Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients.

Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarray-based assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5' domain, complementary to the target sequences and a 3' domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3' reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour DNA Mutations

It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutations in different oncogenes (KRAS, NRAS and BRAF) followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Genetic variants were determined in the plasma of 20 metastatic colorectal cancer (mCRC) patients previously genotyped on tissue biopsy at the diagnosis for medication planning (T0) and following the tumour genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. Our preliminary results show that this combined approach is suitable for routine clinical practice. The microarray platform enables for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while, the ddPCR, consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy

NKG2D engagement on human NK cells leads to DNAM‐1 hypo‐responsiveness through different converging mechanisms

Natural killer (NK) cell activation is regulated by activating and inhibitory receptors that facilitate diseased cell recognition. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. During tumor progression, however, these receptors are frequently downmodulated and rendered functionally inactive. Of note, NKG2D internalization has been associated with the acquisition of a dysfunctional phenotype characterized by the cross-tolerization of unrelated activating receptors. However, our knowledge of the consequences of NKG2D engagement is still incomplete. Here, by cytotoxicity assays combined with confocal microscopy, we demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance

NKG2D down-modulation on human NK cells is followed by DNAM-1 hypo-responsivenss

Natural killer (NK) cells are cytotoxic innate lymphocytes that represent the first line of defense against viral infections and tumor growth. NK cell activation is regulated by activating receptors able to recognize self-molecules up-regulated in stress conditions and inhibitory receptors that mainly bind to major histocompatibility complex class-I (MHC-I) molecules to prevent lysis of normal cells. Among activating receptors, NKG2D and DNAM-1 play a pivotal role in anticancer immune responses since they bind ligands upregulated on transformed cells. The interaction of both NKG2D and DNAM-1 with their ligands promotes a rapid receptor downmodulation that mainly occurs through internalization and lysosomal degradation. Particularly, NKG2D downmodulation has been associated to an exhausted phenotype characterized by down-modulation of the cytolytic machinery and upregulation of inhibitory receptors on both human and murine NK cells. However, our knowledge of the consequences of NKG2D engagement is still incomplete. The aim of the study is to investigate if NKG2D downmodulation could impair DNAM-1 functionality. Primary human NK cells were co-cultured with MICA-transfectants, purified, and used as effector cells in a cytotoxicity assay toward Ba/F3 cell line stably overexpressing MICA and PVR. MICA-experienced NK cells resulted impaired in NKG2D-mediated killing and showed a marked reduction of DNAM-1-triggered cytotoxicity. Analysing the different stages of cellular cytotoxicity by confocal microscopy, we found that MICA-experienced NK cells retained the ability to induce LFA-1 conformational change upon PVR recognition while they fail to polarize perforin-containing granules toward the BaF/3-PVR contact site. We verified the expression of DNAM-1-related inhibitory receptors, CD96 and TIGIT, and found that while CD96 expression remains untouched, TIGIT resulted upregulated in MICA-experienced NK cells both at protein and mRNA levels, as demonstrated by employing FACS and Real-Time PCR analyses. We performed cytotoxicity assays and experiments of confocal microscopy in the presence of anti-TIGIT blocking antibody demonstrating TIGIT contribution in inhibiting the killing of Ba/F3-PVR and a in perforin polarization impairment. However, impairment of MICA-experienced NK cell ability to lyse PVR-expressing target cells was only partially reverted by TIGIT blocking Ab, suggesting that besides TIGIT other mechanisms are responsible for defective lysis. Thus, MICA-experienced NK cells were cross-linked with anti-DNAM-1 antibody and signaling events leading to NK cell killing were assessed. Immunoblotting of cell lysates showed that, Vav1 and Akt phosphorylation resulted almost unaltered. However, DNAM-1 mediated activation of Erk1/2 and Pyk2 was reduced demonstrating that NKG2D stimulation directly affects some of the DNAM-1 activating signals leading to cell cytotoxicity. Collectively, these results demonstrate that NKG2D engagement on human NK cells impairs DNAM-1-mediated killing through two different converging mechanisms: by the upregulation of the checkpoint inhibitory receptor TIGIT, that in turn suppresses DNAM-1-mediated cytotoxic function, and by direct inhibition of DNAM-1-promoted signaling. Our results highlight a novel interplay between NKG2D and DNAM-1/TIGIT receptors that may facilitate neoplastic cell evasion from NK cell-mediated clearance

Small EVs-Associated DNA as Complementary Biomarker to Circulating Tumor DNA in Plasma of Metastatic Colorectal Cancer Patients

It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancer patients is a promising practice to evaluate somatic mutations from solid tumors noninvasively. Recently, it was reported that isolation of extracellular vesicles improves the detection of mutant DNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA in exosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA of eleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarray and droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the same mutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, we found a KRAS mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, to assess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNase I sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is only a small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amount of circulating tumor DNA. The results obtained in our experimental setting suggest that integrating ctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-time assessment of the cancer mutation status

Small EVs-Associated DNA as Complementary Biomarker to Circulating Tumor DNA in Plasma of Metastatic Colorectal Cancer Patients

It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancer patients is a promising practice to evaluate somatic mutations from solid tumors noninvasively. Recently, it was reported that isolation of extracellular vesicles improves the detection of mutant DNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA in exosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA of eleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarray and droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the same mutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, we found a KRAS mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, to assess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNase I sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is only a small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amount of circulating tumor DNA. The results obtained in our experimental setting suggest that integrating ctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-time assessment of the cancer mutation status

Clinical, pathological, and molecular correlates in ferroportin disease: a study of two novel mutations.

Background/AimsClinico-pathological manifestations of ferroportin (Fpn) disease (FD) are heterogeneous, with some patients presenting with iron overload predominantly in macrophages (“M” phenotype), others predominantly in hepatocytes (“H” phenotype). This appears to reflect functional heterogeneity of Fpn mutants, with loss-of-function generally resulting in the M type.MethodsTwo unrelated probands with “non-HFE” hemochromatosis were screened for Fpn mutations. Mutants were functionally characterized by immunofluorescence microscopy, evaluation of their ability to bind hepcidin and export iron, and by expressing them in zebrafish.ResultsTwo novel Fpn mutations were identified: I152F in patient-1, presenting with typical M phenotype; and L233P in patient-2, presenting with ambiguous features (massive overload in both macrophages and hepatocytes). Molecular studies suggested loss of function in both cases. The I152F, normally localized on cell membrane and internalized by hepcidin, showed a unique “primary” deficit of iron export capability. The L233P did not appropriately traffic to cell surface. Loss of function was confirmed by expressing both mutants in vivo in zebrafish, resulting in iron limited erythropoiesis. Clinical manifestations were likely enhanced in both patients by non-genetic factors (HCV, alcohol).ConclusionsThe combination of careful review of clinico-pathological data with molecular studies can yield compelling explanations for phenotype heterogeneity in FD