n = 1,236; 95% CI: 98.5-99.6). There were 38 (2.6%) discordant cases. Ninety percent of results were returned with an analytical turnaround time of within 1 week using the Idylla™ EGFR Mutation Test versus ∼22 days using reference methods. The Idylla™ EGFR Mutation Test performed well versus local methods and had shorter analytical turnaround time. The Idylla™ EGFR Mutation Test can thus support application of personalized medicine in NSCLC.

Functional Investigation of the Tumoural Heterogeneity of Intrahepatic Cholangiocarcinoma by In Vivo PET-CT Navigation: A Proof-of-Concept Study.

Intra-tumoural heterogeneity (IH) is a major determinant of resistance to therapy and outcomes but remains poorly translated into clinical practice. Intrahepatic cholangiocarcinoma (ICC) often presents as large heterogeneous masses at imaging. The present study proposed an innovative in vivo technique to functionally assess the IH of ICC. Preoperative 18F-FDG PET-CT and intraoperative ultrasonography were merged to perform the intraoperative navigation of functional tumour heterogeneity. The tumour areas with the highest and the lowest metabolism (SUV) at PET-CT were selected, identified during surgery, and sampled. Three consecutive patients underwent the procedure. The areas with the highest uptake at PET-CT had higher proliferation index (KI67) values and higher immune infiltration compared to areas with the lowest uptake. One of the patients showed a heterogeneous presence of FGFR2 translocation within the samples. Tumour heterogeneity at PET-CT may drive biopsy to sample the most informative ICC areas. Even more relevant, these preliminary data show the possibility of achieving a non-invasive evaluation of IH in ICC, paving the way for an imaging-based precision-medicine approach.

Predictors of fulvestrant long-term benefit in hormone receptor-positive/HER2 negative advanced breast cancer.

We retrospectively investigated in women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed on formalin-fixed paraffin-embedded tumor samples ESR1 and PI3KCA mutations and miRNAs profiles. 59 patients were evaluable (median age of 67 years, range 32-92). 18-month PFS rate was 27%; the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months, while PI3KCA mutations, found in 36% of patients, were not associated with 18-month PFS. As of miRNAs, miR-549a, miR-644a, miR-16-5p were negatively while let-7c-5p was positively associated with 18-month PFS. In addition, miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a-5p and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of targets modulated by these two latter groups of miRNAs show that in patients achieving 18-month PFS the Hippo and Wnt signaling pathways were predicted to be upregulated while endocrine resistance was potentially repressed by miR-603, miR-181a-5p and miR-199a-miR-199b-3p. Our results provide additional clues on the molecular mechanisms involved in fulvestrant activity and resistance. Underlying pathways should be further elucidated and confirmed in larger cohorts.

Mutation profile and immunoscore signature in thymic carcinomas: An exploratory study and review of the literature.

BACKGROUND: Significant efforts have been made to investigate the molecular pathways involved in thymic carcinogenesis. However, genetic findings have still not impacted clinical practice. The aim of this exploratory trial was to evaluate the immunoscore and molecular profile of a series of thymic carcinomas (TCs), correlating this data with clinical outcome. METHODS: Formalin-fixed, paraffin-embedded (FFPE) TC tissues were retrieved from our center archive. The immunoscore was evaluated according to Angell and Gallon. DNA was extracted from FFPE tumor samples and, when available, from adjacent histologically normal tissues. Next-generation sequencing (NGS) was performed targeting hotspot regions of 50 oncogenes and tumor suppressor genes. RESULTS: A series of 15 TCs were analyzed. After a median follow-up of 82.4 months, the median overall survival was 104.7 months. The immunoscore was >2 in 5/15 patients (33%). Among the investigated genes, absence of mutations was observed in 5/15 patients (33%), whereas three variants in 1/15 (6%) patient, two variants in 4/15 (26%) patients, and one variant in 5/15 patients (33%) were found. The most recurrently mutated genes were FGFR3 (five mutations) and CDKN2A (three mutations, two of which were nonsense). Patients with CDKN2A loss showed a statistically significantly worse survival (P = 0.0013), whereas patients with FGFR3 mutations showed a statistically significantly better survival (P = 0.048). CONCLUSIONS: This study adds data to the few existing reports on the mutational landscape of TCs, providing the first comprehensive analysis to date. Here, we confirm the low rate of mutations in TCs and suggest FGFR3 and CDKN2A mutations as intriguing potential therapeutic targets.

Pulmonary sarcomatoid carcinoma presenting both ALK rearrangement and PD-L1 high positivity: A case report on the therapeutic regimen.

RATIONALE: Pulmonary sarcomatoid carcinoma (PSC) represents <1% of all lung cancers and is characterized by a very poor prognosis. The optimal therapeutic regimen remains unclear. We describe a rare case of PSC with both anaplastic lymphoma kinase (ALK)-arranged and high levels of programmed death ligand 1 (PD-L1) expression. PATIENT CONCERNS: A 46-year-old woman, nonsmoker, came to our attention due to uncontrolled pain in the lower left limb. DIAGNOSIS: PSC with both ALK rearrangement and high levels of PD-L1 expression. INTERVENTIONS: The patient started first-line systemic treatment with pembrolizumab reporting stable disease; at progression, she received second-line treatment with crizotinib. The treatment was not well-tolerated, and the patient then underwent 5 cycles of ceritinib treatment. OUTCOMES: The patient showed a partial response to targeted therapy. At progression, brigatinib was initiated, but the patients reported liver progression soon after the initiation of this therapy. LESSONS: Molecular-driven investigation is necessary in PSC for treatment selection.

Antitumor Activity of a Novel Fibroblast Growth Factor Receptor Inhibitor for Intrahepatic Cholangiocarcinoma.

Fibroblast growth factor receptor 2 (FGFR2) might have an important role in the pathogenesis and biology of cholangiocarcinoma (CCA). We examined FGFR expression in CCA tumor specimens obtained from patients and CCA cell lines, and then determined the effects of the novel FGFR inhibitor, derazantinib (DZB; formally, ARQ 087), which is currently in clinical phase 2 trials for intrahepatic CCA. DZB inhibited the growth of CCA cell lines in a dose-dependent manner, and extracellular signal-regulated kinase 1/2 and AKT. It also activated apoptotic and cell growth arrest signaling. DZB reduced the in vitro invasiveness and the expression of key epithelial-mesenchymal transition genes. The in vitro data correlated with the expression of FGFRs in human CCA specimens by immunohistochemistry (FGFR1, 30% positive; and FGFR2, 65% positive) and the CCA cell lines assayed by Western blot analysis. These correlated in vitro studies suggest that FGFR may play an important role in the pathogenesis and biology of CCA. Our findings support the notion that FGFR inhibitors, like DZB, should be further evaluated at the clinical stage as targeted therapy for CCA treatment.

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

AIMS: 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. METHODS AND RESULTS: Ten resected N/N HCCs arising in cirrhotic background and characterized: (i) as outer lesions by early (n = 3) and G1 (n = 7) HCC; (ii) as inner lesions by G1 (n = 3) and G2 (n = 7) HCC. The largest/smallest diameters of outer and inner nodules were, respectively, 20/6 mm and 16/4 mm. We investigated vascular (CD34 and endocan), hepatocellular VEGF, GS, GPC3, HSP70 and CHC) and molecular (TERT promoter and ß-catenin) changes taking place from the outer neoplastic compartment to the inner neoplastic compartment (INC). A diffuse pattern of CD34+ capillarized vessels and focal endocan immunoreactivity were major distinctive features acquired in the INC; VEGF immunoreactivity was inversely related to CD34 staining. A gain in the number of cells immunoreactive for GPC3, HSP70, and CHC, but not of GS-immunoreactive cells, also occurred in the INC. TERT promoter mutations were seen in half of the cases in both compartments, whereas ß-catenin mutations were more rarely detectable. CONCLUSIONS: Major phenotypic changes take place in the INC of N/N HCC. TERT promoter mutations take place frequently and very early, and, in contrast to ß-catenin mutations, do not appear to be acquired during N/N growth. These findings suggest that inner nodules represent a step further along the pathway of tumour progression, in contrast to earlier, simply initiated, lesions, and that complete neovascularisation predicts a change in HCC biology.

Phase II Study of Everolimus in Patients With Thymoma and Thymic Carcinoma Previously Treated With Cisplatin-Based Chemotherapy.

Purpose No effective salvage treatments are available for patients with advanced/recurrent thymoma (T) or thymic carcinoma (TC) who have progressed after platinum-based chemotherapy. This study evaluated the activity of everolimus in patients with advanced/recurrent T or TC previously treated with cisplatin-containing chemotherapy. Patients and Methods This was a single-arm, single-stage, open-label, multicenter, phase II trial. Patients received oral everolimus 10 mg/d until disease progression, unacceptable toxicity, or patient refusal. A Fleming phase II trial was designed. The null hypothesis of a true disease control rate (DCR) of 40% was tested against a one-sided alternative of a true DCR of 60% (α = ß = 0.10): If disease control were achieved in ≥ 21 of the first 41 evaluable patients, everolimus could be recommended for further evaluation. Progression-free survival, overall survival, and safety were also evaluated. Results From 2011 to 2013, 51 patients were enrolled (T, n = 32; TC, n = 19). Complete remission was observed in one patient with TC, partial response in five patients (T, n = 3; TC, n = 2), and stable disease in 38 patients (T, n = 27; TC, n= 11), with a DCR of 88% (T,: 93.8%; TC, 77.8%). With a median follow up of 25.7 months, median progression-free survival was 10.1 months (T,: 16.6 months; TC, 5.6 months), and median overall survival was 25.7 months (T, not reached; TC, 14.7 months). Fourteen patients had a serious drug-related adverse event; of these patients, nine permanently discontinued treatment. Three patients died of pneumonitis while in the study. Immunohistochemical positivity for p4E-BP1 or insulin-like growth factor-1 receptor was statistically significantly related to a shorter survival. Conclusion Everolimus may induce durable disease control in a high percentage of patients with T or TC, albeit with a potential high risk of fatal pneumonitis.

Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages.

BACKGROUND & AIMS: A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. METHODS: CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. RESULTS: CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. CONCLUSION: CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. LAY SUMMARY: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment.

Morphophenotypic changes in human multistep hepatocarcinogenesis with translational implications.

BACKGROUND & AIMS: Human hepatocarcinogenesis in cirrhosis is thought to be multistep and characterized by a spectrum of nodular lesions, ranging from low to high grade dysplastic nodules (LGDN and HGDN) to early and progressed hepatocellular carcinoma (eHCC and pHCC). The aim of this study was to investigate the morphophenotypical changes of this sequence and their potential translational significance. METHODS: We scored the vascular profile, ductular reaction/stromal invasion and overexpression of five biomarkers (GPC3, HSP70, GS, CHC, and EZH2), in a series of 100 resected nodules (13 LGDN, 16 HGDN, 42 eHCC and 29 small pHCC). RESULTS: The score separated the four groups of nodules as individual entities (p<0.01). In the sequence, biomarker's overexpression progressively increased with parallel decrease of ductular reaction; the vascular remodeling started very early (LGDN) but did not further develop in a proportion of HCC. eHCC was the most heterogeneous entity, with marginal overlap with HGDN and pHCC. Liver environment (fibrosis, etiology) did not impact on the phenotype of the different nodules. A subclass of eHCC (16/42) without evidence of stromal invasion was identified, suggesting a "preinvasive stage" (p<0.05). For diagnosis, the application of four and five biomarkers (rather than the usual three) improved the sensitivity of the assay for the detection of eHCC (76% and 93% vs. 52%); biomarkers in alternative combinations, and also increased the sensitivity of the assay (GS+CHC+EZH2: 76%; GS+CHC+EZH2+HSP70: 90%). CONCLUSIONS: This study supports the multistep nature of human hepatocarcinogenesis, and suggests that eHCC is more heterogeneous than previously thought. This provides further information of the potential translational significance into clinical practice.

FOLFIRI and Cetuximab Every Second Week for First-Line Treatment of KRAS Wild-Type Metastatic Colorectal Cancer According to Phosphatase and Tensin Homolog Expression: A Phase II Study.

BACKGROUND: Retrospective studies have suggested that phosphatase and tensin homolog (PTEN) expression might predict the efficacy of cetuximab in patients with KRAS wild-type metastatic colorectal cancer (mCRC). The present study was designed to prospectively evaluate the efficacy of first-line irinotecan, fluorouracil, and folinate (FOLFIRI) plus cetuximab every second week according to PTEN expression. PATIENTS AND METHODS: Originally, patients with KRAS wild-type mCRC were randomly assigned to receive either FOLFIRI or cetuximab plus FOLFIRI (FOLFIRI-C). After a protocol amendment, the FOLFIRI arm was discontinued, and additional patients received FOLFIRI-C. Cox proportional hazard models were used to investigate the effect of PTEN and MET expression and BRAF and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α mutations on progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 35 and 54 patients received FOLFIRI and FOLFIRI-C, respectively. For the patients assigned to FOLFIRI and FOLFIRI-C, the median OS was 17.7 and 23.3 months and the median PFS was 8.2 and 6.6 months, respectively. For patients receiving FOLFIRI-C, the loss of PTEN expression did not affect PFS or OS. Significant interactions for PFS were detected between the MET expression levels (P = .047) and BRAF mutation (P = .018) and treatment. On univariate analysis, BRAF mutation was significantly associated with shorter OS for patients receiving either FOLFIRI-C (P = .016) or FOLFIRI (P = .035). Multivariate analysis confirmed the independent prognostic value of BRAF mutation on OS and that of MET expression levels on PFS (P = .025) and OS (P = .028) but only in the patients receiving FOLFIRI alone. Adverse events with FOLFIRI-C were consistent with those expected from FOLFIRI plus weekly cetuximab. CONCLUSION: Although prospective analysis of PTEN did not allow a validation of the prognostic value of this biomarker, an every second week cetuximab schedule, in addition to first-line FOLFIRI, was effective and well tolerated. The possible predictive value of MET expression levels warrants additional investigation.

An exploratory biomarker study in metastatic tumors from colorectal cancer patients treated with bevacizumab.

AIMS: Inhibition of angiogenesis is an effective treatment option for metastatic colorectal cancer. Predictive biomarkers to select patients who are most likely to benefit from this therapeutic strategy are lacking. We conducted a pilot, retrospective biomarker study in a cohort of metastatic colorectal cancer patients treated with bevacizumab. The objectives of this study were to evaluate the prognostic value of biomarker expression in metastases and to compare their expression in paired tumor specimens. MATERIALS AND METHODS: Eligible patients were treated with a bevacizumab-containing therapy; from these patients, tumor tissue from metastases was available. PTEN, PI3K p110a, c-MET, and CAIX were analyzed by immunohistochemistry. RESULTS: Forty-two patients received bevacizumab, 13 (31%) with first-line and 29 (69%) with second-line chemotherapy. Expression of CAIX, PI3K p110a, and c-MET in metastases did not predict objective response. PTEN loss was associated with response to treatment (p=0.02) and this association remained significant after adjusting for prognostic variables (p=0.006). However, no association with survival outcomes was found. In 32 patients (76%) with available paired specimens, we observed an equal expression between primary tumors and corresponding metastases in 75% of cases for CAIX in epithelial tumor cells, 56% for CAIX in stromal cells, 63% for PTEN, and 87% for c-MET. CONCLUSION: PTEN loss in metastases appears to be associated with response to bevacizumab-based therapy. However, larger studies are necessary to confirm the potential role of the PI3K/AKT/mTOR pathway in modulating the therapeutic effect of bevacizumab. Tumor heterogeneity should be taken into consideration when analyzing tumor tissues for biomarker studies.

Combined hepatocellular carcinoma - cholangiocarcinoma harboring a metastasis of colon adenocarcinoma.

Combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CC) represents a rare form of primary hepatic neoplasia. We report an unusual case of tumor-to-tumor metastasis: a cHCC-CC harboring a metastasis of colon adenocarcinoma developed in a 59 year old patient with alcohol-related liver cirrhosis. To the best of our knowledge, this is the first case of such a simultaneous occurrence.

Molecular determinants of outcome in sorafenib-treated patients with hepatocellular carcinoma.

PURPOSE: Preclinical studies show that sorafenib, a multitarget kinase inhibitor, displays anti-proliferative, anti-angiogenic, and pro-apoptotic properties in hepatocellular carcinoma (HCC). However, the determinants of sorafenib sensitivity in vivo remain largely unknown. METHODS: We assessed the expression of Mcl-1, activated/phosphorylated extracellular signal-regulated kinase (pERK) 1/2, and activated/phosphorylated AKT (pAKT) in pretreatment tumor specimens from 44 patients with advanced HCC who received sorafenib. Furthermore, we assessed MYC and MET gene copy numbers (GCN) by fluorescence in situ hybridization. RESULTS: Poorer overall survival (OS) times were correlated with pERK expression [hazard ratio (HR) 1.013; 95 % CI 1.003-1.035] and Mcl-1 expression (HR 1.016; 95 % CI 1.002-1.030) in pretreatment tumor samples. Expression levels of pERK and Mcl-1, however, were not correlated with time to tumor progression (TTP). Increased pERK expression was positively associated with higher Cancer of Liver Italian Program scores (P = 0.012) and was prognostic in patients with scores 2-6 but not in those with scores 0-1. pERK expression was significantly less frequent in specimens sourced from previous surgical procedures compared to biopsy samples (9.6 vs. 92.3 %, respectively; P < 0.0001). Analysis of pAKT expression, MET and MYC GCN, did not indicate any prognostic nor predictive values for these biomarkers in terms of survival. CONCLUSIONS: Expression levels of Mcl-1 and pERK are associated with reduced OS in HCC patients treated with sorafenib and might be useful markers for risk stratification. However, in contrast to previous findings, pERK expression levels, as well as other biomarkers tested, did not affect TTP.

Genetic and epigenetic alterations in primary colorectal cancers and related lymph node and liver metastases.

BACKGROUND: Colorectal cancer (CRC) prognosis and survival are strictly related to the development of distant metastases. New targeted therapies have increased patient survival, but the objective response rate is still very limited, partially because of a traditional focus on designing treatment according to the molecular profile of the primary tumor regardless the diversity between the primary tumor and metastases. The objective of this study was to evaluate the presence of molecular heterogeneity during metastatic progression and its potential impact on clinical treatment. METHODS: The authors analyzed v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 mutations, the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) thymine to adenine substitution at codon 1788, and tumor protein 53 (p53) mutations and investigated promoter methylation of Ras association (RalGDS/AF-6) domain family member 1 protein (RASSF1a), E-cadherin, and cyclin-dependent kinase inhibitor 2A (p16INK4a) in 101 primary CRCs (67 stage III and 34 stage IV) and related lymph node and liver metastases. RESULTS: Lymph node metastases were characterized by fewer alterations compared with primary tumors and liver metastases, especially KRAS (P = .03) and p16INK4a (P = .05). Genetic changes, when detectable in metastases, mostly were retained from the primary tumor, whereas epigenetic changes more frequently were acquired de novo. Overall, 31 distinct CRC molecular profiles were detected, none of which characterized a particular tumor stage. When the metastatic lesions also were included in the profiles, there were 53 distinct molecular profiles in 67 patients with stage III disease and 34 distinct molecular profiles in 34 patients with stage IV disease. CONCLUSIONS: Lymph node and liver metastases appear to originate in clonally different processes, with more molecular alterations occurring in distant metastases than in lymph node metastases and with elevated heterogeneity of the primary tumor. Thus, potential prognostic targets should be carefully evaluated for their heterogeneity in both primary tumors and distant metastases to avoid erroneous misclassification.

MicroRNA profiling of hepatocarcinogenesis identifies C19MC cluster as a novel prognostic biomarker in hepatocellular carcinoma.

BACKGROUND AND AIMS: Progressive hepatocarcinogenesis is a stepwise process that drives liver transformation. However, the molecular mechanisms of early liver transformation are far from clear. A role for microRNAs (miRNA) as diagnostic and prognostic factors in human tumours, including hepatocellular carcinoma (HCC), is promising. We aimed to identify novel miRNA as biomarkers for differential diagnosis and predictors of disease progression. METHODS: We used a low-density array platform to profile the expression of 664 mature miRNA in a cohort of 60 hepatitis C virus-positive liver lesions representative of all stages of progressive hepatocarcinogenesis. We validated selected miRNA in two independent patient series by qPCR and we characterized the genomic status of the miRNA cluster C19MC by fluorescent in situ hybridization and copy-number variation analyses. RESULTS: A 18-miRNA signature distinguished cirrhosis, dysplastic nodules and HCC lesions. Four miRNAs overexpressed in HCCs belonged to chromosome 19 miRNA cluster (C19MC). Significant overexpression of C19MC in early HCC compared to dysplastic nodules could be confirmed in a second series of hepatitis B virus-related liver lesions (n = 30). In a third series of 61 HCCs, C19MC cluster was overexpressed in HCCs compared to matched cirrhotic parenchyma and regardless of the type of viral infection. High C19MC miRNA levels were correlated with poor clinico-pathological features, increased risk of tumour recurrence and shorter overall survival time. HCCs overexpressing the C19MC cluster showed genetic amplification of the corresponding locus. CONCLUSIONS: C19MC cluster is a novel molecular alteration characteristic of liver cancer and predictor of poor prognosis. C19MC is an attractive candidate for novel HCC therapies.

p95HER2 truncated form in resected non-small cell lung cancer.

INTRODUCTION: Recent studies suggested that p95HER2, the NH2-terminally truncated form of human epidermal growth factor receptor 2 (HER2), could confer resistance to monoclonal antibodies against HER2 (HER2-mab). The aim of this study was to investigate the role of p95HER2 according to HER2 gene copy number (GCN) and HER2 mutation in non-small cell lung cancer (NSCLC). METHODS: The study included 447 resected NSCLC patients evaluated for P95HER2 status by immunofluorescence. Data were correlated with HER2 GCN evaluated by fluorescence in situ hybridization (FISH) and HER2 mutations. Tumors were scored as positive for p95HER2 expression if any cytoplasmic staining was detected. RESULTS: P95HER2 was successfully evaluated in 431 patients and was positive (p95HER2+) in 33 (7.6%) cases. HER2 GCN was evaluable in 439 patients, and increased GCN (at least four copies in at least 40% cells) was found in 60 cases, of which 22 (5.0%) displayed gene amplification (GA). Among the 22 patients with HER2 amplification, only one resulted P95HER2+. To further investigate whether the receptor is truncated in presence of gene mutation, in addition to the study cohort, we analyzed p95HER2 status in eight NSCLC samples harboring HER2 mutation, and only one case resulted p95HER2+. In the whole population, p95HER2- patients had numerically higher risk of death than p95HER2+ (hazard ratio = 1.4, p = 0.2). No difference in survival was observed between patients with or without HER2 GA (median 38 versus 41 months, p = 0.46). HER2 GA was significantly associated with EGFR and MET GA, with no effect on survival. CONCLUSIONS: HER2 truncation and HER2 increased GCN are not prognostic in resected NSCLC. P95HER2 is a very rare event in individuals displaying HER2 gene amplification or mutation.

Detection of somatostatin receptor subtypes 2 and 5 by somatostatin receptor scintigraphy and immunohistochemistry: clinical implications in the diagnostic and therapeutic management of gastroenteropancreatic neuroendocrine tumors.

AIMS AND BACKGROUND: Somatostatin receptor scintigraphy (SRS) is the standard method for the detection of somatostatin receptors (SSTRs). It is commonly used in gastroenteropancreatic neuroendocrine tumor (GEP-NET) staging, and represents the criterion of choice for treatment with somatostatin (SST) analogs. Immunohistochemistry (IHC) was reported as a reliable method for the detection of SSTRs with theoretically superior sensitivity over SRS. METHODS AND STUDY DESIGN: We retrospectively analyzed the sensitivity and specificity of IHC in the detection of SSTRs in a cohort of consecutive patients with GEP-NETs attending our Institute from 1997 to 2007. IHC analysis was restricted to SSTR2 and SSTR5, and the results were interpreted according to two different scoring systems. SRS was used as the gold standard. Results. Forty-four patients were enrolled; 24 (55%) had foregut carcinoids, 9 (20%) midgut carcinoids, 2 (5%) hindgut carcinoids, and 9 (20%) had GEP-NETs of unknown primary sites. A high concordance rate between IHC and SRS was shown, irrespective of the IHC scoring system applied (73% and 70%). The sensitivity of IHC was 89.3% and 78.6% and the specificity 43.8% and 50%, depending on the scoring system used. CONCLUSIONS: Although SSTR2 was shown to be expressed by IHC in up to 50% of tumors not visualized by SRS, SRS still remains the method of choice in the diagnostic and therapeutic management of GEP-NETs. More pathological and clinical data are needed to properly understand the clinical relevance of immunohistochemical detection of SSTR expression in the absence of tumor uptake at SRS.

Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab.

Cetuximab, an antibody directed against the epidermal growth factor receptor, is an effective clinical therapy for patients with colorectal, head and neck, and non-small cell lung cancer, particularly for those with KRAS and BRAF wild-type cancers. Treatment in all patients is limited eventually by the development of acquired resistance, but little is known about the underlying mechanism. Here, we show that activation of ERBB2 signaling in cell lines, either through ERBB2 amplification or through heregulin up-regulation, leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients who exhibit either de novo or acquired resistance to cetuximab-based therapy has ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximab, represent a rational therapeutic strategy that should be assessed in patients with cetuximab-resistant cancers.

The critical role of agrin in the hematopoietic stem cell niche.

Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called "hematopoietic niches," through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the α-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells.