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Background: There is compelling need for novel biomarkers to predict response to PARP inhibitors (PARPi) in BRCA wild-type (WT) ovarian cancer (OC). Methods: MITO 37 is a multicenter retrospective study aiming at correlating Ki67 expression at diagnosis with a clinical outcome following platinum treatment and PARPi maintenance. Clinical data were collected from high grade serous or endometroid BRCAWT OC treated with niraparib or rucaparib maintenance between 2010–2021 in 15 centers. Ki67 expression was assessed locally by certified pathologists on formalin-fixed paraffin embedded (FFPE) tissues. Median Ki67 was used as a cut-off. Results: A total of 136 patients were eligible and included in the analysis. Median Ki67 was 45.7% (range 1.0–99.9). The best response to platinum according to median Ki67 was 26.5% vs. 39.7% complete response (CR), 69.1% vs. 58.8% partial response (PR), 4.4% vs. 1.5% stable disease (SD). The best response to PARPi according to median Ki67 was 19.1% vs. 36.8% CR, 26.5% vs. 26.5% PR, 26.5 vs. 25% SD, 27.9% vs. 16.2% progressive disease (PD). No statistically significant differences in progression free survival (PFS) and overall survival (OS) were identified between low and high Ki67. PFS and OS are in line with registration trials. Conclusions: Ki67 at diagnosis did not discriminate responders to PARPi.

Simple Summary Immunotherapy has revolutionized cancer treatment, but not for all tumor types. Indeed, sarcomas are considered “immune-cold” tumors, which are relatively unresponsive to immunotherapy. One strategy to potentiate immunotherapy efficacy is to increase tumor immunogenicity, for instance by boosting the number of candidate targets (neoantigens) to be recognized by the immune system. Tumor mutational burden indicates the number of somatic mutations identified in the tumor and normalized per megabase. Tumor mutational burden is considered as an acceptable, measurable surrogate of tumor neoantigens. Here, we explored whether the combination of two DNA-damaging agents, trabectedin and olaparib, could increase tumor mutational burden in sarcomas, to prime subsequent immunotherapy. We found no variation in tumor mutational burden after trabectedin + olaparib in preclinical and clinical samples. Therefore, other aspects should be considered to increase sarcoma immunogenicity, by exploiting different pathways such as the potential modulation of the tumor microenvironment induced by trabectedin + olaparib. Abstract Drug-induced tumor mutational burden (TMB) may contribute to unleashing the immune response in relatively “immune-cold” tumors, such as sarcomas. We previously showed that PARP1 inhibition perpetuates the DNA damage induced by the chemotherapeutic agent trabectedin in both preclinical models and sarcoma patients. In the present work, we explored acquired genetic changes in DNA repair genes, mutational signatures, and TMB in a translational platform composed of cell lines, xenografts, and tumor samples from patients treated with trabectedin and olaparib combination, compared to cells treated with temozolomide, an alkylating agent that induces hypermutation. Whole-exome and targeted panel sequencing data analyses revealed that three cycles of trabectedin and olaparib combination neither affected the mutational profiles, DNA repair gene status, or copy number alterations, nor increased TMB both in homologous recombinant-defective and proficient cells or in xenografts. Moreover, TMB was not increased in tumor specimens derived from trabectedin- and olaparib-treated patients (5–6 cycles) when compared to pre-treatment biopsies. Conversely, repeated treatments with temozolomide induced a massive TMB increase in the SJSA-1 osteosarcoma model. In conclusion, a trabectedin and olaparib combination did not show mutagenic effects and is unlikely to prime subsequent immune-therapeutic interventions based on TMB increase. On the other hand, these findings are reassuring in the increasing warning of treatment-induced hematologic malignancies correlated to PARP1 inhibitor use.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Amyloidosis is a heterogeneous group of diseases characterized by the extracellular deposition of insoluble proteins whose pathogenesis is not yet fully understood. The deposition of amyloid proteins can be systemic or localized, idiopathic or related to systemic diseases, mostly multiple myeloma or chronic inflammatory diseases. Localized head and neck amyloidosis is a rare entity, mainly involving the larynx. Given the rarity of the disease and the absence of a lasting follow-up protocol, there is no standard treatment defined for localized amyloidosis. We report a rare case of localized nasopharyngeal amyloidosis, treated with complete transoral resection and confirmed by histological examination.

11571 Background: Despite surgically resectable pulmonary metastases may lead to cure patients with B-STS (Chudgar NP 2017), a substantial proportion of patients will eventually relapse. Presently, patient selection is based on unique organ involvement, number of metastases, interval between previous surgery and pulmonary progression or relapse. We assessed the impact of anatomical site of metastasis into the lung (as if the pleural site might ease further tumor spreading) and nodule growth rate as additional predictive/prognostic factors of lung progression-free survival (L-PFS) and overall survival (OS). Methods: In our prospectively collected database, we retrospectively evaluated patients operated for B-STS pulmonary progression at 3 different centers from 2005 to 2019. Beyond patients’ clinical features at both baseline and disease progression in the lungs, we focused on whether the relapse occurred into the parenchyma or nearby the pleura (Welter S 2012); secondly, we estimated lung metastasis growth rate, defined as tumor doubling time (TDT) (Nakamura T 2011). Statistical analyses were carried out with IBM SPSS (v. 20.0). Survival outcomes were estimated by Kaplan-Meier method. Hazard ratios (HR) were estimated by Cox regression. Multivariate analysis was performed for both L-PFS and OS according to Cox proportional hazard model. All tests were 2-sided with their corresponding 95% confidence intervals (CI95%). Results: We identified 138 patients who underwent lung metastasectomy [(F=66 (48%); median age at surgery 50 (14-78)]. Median PFS and L-PFS were 8.7 months (CI95% 6.6-10.9) and 8.6 months (CI95% 6.2-11.0), respectively. Median OS was 40.6 months (CI 95% 32.8-48.5). Univariate analysis showed a statistically significant impact of the following variables for both L-PFS and OS: ECOG 0, nodule number 40 days. Disease-free interval ≤ 24 months and absence of metastases at diagnosis showed significant correlation with L-PFS and OS, respectively. At multivariate analyses the following variables retained statistical significance for L-PFS: TDT >40 days (HR 0.53, CI95% 0.31-0.93, p=0.028); nodule number 40 days (HR 0.36, CI95% 0.18-0.72, p=0.004), nodule number

Background: The aim of this retrospective study was to identify different radiological features in intermediate&ndash, advanced laryngeal cancer (LC) associated with arytenoid fixation, in order to differentiate cases still safely amenable to conservative treatment by partial laryngectomy or chemoradiotherapy. Methods: 29 consecutive patients who underwent open partial horizontal laryngectomies (OPHLs), induction chemotherapy followed by radiotherapy in the case of &gt, 50% response (IC + RT) or total laryngectomy were classified as: pattern I (supraglottic LC fixing the arytenoid due to weight effect), pattern II (glottic LC involving the posterior paraglottic space and spreading toward the crico-arytenoid joint and infraglottic extension &lt, 10 mm), pattern III (glottic&mdash, infraglottic LC involving the crico-arytenoid joint and infraglottic extension &gt, 10 mm) and pattern IV (transglottic and infraglottic LC with massive crico-arytenoid unit involvement, reaching the hypopharyngeal submucosa). All glottic cancers treated with surgery were studied by a cross sectional approach. Results: A substantial agreement between the work-up and the pathology results has been obtained in each of the subcategories. Three-year disease-free survivals, local control and freedom from laryngectomy were significantly better in pattern II compared to pattern III&ndash, IV. Conclusions: LC showing fixed arytenoid due to weight effect or posterior paraglottic space involvement with infraglottic extension &lt, 10 mm assessed at the true vocal cord midline are still safely manageable by OPHL or IC + RT.

The application of COThe current video shows three examples of open partial horizontal laryngectomies (OPHLs) performed using COCOIn our experience, the application of CO

Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments. We used patient derived models to accelerate the discovery of treatment options. We developed a platform of Patient Derived Xenografts (PDX), by implanting and propagating patient's tumor samples collected at surgery in severely immunocompromised mice. From each PDX line we derived short term cultures of PDX Derived Tumor Cells (PDTCs). We envisioned that the weakness of PDXs and PDTCs, i.e. lack of human stromal and immune cells, might be instrumental to link tumor biomarkers to treatments. We have successfully propagated 49 PDX lines from metastatic EOC, which were fully characterized as far as histology, immunohistochemistry of epithelial and tissue specific markers and presence of TP53 and BRCA1/2 mutations. On PDTCs cultures we first assessed sensitivity to Carboplatin, currently used as first-line drug in ovarian cancer treatment. Of PDX lines derived from naïve metastatic HGS-EOC copy number variations and whole exome sequencing analyses were carried out, in order to identify putative and actionable cancer genes. Thus, on PDTCs we assayed also approved or experimental targeted drugs as monotherapy or in combinations. In one PDX line we identified a possibly loss-of-function mutation (W624R) of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) with an allele frequency of 0.9, which could result in activation of the PI3K pathway. Several PI3K inhibitors were assayed on PDTCs of this PDX line harboring the PIK3R1W624R. Buparlisib (a Pan Class I PI3Ki) showed the ability to block proliferation of the PDTCs and the growth of the relevant PDXs in vivo. Altogether these data show that Patient Derived models are invaluable tools to unveil actionable pathways for the treatment of advanced/metastatic HGS-EOC. Citation Format: Concetta D'Ambrosio, Jessica Erriquez, Maddalena Arigoni, Sonia Capellero, Gloria Mittica, Eleonora Ghisoni, Fulvio Borella, Dionyssios Katsaros, Silvana Privitera, Marisa Ribotta, Elena Maldi, Giovanna Di Nardo, Enrico Berrino, Tiziana Venesio, Riccardo Ponzone, Marco Vaira, Douglas Hall, Mercedes Jimenez-Linan, Anna Paterson, Giorgio Valabrega, Raffaele Calogero, James Brenton, Mariaflavia Di Renzo, Martina Olivero. Assays of conventional chemotherapeutics and targeted drugs for ovarian cancer using patient derived models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1677.

Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs).Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05-100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence.In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation.Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli.

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

The paper presents a case of a zoonotic trasmission of orf virus infection during religious slaughtering. The patient seeked medical assistance due to the cutaneous papular rash on the trunk, arms and the extremities resembling erythema multiforme. The definitive diagnosis, of a complicated form of orf virus infection, was the consequence of a tight collaboration between physicians and veterinarians. The results confirm that orf virus infection is one of the causes of erythema multiforme in humans. This further case of complicated orf virus infection, after religious slaughtering, lead us to speculate that predisposing factors related to the ethnic background may contribute to the condition.

NAD is an essential coenzyme involved in numerous metabolic pathways. Its principal role is in redox reactions, and as such it is not heavily "consumed" by cells. Yet a number of signaling pathways that bring about its consumption have recently emerged. This has brought about the hypothesis that the enzymes that lead to its biosynthesis may be targets for anticancer therapy. In particular, inhibition of the enzyme nicotinamide phosphoribosyl transferase has been shown to be an effective treatment in a number of preclinical studies, and two lead molecules [N-[4-(1-benzoyl-4-piperidinyl)butyl]-3-(3-pyridinyl)-2E-propenamide (FK866) and (E)-1-[6-(4-chlorophenoxy)hexyl]-2-cyano-3-(pyridin-4-yl)guanidine (CHS 828)] have now entered preclinical trials. Yet, the full potential of these drugs is still unclear. In the present study we have investigated the role of FK866 in neuroblastoma cell lines. We now confirm that FK866 alone in neuroblastoma cells induces autophagy, and its effects are potentiated by chloroquine and antagonized by 3-methyladenine or by down-regulating autophagy-related protein 7. Autophagy, in this model, seems to be crucial for FK866-induced cell death. On the other hand, a striking potentiation of the effects of cisplatin and etoposide is given by cotreatment of cells with ineffective concentrations of FK866 (1 nM). The effect of etoposide on DNA damage is potentiated by FK866 treatment, whereas the effect of FK866 on cytosolic NAD depletion is potentiated by etoposide. Even more strikingly, cotreatment with etoposide/cisplatin and FK866 unmasks an effect on mitochondrial NAD depletion.

We report a case of two patients with foot drop due to peroneal nerve infarct as early sign of two different forms of primary systemic vasculitides: a predominantly small-vessel p-antineutrophil cytoplasmic antibody-positive vasculitis (microscopic polyangiitis) and a predominantly medium-vessel vasculitis (polyarteritis nodosa).

Extra-osseous Ewing sarcomas/peripheral primitive neuroectodermal tumors (EOES/pPNETs) are high-grade malignant tumors found in various organs, such as the lung, skin, intestine, kidney and female genital tract; however, to the best of our knowledge, only two cases have previously been identified in the thyroid gland. We describe a case of primary EOES/PNET of the thyroid gland in a 66-year-old man with a previous history of large B cell lymphoma. During a routine follow-up examination, the patient underwent an ultrasound cervical scan showing a solid nodule of the left thyroid lobe. The fine-needle aspiration biopsy of the nodule suggested a neuroendocrine tumor. Histological and immunohistochemical examination of the surgical specimen supported a diagnosis of EOES/PNET, which was further confirmed by the demonstration of EWSR1 gene translocation by means of fluorescent in situ hybridization and by the detection of glycogen particles and neurosecretory granules by means of electron microscopy. Total body computed tomography and magnetic resonance imaging excluded the involvement of other sites, and therefore a diagnosis of primary EOES/PNET of the thyroid gland was made.This paper also discusses the main differential diagnoses, including lymphoma recurrence, other small round cell tumors (primary or metastatic), and a thyroid localization of an EWS/PNET from another organ.

Aims: To study caveolin-1 (Cav-1) expression in metastatic lung carcinomas. Methods and results: Cav-1 expression was investigated in a series of 121 lung carcinomas and it was shown that 18/121 tumours (14.9%) were Cav-1+. None of the pure bronchioloalveolar carcinomas proved to be positive, vs. 42.8% of the large cell carcinomas (neuroendocrine subtype excluded). Adenocarcinomas (8.5%), large cell neuroendocrine carcinomas (20%) and squamous cell carcinomas (29.6%) displayed an intermediate percentage of positive cases, suggesting a gradient of Cav-1 expression according to tumour histotype-related aggressiveness. Moreover, the percentage of Cav-1+ tumours with distant metastases was almost double that of non-metastatic tumours (17.8% vs. 8.1%), irrespective of the histotype. In 34 tumours metastatic to the brain, primary and secondary lesions were compared and 53% of brain metastases were Cav-1+ vs. 20.6% of primaries, indicating a de novo acquisition of Cav-1 expression. This pattern was exclusive to the brain, as it was not acquired in adrenal metastases. In our series, the presence of epidermal growth factor receptor amplification, determined by fluorescence in situ hybridization, was not related to Cav-1 reactivity. Conclusions: Cav-1 immunoreactivity in lung carcinoma is histotype-dependent and acquired de novo in brain metastases, suggesting a site-specific phenotypic shift in secondary lesions.

Different proteins regulate iron metabolism at the level of various tissues. Among these is a second transferrin receptor (TfR2) that seems to play a key role in the regulation of iron homeostasis. Although TfR2 expression in normal tissues is restricted at the level of the liver, we observed that TfR2 is frequently expressed in cancer cell lines. Taking advantage of this observation we investigated TfR2 expression in primary colon cancers, and showed that this receptor is expressed in about 26% of cases. TfR2 expression in colon cancer is not related to histological grade, but is preferentially associated with mucinous tumors. In colon cancer cell lines, TfR2 is localized in membrane lipid rafts, induces ERK1/ERK2 phosphorylation, when activated by its ligand transferring, and is preferentially expressed during S-M phases of the cell cycle. The presence of TfR2 on the membrane of colon cancer cells may contribute the growth advantage to these cells.

Caveolin 1 (cav-1) is the basic component of the flask-shaped membrane microdomains known as caveolae that are involved in various cell functions. Caveolin 1 can be overexpressed in tumors,suggesting a proneoplastic role, or it can be downregulated. We previously reported that cav-1 expression increases with tumor grade in astrocytomas. Here, we studied cav-1 immunoreactivity in brain umors with an oligodendroglial component to determine the prognostic value of cav-1 expression and to correlate it with 1p/19q deletions. Fifty-four oligodendrogliomas, 26 mixed oligoastrocytomas,and 7 glioblastomas with an oligodendroglial component were assessed for cav-1 expression by immunohistochemistry and for 1p/19q status by fluorescence in situ hybridization. Caveolin-1 was detected in a minority of cases (22%) and was associated mostly with Grade III mixed oligoastrocytomas and glioblastomas with anoligodendroglial component; cav-1 expression was significantly correlated with the absence of a 1p/19q deletion (p = 0.0002). In the 63 cases in which survival data were available, cav-1 expression was also significantly associated with shorter survivals, whereas 1p/19q deletion was associated with longer survivals. Among high-grade tumors, cav-1 expression was the only factor that retained a statistical significance after multivariate analysis for the prediction ofa short survival (p G 0.015). These data are the first evidence that cav-1 immunohistochemistry is an independent prognostic marker in tumors with an oligodendroglial component regardless of the 1p/19q status.

10570 Background: In soft tissue sarcomas (STS) after complete (marginal/wide/radical margins) surgery, surveillance should be tailored to individual patient’s (pts) risk that depends on histotype,...

NAD is an essential coenzyme involved in numerous metabolic pathways and it has been demonstrated that a number of signalling pathways bring about its consumption. Different pathways leading to the formation of NAD are present in cells. Nicotinamide phosphoribosyl transferase (NAMPT), which forms nicotinamide mononucleotide (NMN) from nicotinamide (NM) and PRPP, plays a crucial role in cells to re-use nicotinamide released by NAD-metabolizing enzymes. Moreover, NAMPT has also been described as a cytokine released by immune cells and adipocytes, however the role of NAMPT in the extracellular space is still unclear. The link between NAMPT and cancer is rapidly strengthening. NAMPT has been shown to be involved in angiogenesis and to be up-regulated in a number of solid tumours. Moreover, an important role in tumorigenesis has been postulated for a number of NAD-utilizing enzymes and inhibitors of NAMPT, named FK866 and CHS 828 have entered clinical trails for cancer treatment. In particular, FK866 has entered phase II trial for metastatic melanoma. The aim of our work was to determine the role of NAMPT in melanoma progression and the possibility to use NAMPT inhibitors as anti-cancer agents in melanoma. We investigated the expression of NAMPT in normal nevi, dysplastic nevi and melanoma human samples. Surprisingly, in all melanoma samples and in dysplastic nevi NAMPT is over-expressed, suggesting a possible contribution of this enzyme in melanoma progression. To confirm this data, we investigated the expression of NAMPT in six different melanoma cell lines. All melanoma cells show high levels of NAMPT expression compared to melanocytes. To test if the inhibition of NAMPT was able to decrease melanoma cells viability, we capitalize the action of FK866. However, only one to six melanoma cell line is sensitive to FK866 treatment. To understand why melanoma cells are insensitive to NAMPT inhibition, we investigate MDR expression and the possibility that NAMPT is mutated. Unfortunately, verapamil was not able to increase the sensitivity to FK866 and NAMPT is not mutated in all melanoma cell lines tested, moreover the treatment with FK866 is able to decrease the intracellular NAD level, suggesting that this agent is able to enter cells but not to induce cell death. As NAMPT was described also as a cytokine, we speculate the possibility that melanoma cells are able to release NAMPT in the extracellular space. Indeed, in starvation condition NAMPT is released by melanoma cells in a time-dependent manner. In conclusion, we have demonstrated that NAMPT is over-expressed in human melanoma samples and in melanoma cells, however NAMPT inhibition is not able to affect melanoma cells growth and viability, suggesting that NAMPT up-regulation does not correlate with a pharmacological response. Moreover, NAMPT is released by melanoma cells as a cytokine, we can speculate that NAMPT has a role in the angiogenic process of melanoma progression Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5146. doi:1538-7445.AM2012-5146