Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

NK cells to cure cancer.

Natural Killer (NK) cells are innate lymphocytes able to mediate immune-surveillance and clearance of viral infected and tumor-transformed cells. Growing experimental and clinical evidence highlighted a dual role of NK cells either in the control of cancer development/progression or in promoting the onset of immune-suppressant tumor microenvironments. Indeed, several mechanisms of NK cell-mediated tumor escape have been described and these includes cancer-induced aberrant expression of activating and inhibitory receptors (i.e. NK cell immune checkpoints), impairments of NK cell migration to tumor sites and altered NK cell effector-functions. These phenomena highly contribute to tumor progression and metastasis formation. In this review, we discuss the latest insights on those NK cell receptors and related molecules that are currently being implemented in clinics either as possible prognostic factors or therapeutic targets to unleash NK cell anti-tumor effector-functions in vivo. Moreover, we address here the major recent advances in regard to the genetic modification and ex vivo expansion of anti-tumor specific NK cells used in innovative adoptive cellular transfer approaches.

Comprehensive Phenotyping of Human PB NK Cells by Flow Cytometry.

The NK cell compartment provides powerful innate defenses against virus-infected and tumor cells. Specific NK cell receptors control this process and maintain the immune system homeostasis and prevent autoimmunity. A wide variety of NK cell subsets with different functional capabilities exist and this reflects not only the different maturation stages of NK cells but also different microenvironments in which they can operate. In this review, we will give an overview on the various NK cell subsets present in peripheral blood of healthy donors in order to clearly and univocally identify them on the basis of their phenotypic traits using flow cytometry. © 2020 International Society for Advancement of Cytometry.

Strengthening the AntiTumor NK Cell Function for the Treatment of Ovarian Cancer.

The crosstalk between cancer cells and host cells is a crucial prerequisite for tumor growth and progression. The cells from both the innate and adaptive immune systems enter into a perverse relationship with tumor cells to create a tumor-promoting and immunosuppressive tumor microenvironment (TME). Epithelial ovarian cancer (EOC), the most lethal of all gynecological malignancies, is characterized by a unique TME that paves the way to the formation of metastasis and mediates therapy resistance through the deregulation of immune surveillance. A characteristic feature of the ovarian cancer TME is the ascites/peritoneal fluid, a malignancy-associated effusion occurring at more advanced stages, which enables the peritoneal dissemination of tumor cells and the formation of metastasis. The standard therapy for EOC involves a combination of debulking surgery and platinum-based chemotherapy. However, most patients experience disease recurrence. New therapeutic strategies are needed to improve the prognosis of patients with advanced EOC. Harnessing the body's natural immune defenses against cancer in the form of immunotherapy is emerging as an innovative treatment strategy. NK cells have attracted attention as a promising cancer immunotherapeutic target due to their ability to kill malignant cells and avoid healthy cells. Here, we will discuss the recent advances in the clinical application of NK cell immunotherapy in EOC.

Human NK cell response to pathogens.

NK cells represent important effectors of the innate immunity in the protection of an individual from microbes. During an NK-mediated anti-microbial response, the final fate (survival or death) of a potential infected target cell depends primarily on the type and the number of receptor/ligand interactions occurring at the effector/target immune synapse. The identification of an array of receptors involved in NK cell triggering has been crucial for a better understanding of the NK cell biology. In this context, NCR play a predominant role in NK cell activation during the process of natural cytotoxicity. Regarding the NK-mediated pathogen recognition and NK cell activation, an emerging concept is represented by the involvement of TLRs and activating KIRs. NK cells express certain TLRs in common with other innate cell types. This would mean that specific TLR ligands are able to promote the simultaneous and synergistic stimulation of these innate cells, providing a coordinated mechanism for regulating the initiation and amplification of immune responses. Evidences have been accumulated indicating that viral infections may have a significant impact on NK cell maturation, promoting the expansion of phenotypically and functionally aberrant NK cell subpopulations. For example, during chronic HIV-infection, an abnormal expansion of a dysfunctional CD56neg NK cell subset has been detected that may explain, at least in part, the defective NK cell-mediated antiviral activity. An analogous imbalance of NK cell subsets has been detected in patients receiving HSCT to cure high risk leukemias and experiencing HCMV infection/reactivation. Remarkably, NK cells developing after CMV reactivation may contain "memory-like" or "long-lived" NK cells that could exert a potent anti-leukemia effect.

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization.

BACKGROUND: Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. OBJECTIVE: We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. METHODS: We performed multiparametric cytofluorimetric analysis of PD-1+ NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. RESULTS: We provide unequivocal evidence that PD-1 is highly expressed (PD-1bright) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56dim but not CD56bright NK cells and is confined to fully mature NK cells characterized by the NKG2A-KIR+CD57+ phenotype. Proportions of PD-1bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. CONCLUSIONS: We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction.

TLR-Stimulated Neutrophils Instruct NK Cells To Trigger Dendritic Cell Maturation and Promote Adaptive T Cell Responses.

Polymorphonuclear neutrophils (PMNs) are innate effector cells with pivotal roles in pathogen recognition, phagocytosis, and eradication. However, their role in the development of subsequent immune responses is incompletely understood. This study aimed to identify mechanisms of relevance to the cross talk between human neutrophils and NK cells and its potential role in promoting adaptive immunity. TLR-stimulated PMNs were found to release soluble mediators to attract and activate NK cells in vitro. PMN-conditioned NK cells displayed enhanced cytotoxicity and cytokine production, and responded vigorously to ensuing stimulation with exogenous and endogenous IL-12. The neutrophil-induced activation of NK cells was prevented by caspase-1 inhibitors and by natural antagonists to IL-1 and IL-18, suggesting a role for the NOD-like receptor family pyrin domain containing-3 inflammasome. In addition, PMN-conditioned NK cells triggered the maturation of monocyte-derived dendritic cells, which promoted T cell proliferation and IFN-γ production. These data imply that neutrophils attract NK cells to sites of infection to convert these cells into an active state, which drives adaptive immune responses via maturation of dendritic cells. Our results add to a growing body of evidence that suggests a sophisticated role for neutrophils in orchestrating the immune response to pathogens.

KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts.

Natural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7(+) cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2-specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7(+) cells. Co-incubation of KIR2DS1(+) fresh NK cells or NK-cell clones with HLA-C2(+) CCR7(+) lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2(+) allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1(+) NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions.

Human NK Cells induce neutrophil apoptosis via an NKp46- and Fas-dependent mechanism.

Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell-cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.

NK cell receptors in anti-tumor and healthy tissue protection: Mechanisms and therapeutic advances.

Natural Killer (NK) cells are integral to the innate immune system, renowned for their ability to target and eliminate cancer cells without the need for antigen presentation, sparing normal tissues. These cells are crucial in cancer immunosurveillance due to their diverse array of activating and inhibitory receptors that modulate their cytotoxic activity. However, the tumor microenvironment can suppress NK cell function through various mechanisms. Over recent decades, research has focused on overcoming these tumor escape mechanisms. Initially, efforts concentrated on enhancing T cell activity, leading to impressive results with immunotherapeutic approaches aimed at boosting T cell responses. Nevertheless, a substantial number of patients do not benefit from these treatments and continue to seek effective alternatives. In this context, NK cells present a promising avenue for developing new treatments, given their potent cytotoxic capabilities, safety profile, and activity against T cell-resistant tumors, such as those lacking HLA-I expression. Recent advancements in immunotherapy include strategies to restore and amplify NK cell activity through immune checkpoint inhibitors, cytokines, adoptive NK cell therapy, and CAR-NK cell technology. This review provides a comprehensive overview of NK cell receptors, the tumor escape mechanisms that hinder NK cell function, and the evolving field of NK cell-based cancer immunotherapy, highlighting ongoing efforts to develop more effective and targeted cancer treatment strategies.

Crosstalk and communication of cancer-associated fibroblasts with natural killer and dendritic cells: New frontiers and unveiled opportunities for cancer immunotherapy.

Natural killer (NK) cells and dendritic cells (DCs) are critical mediators of anti-cancer immune responses. In addition to their individual roles, NK cells and DCs are involved in intercellular crosstalk which is essential for the initiation and coordination of adaptive immunity against cancer. However, NK cell and DC activity is often compromised in the tumor microenvironment (TME). Recently, much attention has been paid to one of the major components of the TME, the cancer-associated fibroblasts (CAFs), which not only contribute to extracellular matrix (ECM) deposition and tumor progression but also suppress immune cell functions. It is now well established that CAFs support T cell exclusion from tumor nests and regulate their cytotoxic activity. In contrast, little is currently known about their interaction with NK cells, and DCs. In this review, we describe the interaction of CAFs with NK cells and DCs, by secreting and expressing various mediators in the TME of adult solid tumors. We also provide a detailed overview of ongoing clinical studies evaluating the targeting of stromal factors alone or in combination with immunotherapy based on immune checkpoint inhibitors. Finally, we discuss currently available strategies for the selective depletion of detrimental CAFs and for a better understanding of their interaction with NK cells and DCs.

The landscape of combining immune checkpoint inhibitors with novel Therapies: Secret alliances against breast cancer.

This review focuses on the immune checkpoint inhibitors (ICIs) in the context of breast cancer (BC) management. These innovative treatments, by targeting proteins expressed on both tumor and immune cells, aim to overcome tumor-induced immune suppression and reactivate the immune system. The potential of this approach is the subject of numerous clinical studies. Here, we explore the key studies and emerging therapies related to ICIs providing a detailed analysis of their specific and combined use in BC treatment.

Evaluation of milk powder quality by protein oxidative modifications.

The objective of the present research was to evaluate commercially available milk powders according to their protein oxidative modifications and antioxidant capacity, and to evaluate if these characteristics are related to physical quality parameters such as dispersibility or stability during storage. Fifteen commercially processed spray-dried milk powders were evaluated: 6 whole milk powders (WMP), 4 skim milk powders (SMP), and 5 infant formula powders (IFP). Protein oxidative status was measured as protein carbonyl (PC) content, dityrosine content, and extent of protein polymerization. The level of PC was slightly lower in SMP than in WMP, whereas IFP had more than twice as much PC as WMP (2.8 ± 0.4, 2.1 ± 0.2, and 6.5 ± 1.3 nmol/mg of protein for WMP, SMP, and IFP, respectively). No differences were detected in dityrosine accumulation. Although all the possible pairs of parameters were tested for correlations, we found that 4 parameters were linked: PC, whey content, protein aggregate level, and dispersibility. After 9 mo of storage at -20°C or room temperature, all milk samples were analyzed to evaluate changes in protein oxidative status (PC, dityrosine, and protein integrity) and related parameters. Compared with the initial condition, PC increased in all tested samples after 9 mo of storage at -20°C or at room temperature. Stored milk powders had increased PC and decreased dispersibility compared with prestorage levels. Our results highlight the importance of protein oxidative status in milk powder and its relationship to other related quality parameters, such as protein integrity and dispersibility. Our findings suggest that the understanding of such relationships could help in developing quality differentiation for different types of milk powders in the product market.

Identification of a novel cord blood NK cell subpopulation expressing functional programmed death receptor-1.

Background: Natural Killer cells (NKs) represent the innate counterpart of TCRαß lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection. Methods: In this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis. Results: We identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered. Conclusion: To our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

The fading guardian: clinical relevance of TP53 null mutation in high-grade serous ovarian cancers.

Background: we evaluated the concordance between immunohistochemical p53 staining and TP53 mutations in a series of HGSOC. Moreover, we searched for prognostic differences between p53 overexpression and null expression groups. Methods: patients affected by HGSOC were included. For each case p53 immunohistochemical staining and molecular assay (Sanger sequencing) were performed. Kaplan-Meier survival analyses were undertaken to determine whether the type of TP53 mutation, or p53 staining pattern influenced overall survival (OS) and progression free survival (PFS). Results: 34 HGSOC were considered. All cases with a null immunohistochemical p53 expression (n=16) showed TP53 mutations (n=9 nonsense, n=4 in-frame deletion, n=2 splice, n=1 in-frame insertion). 16 out of 18 cases with p53 overexpression showed TP53 missense mutation. Follow up data were available for 33 out of 34 cases (median follow up time 15 month). We observed a significant reduction of OS in p53 null group [HR = 3.64, 95% CI 1.01-13.16]. Conclusion: immunohistochemical assay is a reliable surrogate for TP53 mutations in most cases. Despite the small cohort and the limited median follow up, we can infer that HGSOC harboring p53 null mutations are a more aggressive subgroup.

NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells.

BACKGROUND: The natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at determining to what extent NKC haplotypes impact on NK cell repertoire and function, and whether such gene variants impact on outcome of IL-2-based immunotherapy in acute myeloid leukemia (AML). METHODS: Genotype status of NKG2D rs1049174 and NKG2A rs1983526 was determined using the TaqMan-Allelic discrimination approach. To dissect the impact of single nucloetide polymorphim (SNP) on NK cell function, we engineered the K562 cell line with CRISPR to be killed in a highly NKG2D-dependent fashion. NK cells were assayed for degranulation, intracellular cytokine production and cytotoxicity using flow cytometry. RESULTS: In AML patients receiving immunotherapy, the NKG2A gene variant, rs1983526, was associated with superior leukemia-free survival and overall survival. We observed that superior NK degranulation from individuals with the high-cytotoxicity NKG2D variant was explained by presence of a larger, highly responsive NKG2A+ subset. Notably, NK cells from donors homozygous for a favorable allele encoding NKG2A mounted stronger cytokine responses when challenged with leukemic cells, and NK cells from AML patients with this genotype displayed higher accumulation of granzyme B during histamine dihydrochloride/IL-2 immunotherapy. Additionally, among AML patients, the NKG2A SNP defined a subset of patients with HLA-B-21 TT with a strikingly favorable outcome. CONCLUSIONS: The study results imply that a dimorphism in the NKG2A gene is associated with enhanced NK cell effector function and improved outcome of IL-2-based immunotherapy in AML.

A new method for oral cancer biomarkers detection with a non-invasive cyto-salivary sampling and rapid-highly sensitive ELISA immunoassay: a pilot study in humans.

Introduction: Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies and has a 5-year mortality rate close to 50%. A consistent part (70%) of all oral cancers is diagnosed at an advanced stage since available screening techniques are ineffective. Therefore, it would be urgent to improve them. The diagnostic gold standard is tissue biopsy with histological and immunohistochemical assessment. This method presents some limitations. Biopsy is invasive and the histopathological evaluation is semi-quantitative, and the absolute abundance of the target cannot be reliably determined. In addition, tissue is highly processed and may lead to loss of information of the natural state. The search for classical and new clinical biomarkers on fragments of tissue/cells collected with a cytobrush is a highly hopeful technique for early detection and diagnosis of OSCC, because of its non-invasive sampling and easy collection method. Methods: Here we analyzed cytobrush biopsies samples collected from the oral cavity of 15 patients with already diagnosed OSCC by applying an innovative high-sensitivity ELISA technique, in order to verify if this approach may provide useful information for detection, diagnosis, and prognosis of OSCC. To this end, we selected six biomarkers, already used in clinical practice for the diagnosis of OSCC (EGFR, Ki67, p53) or selected based on recent scientific and clinical data which indicate their presence or over-expression in cells undergoing transformation and their role as possible molecular targets in immunecheckpoints blockade therapies (PD-L1, HLA-E, B7-H6). Results: The selected tumor biomarkers were highly expressed in the tumor core, while were virtually negative in healthy tissue collected from the same patients. These differences were highly statistically significant and consistent with those obtained using the gold standard test clearly indicating that the proposed approach, i.e. analysis of biomarkers by a custom ELISA technique, is strongly reliable. Discussion: These preliminary data suggest that this non-invasive rapid phenotyping technique could be useful as a screening tool for phenotyping oral lesions and support clinical practice by precise indications on the characteristics of the lesion, also with a view to the application of new anti-tumor treatments, such as immunotherapy, aimed at OSCC patients.

Harnessing Immune Response in Acute Myeloid Leukemia.

Despite the results achieved with the evolution of conventional chemotherapy and the inclusion of targeted therapies in the treatment of acute myeloid leukemia (AML), survival is still not satisfying, in particular in the setting of relapsed/refractory (R/R) disease or elderly/unfit patients. Among the most innovative therapeutic options, cellular therapy has shown great results in different hematological malignancies such as acute lymphoblastic leukemia and lymphomas, with several products already approved for clinical use. However, despite the great interest in also expanding the application of these new treatments to R/R AML, no product has been approved yet for clinical application. Furthermore, cellular therapy could indeed represent a powerful tool and an appealing alternative to allogeneic hematopoietic stem cell transplantation for ineligible patients. In this review, we aim to provide an overview of the most recent clinical research exploring the effectiveness of cellular therapy in AML, moving from consolidated approaches such as post- transplant donor's lymphocytes infusion, to modern adoptive immunotherapies such as alloreactive NK cell infusions, engineered T and NK cells (CAR-T, CAR-NK) and novel platforms of T and NK cells engaging (i.e., BiTEs, DARTs and ANKETTM).

NK/DC crosstalk in anti-viral response.

In recent years, it has been emphasized the role of the crosstalk between natural killer (NK) cells and monocyte-derived dendritic cells dendritic cells (moDCs) in the regulation of the early phases of innate immunity innate immunity and of the subsequent adaptive immune responses. NK cells and DCs coordinate their response communicating through direct cell-to-cell contact and soluble factors. NK cells appear to contribute to the quality control of immature DCs (iDCs) undergoing maturation. On the other hand, DCs may shape the magnitude of innate immune responses by modulating the NK-mediated cytolytic activity against tumors or infected cells. Recent studies suggest that the cooperation between NK cells and DCs is also critical in several anti-viral responses. In particular, NK cells are capable of effectively counteracting viral immune evasion immune evasion strategies by eliminating infected DCs, that display impaired antigen presenting functions, thus indirectly favoring the development of adaptive immune responses to viral antigens cross-presented by healthy DCs.

Case report: Variable response to immunotherapy in ovarian cancer: Our experience within the current state of the art.

Despite recent advances in ovarian cancer (OC) treatment, including the introduction of bevacizumab and PARP-inhibitors, OC remains a lethal disease. Other therapeutic options are being explored, such as immunotherapy (IT), which has been proved effective in many solid tumors. Findings about tumor-infiltrating cytotoxic and regulatory T cells, together with the expression of PD-1 on immune cells and of PD-L1 on tumor cells, gave the rationale for an attempt to the use of IT also in OC. We treated two patients with avelumab, an anti-PD-L1 monoclonal antibody, after the first line of chemotherapy: Patient A underwent 19 cycles of maintenance therapy with avelumab with a disease-free interval of 12 months, whereas patient B showed a slight progression of disease after only eight cycles. A higher PD-L1 expression in tumor cells of patient A was detected. She also underwent a genomic assessment that described the presence of a high Tumor Mutational Burden (TMB) and a status of Loss of Heterozygosity (LoH). This different response to the same treatment puts in evidence that some genomic and immune features might be investigated.