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Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment.

Colorectal cancer (CRC) is the second most frequently diagnosed cancer worldwide and represents a major challenge for public health. Despite advances in molecular profiling, important gaps remain in our understanding of tumorigenesis and the regulatory mechanisms underlying CRC progression. The most widely adopted classification system is the Consensus Molecular Subtypes (CMS), which stratifies CRC into four biologically distinct subtypes. We investigated the role of A-to-I RNA editing across CMS subtypes in a cohort of 100 CRC patients at various disease stages. Bulk RNA-seq data were analyzed using REDItools to detect editing events, focusing on both recoding sites and edits within repetitive elements, such as Alu sequences. Furthermore, expression levels of the ADAR enzyme family were assessed, and deconvolution analyses were performed on single-cell RNA-seq data from an independent cohort of stage II CRC patients to characterize editing activity within the tumor microenvironment (TME). Competitive endogenous RNA (ceRNA) networks, specific to each CMS subtype, were constructed based on editing events in repetitive elements. A multivariate Cox proportional hazards model was applied to evaluate associations with overall survival (OS). We observed statistically significant differences in ADARB1 expression across CMS subtypes. Single-cell RNA-seq data revealed subtype-specific distribution patterns of ADAR enzymes within the TME. Analysis of editing events showed subtype-specific signatures in both known cancer-related genes (e.g., COPA , CADPS , IGFBP7 ) and novel candidates ( ZNF552 , RALGPS1 ). Editing in repetitive elements informed the construction of distinct ceRNA networks for each CMS subtype, suggesting different post-transcriptional regulatory mechanisms. Survival analysis identified three variables significantly associated with OS, independent of CMS classification and clinical stage: ADARB1 expression, and editing events in NOP14-AS1 (chr4:2960236; p  = 0.036; HR = 0.0069), previously linked to 5-FU sensitivity, and ST7-AS2 (chr4:117120557). This study underscores the biological relevance of RNA editing in CRC, highlighting its impact on chemoresistance, the tumor microenvironment, and subtype-specific gene regulation. Our findings suggest that RNA editing represents a critical post-transcriptional regulatory layer in CRC and holds potential as a biomarker and therapeutic target.

Non-small-cell lung cancer, histologically classified into adenocarcinoma (AD) and squamous cell carcinoma, is one of the most deadly malignancies worldwide. Lung AD (LUAD) could benefit of a plethora of target therapies and, in the last few years, also of immunotherapies. Here we focused on a real-life cohort of LUAD and The Cancer Genome Atlas (TCGA)-LUAD dataset aiming to gain insights into the immune contexture of such a malignancy. We explored the mutational status of 41 genes and the expression of 94 genes, related to immune-checkpoint, inflammation, and stromal microenvironment. Surprisingly, we found that our cohort has a very low mutational burden if we consider our panel as its surrogate. Regarding gene expression data, we identified 31 genes significantly deregulated in tumor tissues compared with a pool of normal samples. Unsupervised hierarchical clustering of the deregulated genes is able to identify two clusters of tumor samples, differently enriched in alterations in actionable genes. In particular, we identified a cluster enriched in patients carrying KRAS alterations. In silico deconvolution, that is the inferring of tumor microenvironment composition by gene expression data, through TIMER algorithm has been performed to explore immune microenvironment. Estimation performed on our gene expression matrix showed that B cell infiltration is lower in the KRAS-mutated enriched cluster, as in the TCGA-LUAD dataset. Such a finding has been validated in situ through immunohistochemistry in an independent cohort. Moreover, cases in LUAD-TCGA with low B cell infiltration have a significantly worse overall survival than those with higher levels. In the real-life cohort we observed that cases belonging to cluster enriched in KRAS-mutated patients have a poor outcome. LUAD driven by KRAS mutation represents an unmet clinical need, being refractory to pharmacological inhibition. Our results link KRAS mutations to B cell infiltration. Thus, the present findings could be helpful in a better definition of immunotherapeutic approaches for KRAS mutated patients.

Background BRAF/MEK inhibitors have improved the outcome in metastatic melanoma (MM) patients harboring a BRAF mutation, but no biomarker predictive of response has been identified. Methods We conducted a retrospective analysis on 264 MM patients that had received first-line targeted therapy with BRAF/MEK inhibitors. Next-generation sequencing (NGS) was performed on tissue biopsies, and samples with > 30% tumor cellularity were included in the study. The impact of BRAF variant allele frequency (BRAF-VAF) on clinical treatment outcomes was analyzed. Results BRAF-VAF was dichotomized using two approaches. (1) The “surv_cutpoint” function identified two different cut-off for progression-free survival (PFS: 44.05%) and overall survival (OS:45.1%). Patients with BRAF-VAF > 44.05% showed a significantly lower PFS (median PFS: 10 months, 95% CI: 7–13 months), compared to patients with BRAF-VAF < 44.05% (median PFS: 13 months, 95% CI: 12–21 months). Moreover, patients with higher VAF (> 45.1%) experienced a lower OS (median OS: 26 months, 95% CI: 19–38 months), compared with patients with VAF < 45.1% (median OS: 29 months, 95% CI: 29–51 months). (2) The ROC analysis significantly predicted PFS but not OS. BRAF-VAF normalized with neoplastic cellularity (nVAF) showed a strong association with both PFS, and OS compared to BRAF-VAF alone. nVAF also emerged as an independent predictor for PFS in the multivariate analysis (HR: 3.88, 95% CI: 1.84–8.20), with a higher nVAF score associated with a 3.88-fold increased risk of progression. Conclusions Our study demonstrated the role of the BRAF-VAF as predictor of response in MM patients treated with BRAF/MEK inhibitors. Moreover, VAF normalization predicts PFS better than BRAF-VAF alone. Highlights The role of BRAF-VAF in predicting response to BRAF/MEK inhibitors therapy in melanoma has not been elucidated yet. In 264 metastatic melanoma patients treated with first-line targeted therapy, high BRAF-VAF values correlated with worse clinical outcomes. This evidence is further strengthened when BRAF-VAF was normalized using neoplastic cellularity (nVAF). BRAF-VAF can be used as predictor of clinical outcomes in metastatic melanoma patients treated with first-line targeted therapy.

Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by high immunogenicity which makes it particularly suitable for immune checkpoint inhibitors (ICIs) treatment. We report the case of a 53-year-old man with metastatic DM successfully treated with the combination of anti-CTLA-4 and anti-PD-1 antibodies, who developed serious immune-related adverse events (irAEs). The primary tumor was characterized by absent PD-L1 expression and no-brisk lymphocytes infiltration. NGS showed absence of BRAF mutation, a high tumor mutational burden, and an UV-induced DNA damage signature. Metastatic lesions regressed rapidly after few cycles of ICIs until complete response, however the patient developed serious irAEs including hypothyroidism, adrenal deficiency, and acute interstitial nephritis which led to the definitive suspension of treatment. Currently, the patient has normal renal functionality and no disease relapse after 26 months from starting immunotherapy, and after 9 months from its definitive suspension. Efficacy and toxicity are two sides of the same coin of high sensitivity to ICIs in DM. For this reason, these patients should be closely monitored during ICIs therapy to promptly identify serious side effects and to correctly manage them.

The number of treatment options for melanoma patients has grown in the past few years, leading to considerable improvements in both overall and progression-free survival. Targeted therapies and immune checkpoint inhibitors have opened a new era in the management of melanoma patients. Despite the clinical advances, further research efforts are needed to identify other “druggable” targets and new biomarkers to improve the stratification of melanoma patients who could really benefit from targeted and immunotherapies. To this end, many studies have focused on the role of microRNAs (miRNAs) that are small non-coding RNAs (18-25 nucleotides in length), which post-transcriptionally regulate the expression of their targets. In cancer, they can behave either as oncogenes or oncosuppressive genes and play a central role in many intracellular pathways involved in proliferation and invasion. Given their modulating activity on the transcriptional landscape, their biological role is under investigation to study resistance mechanisms. They are able to mediate the communication between tumor cells and their microenvironment and regulate tumor immunity through direct regulation of the genes involved in immune activation or suppression. To date, a very promising miRNA-based strategy is to use them as prognosis and diagnosis biomarkers both as cell-free miRNAs and extracellular-vesicle miRNAs. However, miRNAs have a complex role since they target different genes in different cellular conditions. Thus, the ultimate aim of studies has been to recapitulate their role in melanoma in biological networks that account for miRNA/gene expression and mutational state. In this review, we will provide an overview of current scientific knowledge regarding the oncogenic or oncosuppressive role of miRNAs in melanoma and their use as biomarkers, with respect to approved therapies for melanoma treatment.

Background It is frequently asked whether chemotherapy can still play a role in metastatic melanoma considering the effectiveness of the available drugs today, including antiCTLA4/antiPD1 immunotherapy and antiBRAF/antiMEK inhibitors. However, only approximately half of patients respond to these drugs, and the majority progress after 6–11 months. Therefore, a need for other therapeutic options is still very much apparent. We report the first large trial of a sequential full dose of fotemustine (FM) preceded by a low dose of temozolomide (TMZ) as a chemo-modulator in order to inactivate the DNA repair action of O(6)-methylguanine DNA-methyltransferase (MGMT). Primary endpoints were overall response and safety. We also evaluated specific biological parameters aiming to tailor these chemotherapies to selected patients. Methods A total of 69 consecutive patients were enrolled. The main features included a median age of 60 years (21–81) and M1c stage, observed in 74% of the patients, with brain metastases in 15% and high LDH levels in 42% of the patients. The following schedule was used: oral TMZ 100 mg/m 2 on days 1 and 2 and FM iv 100 mg/m 2 on day 2, 4 h after TMZ; A translational study aiming to analyse MGMT methylation status and base-excision repair (BER) gene expression was performed in a subset of 14 patients. Results We reported an overall response rate of 30.3% with 3 complete responses and a disease control rate of 50.5%. The related toxicity rate was low and mainly of haematological types. Although our population had a very poor prognosis, we observed a PFS of 6 months and an OS of 10 months. A non-significant correlation with response was found with the mean expression level of the three genes involved in the BER pathway (APE1, XRCC1 and PARP1), whereas no association was found with MGMT methylation status. Conclusion This schedule could represent a good alternative for patients who are not eligible for immune or targeted therapy or whose previous therapies have failed. Trial registration EUDRACT 2009–016487-36l ; date of registration 23 June 2010.

Selective inhibitors of BRAF, vemurafenib and dabrafenib are the standard of care for metastatic melanoma patients with BRAF V600, while chemotherapy continued to be widely used in BRAF wild type patients. In order to discover novel candidate biomarkers predictive to treatment, serum of 39 metastatic melanoma vemurafenib (n = 19) or chemotherapy (n = 20) treated patients at baseline, at disease control and at progression, were analyzed using SELDI-TOF technology. In silico analysis was used to identify more significant peaks. In patients with different BRAF status, we found 5 peptides significantly deregulated, with the down-regulation of the m/z 9176 peak strongly associated with BRAF mutation. At baseline as predictive biomarkers we identified 2 peptides - m/z 6411, 4075 - as significantly up-regulated in responders to chemotherapy and 4 peaks - m/z 5900, 12544, 49124 and 11724 - significantly up-regulated in longer vs shorter responders to vemurafenib. After response, 3 peptides (m/z 4658, 18639, and 9307) resulted significantly down regulated while 3 peptides m/z 9292, 7765 and 9176 appeared up-regulated respectively in chemotherapy and vemurafenib responder patients. In vemurafenib treated patients, 16 peaks appeared deregulated at progression compared to baseline time. In silico analysis identified proteins involved in invasiveness (SLAIN1) and resistance (ABCC12) as well as in the pathway of detoxification (NQO1) and apoptosis (RBM10, TOX3, MTEFD1, TSPO2). Proteins associated with the modulation of neuronal plasticity (RIN1) and regulatory activity factors of gene transcription (KLF17, ZBTB44) were also highlighted. Our exploratory study highlighted some factors that deserve to be further investigated in order to provide a framework for improving melanoma treatment management through the development of biomarkers which could act as the strongest surrogates of the key biological events in stage IV melanoma.

Cancer development is related not only to genetic alterations but also to aberrant epigenetic changes that could lead to heritable gene patterns critical for neoplastic initiation and progression. Knowledge of epigenetic regulation in cancer cells is useful for both the understanding of carcinogenesis and for the possibility of using epigenetic drugs. HOX genes deregulation have a crucial role in oncogenesis process and tumor suppression. In this report, the methylation of HOXA1, HOXA9, HOXA10, HOXB13, HNF1B, OTX1, TLX1 genes have been analyzed in patients with hereditary breast cancer. This is the first study analyzing BRCA mutational status of patients with respect to methylation of HOX genes. HOXA10 has been found to be methylated in all patients analyzed but never in healthy subjects. With respect to clinical pathological information, hypermethylation of all studied genes, with the exception of OTX1, was significantly associated with absence of HER2 neu expression (P<0.05). Moreover, hypermethylation of HOXB13, HOXA10 and HOXA1 was associated with a high proliferation index (Mib1≥10%, P<0.05) and hypermethylation of HOXB13 and HOXA10 also with high expression of estrogen and progesterone receptors. These preliminary data suggest a possible involvement of HOX genes in familial breast cancer as marker helpful to identify high-risk patients.