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Background Immune checkpoint inhibitors (ICIs) have particular, immune-related adverse events (irAEs), as a consequence of interfering with self-tolerance mechanisms. The incidence of irAEs varies depending on ICI class, administered dose and treatment schedule. The aim of this study was to define a baseline (T0) immune profile (IP) predictive of irAE development. Methods A prospective, multicenter study evaluating the immune profile (IP) of 79 patients with advanced cancer and treated with anti-programmed cell death protein 1 (anti-PD-1) drugs as a first- or second-line setting was performed. The results were then correlated with irAEs onset. The IP was studied by means of multiplex assay, evaluating circulating concentration of 12 cytokines, 5 chemokines, 13 soluble immune checkpoints and 3 adhesion molecules. Indoleamine 2, 3-dioxygenase (IDO) activity was measured through a modified liquid chromatography–tandem mass spectrometry using the high-performance liquid chromatography-mass spectrometry (HPLC–MS/MS) method. A connectivity heatmap was obtained by calculating Spearman correlation coefficients. Two different networks of connectivity were constructed, based on the toxicity profile. Results Toxicity was predominantly of low/moderate grade. High-grade irAEs were relatively rare, while cumulative toxicity was high (35%). Positive and statistically significant correlations between the cumulative toxicity and IP10 and IL8, sLAG3, sPD-L2, sHVEM, sCD137, sCD27 and sICAM-1 serum concentration were found. Moreover, patients who experienced irAEs had a markedly different connectivity pattern, characterized by disruption of most of the paired connections between cytokines, chemokines and connections of sCD137, sCD27 and sCD28, while sPDL-2 pair-wise connectivity values seemed to be intensified. Network connectivity analysis identified a total of 187 statistically significant interactions in patients without toxicity and a total of 126 statistically significant interactions in patients with toxicity. Ninety-eight interactions were common to both networks, while 29 were specifically observed in patients who experienced toxicity. Conclusions A particular, common pattern of immune dysregulation was defined in patients developing irAEs. This immune serological profile, if confirmed in a larger patient population, could lead to the design of a personalized therapeutic strategy in order to prevent, monitor and treat irAEs at an early stage.

Background: Immunotherapy has revolutionized the approach to metastatic triple-negative breast cancers (mTNBC). Atezolizumab was approved for patients with mTNBC whose tumours express PD-L1, determined by SP 142 assay. To assess the availability and practice of SP142 test we administered a survey to all the 15 Pathology Departments of the Lazio Region during a 6-months period.Methods: The survey comprised 12 questions regarding the availability of SP142 in the Pathology Departments, the percentage of positive tests, the difficulties of pathologists in case close to cut-off value and the tested samples.Results: The SP142 assay was available only in 8 Centers. In case of positive result, most Centers (5/8, 62.5%) reported values of PD-L1 expression ranging from > 1 to Conclusion: Our results raise some important issues concerning the evaluation of PD-L1 in the “real-life” setting, providing strategies for its implementation.

Background: Several randomized clinical trials clearly demonstrated that adding palbociclib (P) to endocrine therapies (ET), such as letrozole (LT) or fulvestrant (FLV), significantly improves outcome both in first-line/endocrine sensitive and second-line/endocrine resistant hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2) metastatic breast cancer (MBC) patients (pts). The aims of the present study were to assess efficacy, activity and toxicity of P combined with either LT or FLV in a real world setting, and to study the efficacy and activity of treatments administered after progression to P + ET. Methods: Records of 245 consecutive HR+/HER2- MBC patients from 14 Italian cancer centers were reviewed in this observational study. Primary end-point was progression-free survival (PFS) obtained with P+ET; secondary end-points were overall response rates (ORR) and clinical benefit rate (CBR) obtained with P+ET and with post-progression treatments, as well as overall survival (OS), PFS to subsequent treatment lines and post-progression survival (PPS). Results: Overall 245 pts were treated with P+ET from April 2014. Of them, 116 (47%) received it in first-line setting, 70 (28%) in second-line and the rest (68 pts, 25%) in subsequent lines. Median age was 60 (35-80) years and median ECOG performance status was 0. Seventy-three pts (30%) had more than two metastatic sites and 75 (31%) had visceral metastasis. Ninenty-one (37%) pts received P+LT and 92 (37%) were treated with P+FLV. For 15 (6%) premenopausal pts an LHRH analogue was added to P+ET. In 77 (41%) patients a biopsy of a metastatic lesion was performed. Among the 116 pts treated in first-line setting, median PFS was 14.7 (95% CI 12.0-17.3) and 15.1 (95%CI 8.6-21.6) months for pts receiving P+LT and P+FLV, respectively. ORR was 48% (95% CI: 39-57%) and CBR was 75% (95% CI, 66-82%). Four (3%) complete responses (CR), 52 (45%) partial responses (PR), 31 (27%) stable disease (SD) and 11 (9%) progression disease (PD) were observed as best response; 18 pts were not yet evaluable. Among the 70 pts treated in second-line setting, median PFS was 10.8 (95%CI 0-24.3) and 7.9 (95%CI 3.3-12.5) months, for pts receiving P+LT and P+FLV, respectively. The ORR was 26% (95% CI: 16-38) and the CBR was 66% (95% CI, 53-76). One CR (1%), 17 PR (24%), 28 SD (40%) and 13 PD (18.5%) were observed as best response, while 9 were not yet evaluable. Best response was achieved after a median of 5 cycle. At time of the current analysis, 50 pts experienced PD during P+ET. Of them, 38 (76%) received chemotherapy (capecitabine, eribulin, nab-paclitaxel, paclitaxel+bevacizumab, vinoreline) and 12 (24%) ET. To date, only 5 pts progressed during these therapies, preventing us for evaluating secondary end-points regarding efficacy and activity. The most frequent adverse event (AE) was grade 3-4 neutropenia (31%), with febrile neutropenia reported in 5 (2%) cases. Main non-hematological AEs were fatigue and gastrointestinal symptoms (diarrhea and stypsis). Conclusions: Our findings confirm the efficacy and safety of P+ET as first/second-line treatment for HR+/HER2- MBC pts, even in a non-selected, real world population. Since data regarding the efficacy and activity of post-progression therapies were not mature at the time of the current analysis, they will be subsequently presented. Citation Format: Alessandra Fabi, Michelangelo Russillo, Mariangela Ciccarese, Simone Scagnoli, Francesco Schettini, Giuseppe Buono, Vito Lo Russo, Grazia Arpino, Rosalba Rossella, Giuseppina Sarobba, Marianna Giampaglia, Patrizia Pellegrini, Simonetta Stani, Michela Palleschi, Vincenzo Adamo, Francesca Morelli, Maria Agnese Fabbri, Cecilia Nistico, Gianluigi Ferretti, Giovanna Catania, Simona Pisegna, Diana Giannarelli, Francesco Cognetti. Real-world evidence of efficacy and activity of palbociclib plus endocrine therapy and post-progression treatments in HR+/HER2- metastatic breast cancer patients: The PALPract study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-18.

2553 Background: Immune checkpoint inhibitors (ICIs) have peculiar, immune-related adverse events (irAEs), as a consequence of interfering with self-tolerance mechanisms. The incidence of irAEs varies by ICI class, administered dose and treatment schedule. The aim of the study was to define a baseline (T0) immune profile (IP) predictive of irAE development. Methods: A prospective, multicenter study evaluating the IP of 79 patients with advanced cancer, treated with anti-PD-1drugs as a first- or second-line setting was performed. The results were then correlated with irAEs onset. The IP was studied by means of multiplex assay, evaluating circulating concentration of 12 cytokines, 5 chemokines, 13 soluble immune checkpoints and 3 adhesion molecules. IDO levels were evaluated through a modified liquid chromatography–tandem mass spectrometry. Data were first pre-processed by performing logarithmic transformation and Shapiro-Wilk-test. A connectivity heatmap was obtained by calculating Spearman correlation coefficients. Two different networks of connectivity were constructed, based on the toxicity profile. Results: Toxicity was predominantly of low/moderate grade. High-grade irAEs were relatively rare, while cumulative toxicity was high (23%). A positive and statistically significant correlation between the cumulative toxicity and IP10 and IL8, sLAG3, sPDL-2 sHEVM, sCD137, sCD27 and sICAM1 was found. Moreover, patients who experienced irAEs had a markedly different connectivity pattern characterized by disruption of most of the paired connections between cytokines (all the connections of the cytokine IL8, most of the connections between the pro-inflammatory chemokines, or all the connections of CD137, CD27, and CD28), while sPDL-2 pair-wise connectivity values seemed to be intensified. Network connectivity analysis identified a total of 187 statistically significant interactions in patients without toxicity and a total of 126 interactions in patients with toxicity. Ninety-eight interactions were common to both networks, while 29 were specifically observed in patients with toxicity. Conclusions: A peculiar pattern of immune dysregulation in patients who develop irAEs was defined. This immune serological profile could lead to the design of a personalized therapeutic strategy in order to prevent, monitor and treat irAEs at an early stage.

Background: To date, a consensus has not yet been reached about the therapy sequence after disease progression (PD) on CDK4/6 inhibitors in patients with HR+/HER2- metastatic breast cancer (MBC). Objectives: The present study assesses, in a real-world setting, the activity of different subsequent therapies in patients who experienced a PD on palbociclib (P) + endocrine therapy (ET), to evaluate the best therapy sequence. Methods: This is a multicenter retrospective observational study. Records of consecutive HR+/HER2- MBC patients from January 2017 to May 2019 were reviewed. The primary endpoint was the evaluation of progression-free survival (PFS) according to subsequent treatment lines after progression on P+ET. Toxicity data were also collected. Results: The outcomes were analyzed in 89 MBC patients that had progressed on previous P+ET: 17 patients were on hormone therapy (HT) and 31 patients on chemotherapy (CT) as second-line treatments; seven patients were on HT and 34 on CT as third-line therapies. PFS of patients treated with HT as second-line therapy is significantly improved when compared with patients treated with CT (p=0.01). Considering third-line settings, the difference in PFS was not statistically different between HT and CT. A better outcome in terms of toxicity is observed among HT patients for both second- and third-line therapies. Conclusions: patients who were progressive on P+ET could still benefit from a subsequent ET. In patients who experienced a good efficacy from prior ET, without visceral metastatic sites, HT seems the most suitable option, when compared to CT, also in terms of safety.

Although molecular profiling at diagnosis has traditionally relied on direct sampling of neoplastic tissue, cancer clonal evolution represents a critical obstacle to use primary tissue biopsies to guide clinical decision-making at the time of progressive disease. Liquid biopsies might offer enormous advantages over tissue biopsies, tracking in real-time temporal-based tumor dynamics following each line of treatment. Here, we compared two liquid biopsy assays, specifically real-time polymerase chain reaction and next-generation sequencing, to track the KRAS G12C mutation at onset of progression from previous lines of therapy. The KRAS G12C mutation was acquired at the time of progressive disease in 24% of patients. Furthermore, all patients with KRAS G12C mutation-positive tissue became negative in ctDNA at progressive disease. The presence of other somatic mutations in all these samples confirmed the tumor origin of the circulating DNA. This pilot study suggests that in the assessment of the plasma KRAS G12C mutation as a druggable target, real-time PCR assay Idylla might be a suitable approach to better match patients to interventional biomarker-targeted therapies.

Simple Summary Vulvar cancer is a difficult clinical condition to treat. Although it is not one of the most frequently diagnosed cancers, its incidence is not negligible. Treatment depends on the extent of the disease and is currently based on surgery, radiotherapy and chemotherapy. The combination of these possible treatments, in the context of multidisciplinary discussions, is crucial. In this paper we present a review of the data available in the literature on the role of chemotherapy in the treatment of vulvar cancer, with a look at future perspectives. Abstract The actual role of chemotherapy in vulvar cancer is undeniably a niche topic. The low incidence of the disease limits the feasibility of randomized trials. Decision making is thus oriented by clinical and pathological features, whose relevance is generally weighted against evidence from observational studies and clinical practice. The therapeutic management of vulvar cancer is increasingly codified and refined at an individual patient level. It is of note that the attitude towards evidence sharing and discussion within a multidisciplinary frame is progressively consolidating. Viable options included in the therapeutic armamentarium available for vulvar cancer patients are frequently an adaption from standards used for cervical or anal carcinoma. Chemotherapy is more frequently combined with radiotherapy as neo-/adjuvant or definitive treatment. Drugs commonly used are platinum derivative, 5-fluorouracil and mitomicin C, mostly in combination with radiotherapy for radiosensitization. Exclusive chemotherapy in the neo-/adjuvant setting comprises platinum-derivative, combined with bleomicin and methotrexate, 5-fluorouracil, ifosfamide or taxanes. In advanced disease, current regimens include cisplatin-based chemoradiation, with or without 5-fluorouracil, or doublets with platinum in combination with a taxane. Our work is also enriched by a concise excursus on the biologic pathways underlying vulvar cancer. Introductory hints are also provided on targeted agents, a rapidly evolving research field.

Pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) can predict better survival outcomes in patients with early triple negative breast cancer (TNBC). Tumor infiltrating lymphocytes (TILs), Programmed Death-Ligand 1 (PD-L1), and Cluster of Differentiation 73 (CD73) are immune-related biomarkers that can be evaluated in the tumor microenvironment. We investigated if the contemporary expression of these biomarkers combined in a tissue immune profile (TIP) can predict pCR better than single biomarkers in TNBC. Tumor infiltrating lymphocytes (TILs), CD73 expression by cancer cells (CC), and PD-L1 expression by immune cells (IC) were evaluated on pre-NACT biopsies. We defined TIP positive (TIP+) as the simultaneous presence of TILS &ge, 50%, PD-L1 &ge, 1%, and CD73 &le, 40%. To consider the effects of all significant variables on the pCR, multivariate analysis was performed. Akaike information criterion (AIC) and Bayesian information criterion (BIC) were used for model selection. We retrospectively analyzed 60 biopsies from patients with TNBC who received standard NACT. Pathological complete response was achieved in 23 patients (38.0%). Twelve (20.0%) cases resulted to be TIP+. The pCR rate was significantly different between TIP+ (91.7%) and TIP&minus, (25.0%) (p &lt, 0.0001). Using a multivariate analysis, TIP was confirmed as an independent predictive factor of pCR (OR 49.7 (6.30&ndash, 392.4), p &lt, 0.0001). Finally, we compared the efficacy of TIP versus each single biomarker in predicting pCR by AIC and BIC. The combined immune profile is more accurate in predicting pCR (AIC 68.3, BIC 74.5) as compared to single biomarkers. The association between TIP+ and pCR can be proposed as a novel link between immune background and response to chemotherapy in TNBC, highlighting the need to consider an immunological patients&rsquo, profile rather than single biomarkers.

A recently described putative tumor suppressor gene, the cyclin-dependent kinase 4 inhibitor (p16), has been shown to be altered by deletions and/or point mutations in various human cancers. To assess the incidence and clinico-biologic correlations of p16 homozygous deletion in hemopoietic tumors, we studied a panel of 244 DNA samples representative of distinct acute (99 cases) and chronic (57 cases) leukemia subtypes, myelodysplastic (22 cases) and myeloproliferative (15 cases) syndromes, and lymphomas (51 cases). A 361-bp probe complementary to the p16 exon 2 gene sequences was generated by polymerase chain reaction and used in Southern blot hybridization against these tumor DNAs. Homozygous deletions of p16 (p16-/-) were detected in 10 of 58 (17%) cases of acute lymphoblastic leukemia (ALL) of either B or T lineage and in no other tumors. Single-strand conformation polymorphism analysis of p16 exons 1 and 2 was also performed in 40 of the 58 ALL cases and in 16 lymphomas. In no cases were point mutations detected. The comparison of clinical features at presentation in p16-/- and in p16 germline ALL cases showed a greater leukemic cell mass (P = .001) and higher white blood cell counts (P = .01) in the former group. Two ALL cases in which diagnostic and relapse DNA samples were available showed p16-/- in both specimens. We conclude that homozygous p16 gene deletions characterize a subset of ALL with features of aggressive disease.

Natural killer (NK) cells are a component of the innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. Although the synthetic double-stranded (ds) RNA polyinosinic-polycytidylic acid (poly I:C), a mimic of a common product of viral infections, is known to rapidly up-regulate their in vivo functions, NK cell ability to directly respond to dsRNA is still mostly unknown. Our results show that treatment with poly I:C significantly up-regulates both natural and CD16-mediated cytotoxicity of highly purified human NK cells. Poly I:C also induces the novel capability of producing CXCL10 chemokine in human NK cells and synergistically enhances interferon-γ (IFN-γ) production induced by either adaptive or innate cytokines. In accordance with the expression of Toll-like receptor-3 (TLR3) and of TRIF/TICAM-1 adaptor, poly I:C stimulation induces the activation of interferon regulatory factor-3 (IRF-3) transcription factor and of p38 mitogen-activated protein kinase (MAPK) in human NK cells. Finally, we demonstrate that p38 MAPK activity is required for the dsRNA-dependent enhancement of cytotoxicity and CXCL10 production. The occurrence of dsRNA-induced signaling and functional events closely correlates with the TLR3 mRNAprofile in different NK cell populations. Taken together, these data identify p38 as a central component of NK cell ability to directly respond to dsRNA pathogen-associated molecular pattern (PAMP).

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cγ2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.