Interpreting and integrating genomic tests results in clinical cancer care: Overview and practical guidance.

The last decade has seen rapid progress in the use of genomic tests, including gene panels, whole-exome sequencing, and whole-genome sequencing, in research and clinical cancer care. These advances have created expansive opportunities to characterize the molecular attributes of cancer, revealing a subset of cancer-associated aberrations called driver mutations. The identification of these driver mutations can unearth vulnerabilities of cancer cells to targeted therapeutics, which has led to the development and approval of novel diagnostics and personalized interventions in various malignancies. The applications of this modern approach, often referred to as precision oncology or precision cancer medicine, are already becoming a staple in cancer care and will expand exponentially over the coming years. Although genomic tests can lead to better outcomes by informing cancer risk, prognosis, and therapeutic selection, they remain underutilized in routine cancer care. A contributing factor is a lack of understanding of their clinical utility and the difficulty of results interpretation by the broad oncology community. Practical guidelines on how to interpret and integrate genomic information in the clinical setting, addressed to clinicians without expertise in cancer genomics, are currently limited. Building upon the genomic foundations of cancer and the concept of precision oncology, the authors have developed practical guidance to aid the interpretation of genomic test results that help inform clinical decision making for patients with cancer. They also discuss the challenges that prevent the wider implementation of precision oncology.

Recommendations for reporting tissue and circulating tumour (ct)DNA next-generation sequencing results in non-small cell lung cancer.

Non-small cell lung cancer is a heterogeneous disease and molecular characterisation plays an important role in its clinical management. Next-generation sequencing-based panel testing enables many molecular alterations to be interrogated simultaneously, allowing for comprehensive identification of actionable oncogenic drivers (and co-mutations) and appropriate matching of patients with targeted therapies. Despite consensus in international guidelines on the importance of broad molecular profiling, adoption of next-generation sequencing varies globally. One of the barriers to its successful implementation is a lack of accepted standards and guidelines specifically for the reporting and clinical annotation of next-generation sequencing results. Based on roundtable discussions between pathologists and oncologists, we provide best practice recommendations for the reporting of next-generation sequencing results in non-small cell lung cancer to facilitate its use and enable easy interpretation for physicians. These are intended to complement existing guidelines related to the use of next-generation sequencing (solid and liquid). Here, we discuss next-generation sequencing workflows, the structure of next-generation sequencing reports, and our recommendations for best practice thereof. The aim of these recommendations and considerations is ultimately to ensure that reports are fully interpretable, and that the most appropriate treatment options are selected based on robust molecular profiles in well-defined reports.

Multiple predictive biomarker testing in melanoma: Another challenge in identifying the optimal approach on cytological samples.

BACKGROUND: The management of cutaneous melanoma has changed dramatically in recent years thanks to the development of tyrosine kinase and immune-checkpoint inhibitors (ICIs). Thus, multiple biomarker testing is becoming ever more important for the identification of patients who are potentially eligible for these treatments. One reliable approach to the molecular evaluation of metastatic melanoma is fine needle cytology (FNC). To examine the utility of this approach for assessing PD-L1 expression levels, we evaluated the cellular adequacy of residual cell block (CB) material from metastatic melanomas that were previously tested for BRAF and NRAS mutations. METHODS: We retrieved from our internal archives a series of FNC samples of metastatic melanoma that had been subjected to molecular testing on residual CB material or a dedicated needle rinse between January 2016 and July 2022. Real-time polymerase chain reaction was used to assess BRAF and NRAS status, and an SP263 assay was employed to ascertain PD-L1 expression levels. RESULTS: Overall, n = 19 cases were selected. Of these, 11 (57.9%) cases revealed a BRAF exon 15 p.V600E mutation, one case (5.3%) revealed NRAS mutation, and seven cases (36.8%) showed no mutations. Regarding PD-L1 assessment, 16/19 (84.2%) cases were deemed adequate, meaning they contained at least 100 viable cells. CONCLUSIONS: We highlighted the feasibility of assessing PD-L1 expression levels in residual CB material from metastatic melanomas previously tested for BRAF and NRAS mutations. Moreover, we pointed out that FNC needle rinses may be an alternative source of nucleic acids for molecular testing, preserving CB material for immunocytochemistry evaluation.

FGFR2 testing in cholangiocarcinoma: translating molecular studies into clinical practice.

Cholangiocarcinoma (CCA) is a heterogeneous group of neoplasms burdened by a dismal prognosis. Several studies have investigated the genomic profile of CCA and identified numerous druggable genetic alterations, including FGFR2 fusions/rearrangements. Approximately 5-7% of CCAs and 10-20% of intrahepatic iCCAs harbor FGFR2 fusions. With the recent advent of FGFR-targeting therapies into clinical practice, a standardization of molecular testing for FGFR2 alterations in CCA will be necessary. In this review, we describe the technical aspects and challenges related to FGFR2 testing in routine practice, focusing on the comparison between Next-Generation Sequencing (NGS) and FISH assays, the best timing to perform the test, and on the role of liquid biopsy.

The ideal reporting of RAS testing in colorectal adenocarcinoma: a pathologists' perspective.

RAS gene mutational status represents an imperative predictive biomarker to be tested in the clinical management of metastatic colorectal adenocarcinoma. Even if it is one of the most studied biomarkers in the era of precision medicine, several pre-analytical and analytical factors may still impasse an adequate reporting of RAS status in clinical practice, with significant therapeutic consequences. Thus, pathologists should be aware on the main topics related to this molecular evaluation: (i) adopt diagnostic limit of detections adequate to avoid the interference of sub-clonal cancer cell populations; (ii) choose the most adequate diagnostic strategy according to the available sample and its qualification for molecular testing; (iii) provide all the information regarding the mutation detected, since many RAS mutation-specific targeted therapeutic approaches are in development and will enter into routine clinical practice. In this review, we give a comprehensive description of the current scenario about RAS gene mutational testing in the clinic focusing on the pathologist's role in patient selection for targeted therapies.

Think "HER2" different: integrative diagnostic approaches for HER2-low breast cancer.

This work explores the complex field of HER2 testing in the HER2-low breast cancer era, with a focus on methodological aspects. We aim to propose clear positions to scientific societies, institutions, pathologists, and oncologists to guide and shape the appropriate diagnostic strategies for HER2-low breast cancer. The fundamental question at hand is whether the necessary tools to effectively translate our knowledge about HER2 into practical diagnostic schemes for the lower spectrum of expression are available. Our investigation is centered on the significance of distinguishing between an immunohistochemistry (IHC) score 0 and score 1+ in light of the clinical implications now apparent, as patients with HER2-low breast cancer become eligible for trastuzumab-deruxtecan treatment. Furthermore, we discuss the definition of HER2-low beyond its conventional boundaries and assess the reliability of established diagnostic procedures designed at a time when therapeutic perspectives were non-existent for these cases. In this regard, we examine potential complementary technologies, such as gene expression analysis and liquid biopsy. Ultimately, we consider the potential role of artificial intelligence (AI) in the field of digital pathology and its integration into HER2 testing, with a particular emphasis on its application in the context of HER2-low breast cancer.

The Treatment of Advanced Melanoma: Therapeutic Update.

Cutaneous melanoma is the main cause of death for skin cancer. The majority of patients with a diagnosis of melanoma have localized disease, which can be successfully treated with surgical treatment. However, the surgical approach is not curative for advanced melanoma (AM). Indeed, the management of AM is still challenging, since melanoma is the solid tumor with the highest number of mutations and cancer cells have the capacity to evade the immune system. In the past, the treatment of AM relied on chemotherapeutic agents, without showing efficacy data. Recent knowledge on melanoma pathogenesis as well as the introduction of immunotherapies, targeted therapies vaccines, small molecules, and combination therapies has revolutionized AM management, showing promising results in terms of effectiveness and safety. The aim of this review is to assess and to discuss the role of emerging therapies for AM management in order to obtain a complete overview of the currently available treatment options and future perspectives.

B7-H3/CD276 Inhibitors: Is There Room for the Treatment of Metastatic Non-Small Cell Lung Cancer?

The striking clinical outcomes of antibody-based immunotherapy, through the inhibitors of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and the programmed cell death protein-1 (PD-1) and its ligand (PD-L1) axis, have driven research aimed at identifying further clinically relevant tumor antigens that can serve as targets in solid tumors. B7 homolog 3 protein (B7-H3, also known as CD276) is a member of the B7 family overexpressed in tumor tissues, including non-small cell lung cancer (NSCLC), while showing limited expression in normal tissues, becoming an attractive and promising target for cancer immunotherapy. B7-H3 expression in tumors has been demonstrated to be associated with poor prognosis. In addition to its role in immune modulation, B7-H3 also promotes pro-tumorigenic functions such as tumor migration, invasion, metastases, resistance, and metabolism. In this review, we will provide an overview of this newly characterized immune checkpoint molecule and its development in the management of metastatic NSCLC.

Evaluation of the Molecular Landscape in PD-L1 Positive Metastatic NSCLC: Data from Campania, Italy.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) have increased and improved the treatment options for patients with non-oncogene-addicted advanced stage non-small cell lung cancer (NSCLC). However, the role of ICIs in oncogene-addicted advanced stage NSCLC patients is still debated. In this study, in an attempt to fill in the informational gap on the effect of ICIs on other driver mutations, we set out to provide a molecular landscape of clinically relevant oncogenic drivers in programmed death-ligand 1 (PD-L1) positive NSCLC patients. METHODS: We retrospectively reviewed data on 167 advanced stage NSCLC PD-L1 positive patients (≥1%) who were referred to our clinic for molecular evaluation of five driver oncogenes, namely, EGFR, KRAS, BRAF, ALK and ROS1. RESULTS: Interestingly, n = 93 (55.7%) patients showed at least one genomic alteration within the tested genes. Furthermore, analyzing a subset of patients with PD-L1 tumor proportion score (TPS) ≥ 50% and concomitant gene alterations (n = 8), we found that n = 3 (37.5%) of these patients feature clinical benefit with ICIs administration, despite the presence of a concomitant KRAS gene alteration. CONCLUSIONS: In this study, we provide a molecular landscape of clinically relevant biomarkers in NSCLC PD-L1 positive patients, along with data evidencing the clinical benefit of ICIs in patient NSCLC PD-L1 positive alterations.

ctDNA analysis in the personalized clinical management of gastroesophageal adenocarcinoma: turning hope into reality.

Gastroesophageal adenocarcinoma (GEA) is a global health issue with a high fatality-to-case ratio and a 5-year overall survival that has only slightly improved. High-throughput molecular profiling has uncovered a profound complexity and heterogeneity in GEA biology, which limits considerably the treatment advances. Liquid biopsy with circulating tumor (ct)DNA analysis could elucidate GEA molecular heterogeneity and provide diagnostic, prognostic and predictive information to guide clinical decision-making. However, only a handful of studies have shown positive results for the application of ctDNA analysis in GEA clinical management. As a result, no comprehensive information is available to date on this continuously evolving topic. Here, we discuss the current state of knowledge, along with promises and challenges related to ctDNA analysis in GEA.

Immunotherapy in Lung Cancer: Are the Promises of Long-Term Benefit Finally Met?

Over the last few years, agents targeting immune checkpoints have shown potential to improve therapeutic outcomes in patients with lung cancer in multiple clinical settings. Inhibitors of PD-1/PD-L1 have been approved for the treatment of different types of lung cancer by the FDA either alone or in combination with chemotherapy or other immune checkpoint inhibitors, such as anti-CTLA-4 agents. The introduction of these agents in clinical practice has revolutionized the therapeutic approach to lung cancer, keeping the promises of long-term benefit in selected patient populations. The therapeutic indications of immunotherapy in lung cancer are rapidly growing, and multiple combinations entered clinical practice or are under active development. Furthermore, the quest for a reliable predictive biomarker is still ongoing to overcome the limits of currently approved tests for patients' selection. In this review, we summarized the current status and progress of anti-PD-1/PD-L1 agents in lung cancer treatment.

PD-L1 and beyond: Immuno-oncology in cytopathology.

Over the past decade, immunotherapy has emerged as one of the most promising cancer treatments. Several monoclonal antibodies targeting the programmed death 1 (PD-1)/ programmed death ligand-1 (PD-L1) pathway have been integrated into standard-of-care treatments for a wide range of cancer types. Although all the available PD-L1 immunohistochemistry (IHC) assays have been developed on formalin-fixed histological specimens, a growing body of research has recently suggested the feasibility of PD-L1 testing on cytological samples. Although promising results have been reported, several important issues still need to be addressed. Among these are pre-analytical issues, cyto-hystological correlation, and inter-observer agreement. This review will briefly summarise the knowledge gaps and future directions of cytopathology in the immuno-oncology scenario.

Next generation sequencing in cytology.

The application of next generation sequencing (NGS) technology to cytological samples has significantly modified molecular cytopathology practice. Cytological samples represent a valid source of high-quality DNA for NGS analysis, especially for predicting patients' response to targeted treatments and for refining the risk of malignancy in indeterminate cytological diagnoses. However, several pre-analytical factors may influence the reliability of NGS clinical analysis. Here, we briefly review the challenges of NGS in cytology practice, focusing on those pre-analytical factors that may negatively affect NGS success rates and routine diagnostic applications. Finally, we address the future directions of the field.

Novel Emerging Molecular Targets in Non-Small Cell Lung Cancer.

In the scenario of systemic treatment for advanced non-small cell lung cancer (NSCLC) patients, one of the most relevant breakthroughs is represented by targeted therapies. Throughout the last years, inhibitors of the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-Ros oncogene 1 (ROS1), and V-raf murine sarcoma viral oncogene homolog B (BRAF) have been approved and are currently used in clinical practice. However, other promising molecular drivers are rapidly emerging as therapeutic targets. This review aims to cover the molecular alterations with a potential clinical impact in NSCLC, including amplifications or mutations of the mesenchymal-epithelial transition factor (MET), fusions of rearranged during transfection (RET), rearrangements of the neurotrophic tyrosine kinase (NTRK) genes, mutations of the Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), as well as amplifications or mutations of human epidermal growth factor receptor 2 (HER2). Additionally, we summarized the current status of targeted agents under investigation for such alterations. This revision of the current literature on emerging molecular targets is needed as the evolving knowledge on novel actionable oncogenic drivers and targeted agents is expected to increase the proportion of patients who will benefit from tailored therapeutic approaches.

[Liquid biopsy as indication of tumour progression in lung cancer: a potential intervention in tertiary prevention]. / La biopsia liquida quale segnale di progressione tumorale nel carcinoma polmonare: potenziale di intervento in prevenzione terziaria.

In the era of personalised therapies, liquid biopsy is considered an important diagnostic tool in the clinical management of cancer patients. Tissue specimen represents the gold standard for molecular evaluation of specific gene targets alterations that lead cancer patients to benefit of a "tailed therapy" based on molecular features of the tumour. This innovative source of nucleic acids was introduced in clinical setting only for non-small-cell lung cancer (NSCLC) patients to test epidermal grow factor receptor (EGFR) mutations when tissue is not available for a number of reasons (difficult access to the lesion, the presence of other disabling pathologies, especially in elderly patients, rejection by the patient, etcetera) or to monitor acquired resistance mutation after a first line of treatment. The present study aimed at assessing the diagnostic potential of liquid biopsy in balanced tertiary screening modelling. The cases relating to 5 years of activity regarding to molecular diagnostics performed on liquid biopsy specimens in the Predictive diagnostic laboratory of the University hospital "Federico II" of Naples (Campania Region, Southern Italy) were reviewed. Laboratory data were collected through the software SPSS. Non-parametric analysis was performed in order to test the differences between "wild type" patients or not. A multivariate logistic model was performed in order to assess the effect of mutation, age, and gender on the tumour progression. The results of the revision concern 515 total cases (almost of all plasma or peripheral blood), which allowed to evaluate the liquid biopsies for women and men. The average age of the patients is 66.3 years, and the 25° percentile is 59 years. The cases are: 221 basal and 294 by progression. The cases with mutation, as expected, have an odds ratio of 4,15, compared to the basal, to have a tumour progression (95%IC 2,7-6,3), regardless of gender and age. The detected mutations were 131 from different types of pulmonary carcinomas. Working on case data, specifying the characteristics of the patients with mutations will drive a further estimate in tertiary prevention screening designs.

Emerging angiogenesis inhibitors for non-small cell lung cancer.

Introduction: Angiogenesis represents a complex process crucial during embryo development, wound healing, and collateral formation for improved organ perfusion. Numerous stimulatory and inhibitory pathways through their balance regulate angiogenesis and vascular homeostasis. Targeting the pathways implicated in the regulation of angiogenesis and neo-angiogenesis plays an important role in cancer research, treatment, and patients' outcome. Antiangiogenic strategies, including monoclonal antibodies binding vascular endothelial growth factor (VEGF) or the corresponding receptor and small molecules which inhibit the function of different angio-related tyrosine kinase, produced interesting results in cancer treatments including non-small-cell lung cancer (NSCLC). Areas covered: The current state-of-the-art of anti-angiogenesis treatment in the management of NSCLC patients is reviewed and discussed. A structured search of bibliographic databases for peer-reviewed research literature and of main meetings using a focused review question was undertaken in order to discuss about emerging angiogenesis inhibitors in NSCLC. Expert opinion: Targeting angiogenesis remains an important therapeutic strategy in the management of NSCLC. Moreover, VEGF has been recognized having also an immunosuppressive action leading to investigate the potential activity of angiogenic inhibitors in restoring the antitumor immunity by targeting VEGF/VEGF-Receptor. Furthermore, new anti-angiogenic drugs for which there is also the availability of predictive biomarkers are welcome.

Setting up and exploitation of a nano/technological platform for the evaluation of HMGA1b protein in peripheral blood of cancer patients.

Even if cancer specific biomarkers are present in peripheral blood of cancer patients, it is very difficult to detect them with conventional technology because of their low concentration. A potential cancer biomarker is the HMGA1b protein, whose overexpression is a feature of several human malignant neoplasias. By taking advantage of the surface plasmon resonance (SPR) phenomenon, we realized a specific nano/technology-based assay for cancer detection. More in details, anti-HMGA1b monoclonal antibodies, whose affinity was previously defined by ELISA, were immobilized onto metallic surfaces to develop a direct SPR-based assay. After having analyzed blood samples from colorectal cancer patients and healthy people for the presence of HMGA1b, we observed a 2-fold increase of the HMGA1b levels in the blood of cancer patients with respect to the healthy control people. We conclude that the set-up technology might allow to detect a tumoral mass through the evaluation of HMGA1b protein blood levels.

Prognostic Factors and Biomarkers of Responses to Immune Checkpoint Inhibitors in Lung Cancer.

Manipulation of the immune response is a game changer in lung cancer treatment, revolutionizing management. PD1 and CTLA4 are dynamically expressed on different T cell subsets that can either disrupt or sustain tumor growth. Monoclonal antibodies (MoAbs) against PD1/PDL1 and CTLA4 have shown that inhibitory signals can be impaired, blocking T cell activation and function. MoAbs, used as both single-agents or in combination with standard therapy for the treatment of advanced non-small cell lung cancer (NSCLC), have exhibited advantages in terms of overall survival and response rate; nivolumab, pembrolizumab, atezolizumab and more recently, durvalumab, have already been approved for lung cancer treatment and more compounds are in the pipeline. A better understanding of signaling elicited by these antibodies on T cell subsets, as well as identification of biological determinants of sensitivity, resistance and correlates of efficacy, will help to define the mechanisms of antitumor responses. In addition, the relevance of T regulatory cells (Treg) involved in immune responses in cancer is attracting increasing interest. A major challenge for future research is to understand why a durable response to immune checkpoint inhibitors (ICIs) occurs only in subsets of patients and the mechanisms of resistance after an initial response. This review will explore current understanding and future direction of research on ICI treatment in lung cancer and the impact of tumor immune microenvironment n influencing clinical responses.

Targeting Immune-Related Biological Processes in Solid Tumors: We do Need Biomarkers.

Immunotherapy has become the standard-of-care in many solid tumors. Despite the significant recent achievements in the diagnosis and treatment of cancer, several issues related to patients' selection for immunotherapy remain unsolved. Multiple lines of evidence suggest that, in this setting, the vision of a single biomarker is somewhat naïve and imprecise, given that immunotherapy does not follow the rules that we have experienced in the past for targeted therapies. On the other hand, additional immune-related biomarkers that are reliable in real-life clinical practice remain to be identified. Recently, the immune-checkpoint blockade has been approved in the US irrespective of the tumor site of origin. Further histology-agnostic approvals, coupled with with tumor-specific companion diagnostics and guidelines, are expected in this field. In addition, immune-related biomarkers can also have a significant prognostic value. In this review, we provide an overview of the role of these biomarkers and their characterization in the management of lung cancer, melanoma, colorectal cancer, gastric cancer, head and neck cancer, renal cell carcinoma, urothelial cancers, and breast cancer.

Osimertinib.

Epidermal growth factor receptor (EGFR)-mutated (exons 18-21) advanced non-small cell lung cancers (NSCLCs) are generally characterized by exquisite sensitivity to treatment with an EGFR-tyrosine kinase inhibitor (-TKI). First-generation or reversible EGFR-TKIs include gefitinib and erlotinib, while, more recently, second-generation or irreversible EGFR-TKIs have been developed, namely afatinib and dacomitinib, with the aim of overcoming/delaying acquired resistance to treatment. Nevertheless, clinical trials have shown that resistance eventually emerges after a median time of slightly less than one year, regardless of whether first- or second-generation EGFR-TKIs are used. In this context, a secondary EGFR mutation in exon 20, namely T790M, has been found to be responsible for approximately 60% of cases of acquired resistance. Alternatively, T790M resistance mutation can be found de novo, in which case it limits the antitumor activity of both first- or second-generation EGFR-TKIs. Osimertinb is an orally bioavailable, third-generation EGFR-TKI that acts by irreversibly binding both EGFR activating mutations and T790M, while sparing wild-type EGFR. On this basis, osimertinib has proven more efficacious than platinum-based chemotherapy in the setting of EGFR T790M-positive NSCLCs pretreated with a first- or second-generation EGFR-TKI. More recently, in another phase 3 trial, osimertinib outperformed gefitinib or erlotinib as first-line treatment of EGFR-mutated (ex19del or L858R) advanced NSCLCs, thus emerging as a new standard of care in this setting. In the present review, we will discuss the preclinical and clinical development of osimertinib, briefly touching upon its activity in special populations and biomarkers of sensitivity to treatment.