CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Mutated axon guidance gene PLXNB2 sustains growth and invasiveness of stem cells isolated from cancers of unknown primary.

The genetic changes sustaining the development of cancers of unknown primary (CUP) remain elusive. The whole-exome genomic profiling of 14 rigorously selected CUP samples did not reveal specific recurring mutation in known driver genes. However, by comparing the mutational landscape of CUPs with that of most other human tumor types, it emerged a consistent enrichment of changes in genes belonging to the axon guidance KEGG pathway. In particular, G842C mutation of PlexinB2 (PlxnB2) was predicted to be activating. Indeed, knocking down the mutated, but not the wild-type, PlxnB2 in CUP stem cells resulted in the impairment of self-renewal and proliferation in culture, as well as tumorigenic capacity in mice. Conversely, the genetic transfer of G842C-PlxnB2 was sufficient to promote CUP stem cell proliferation and tumorigenesis in mice. Notably, G842C-PlxnB2 expression in CUP cells was associated with basal EGFR phosphorylation, and EGFR blockade impaired the viability of CUP cells reliant on the mutated receptor. Moreover, the mutated PlxnB2 elicited CUP cell invasiveness, blocked by EGFR inhibitor treatment. In sum, we found that a novel activating mutation of the axon guidance gene PLXNB2 sustains proliferative autonomy and confers invasive properties to stem cells isolated from cancers of unknown primary, in EGFR-dependent manner.

Real-world histopathological approach to malignancy of undefined primary origin (MUO) to diagnose cancers of unknown primary (CUPs).

The aim of this study is to envisage a streamlined pathological workup to rule out CUPs in patients presenting with MUOs. Sixty-four MUOs were classified using standard histopathology. Clinical data, immunocytochemical markers, and results of molecular analysis were recorded. MUOs were histologically subdivided in clear-cut carcinomas (40 adenocarcinomas, 11 squamous, and 3 neuroendocrine carcinomas) and unclear-carcinoma features (5 undifferentiated and 5 sarcomatoid tumors). Cytohistology of 7/40 adenocarcinomas suggested an early metastatic cancer per se. In 33/40 adenocarcinomas, CK7/CK20 expression pattern, gender, and metastasis sites influenced tissue-specific marker selection. In 23/40 adenocarcinomas, a "putative-immunophenotype" of tissue of origin addressed clinical-diagnostic examinations, identifying 9 early metastatic cancers. Cell lineage markers were used to confirm squamous and neuroendocrine differentiation. Pan-cytokeratins were used to confirm the epithelial nature of poorly differentiated tumors, followed by tissue and cell lineage markers, which identified one melanoma. In total, 47/64 MUOs (73.4%) were confirmed CUP. Molecular analysis, feasible in 37/47 CUPs (78.7%), had no diagnostic impact. Twenty CUP patients, mainly with squamous carcinomas and adenocarcinomas with putative-gynecologic-immunophenotypes, presented with only lymph node metastases and had longer median time to progression and overall survival (< 0.001), compared with patients with other metastatic patterns. We propose a simplified histology-driven workup which could efficiently rule out CUPs and identify early metastatic cancer.

DNA damage response and repair genes in advanced bone and soft tissue sarcomas: An 8-gene signature as a candidate predictive biomarker of response to trabectedin and olaparib combination.

Background: Advanced and unresectable bone and soft tissue sarcomas (BSTS) still represent an unmet medical need. We demonstrated that the alkylating agent trabectedin and the PARP1-inhibitor olaparib display antitumor activity in BSTS preclinical models. Moreover, in a phase Ib clinical trial (NCT02398058), feasibility, tolerability and encouraging results have been observed and the treatment combination is currently under study in a phase II trial (NCT03838744). Methods: Differential expression of genes involved in DNA Damage Response and Repair was evaluated by Nanostring® technology, extracting RNA from pre-treatment tumor samples of 16 responder (≥6-month progression free survival) and 16 non-responder patients. Data validation was performed by quantitative real-time PCR, RNA in situ hybridization, and immunohistochemistry. The correlation between the identified candidate genes and both progression-free survival and overall survival was investigated in the publicly available dataset "Sarcoma (TCGA, The Cancer Genome Atlas)". Results: Differential RNA expression analysis revealed an 8-gene signature (CDKN2A, PIK3R1, SLFN11, ATM, APEX2, BLM, XRCC2, MAD2L2) defining patients with better outcome upon trabectedin+olaparib treatment. In responder vs. non-responder patients, a significant differential expression of these genes was further confirmed by RNA in situ hybridization and by qRT-PCR and immunohistochemistry in selected experiments. Correlation between survival outcomes and genetic alterations in the identified genes was shown in the TCGA sarcoma dataset. Conclusions: This work identified an 8-gene expression signature to improve prediction of response to trabectedin+olaparib combination in BSTS. The predictive role of these potential biomarkers warrants further investigation.

Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.

PARP1 Inhibitor and Trabectedin Combination Does Not Increase Tumor Mutational Burden in Advanced Sarcomas-A Preclinical and Translational Study.

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.

Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition.

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

HIV Protease Inhibitors Block HPV16-Induced Murine Cervical Carcinoma and Promote Vessel Normalization in Association with MMP-9 Inhibition and TIMP-3 Induction.

Antiretrovirals belonging to the human immunodeficiency virus (HIV) protease inhibitor (HIV-PI) class exert inhibitory effects across several cancer types by targeting tumor cells and its microenvironment. Cervical carcinoma represents a leading cause of morbidity and mortality, particularly in women doubly infected with high-risk human papillomaviruses (HR-HPV) and HIV; of note, combined antiretroviral therapy has reduced cervical carcinoma onset and progression in HIV-infected women. We evaluated the effectiveness and mechanism(s) of action of HIV-PI against cervical carcinoma using a transgenic model of HR-HPV-induced estrogen-promoted cervical carcinoma (HPV16/E2) and found that treatment of mice with ritonavir-boosted HIV-PI, including indinavir, saquinavir, and lopinavir, blocked the growth and promoted the regression of murine cervical carcinoma. This was associated with inhibition of tumor angiogenesis, coupled to downregulation of matrix metalloproteinase (MMP)-9, reduction of VEGF/VEGFR2 complex, and concomitant upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3). HIV-PI also promoted deposition of collagen IV at the epithelial and vascular basement membrane and normalization of both vessel architecture and functionality. In agreement with this, HIV-PI reduced tumor hypoxia and enhanced the delivery and antitumor activity of conventional chemotherapy. Remarkably, TIMP-3 expression gradually decreased during progression of human dysplastic lesions into cervical carcinoma. This study identified the MMP-9/VEGF proangiogenic axis and its modulation by TIMP-3 as novel HIV-PI targets for the blockade of cervical intraepithelial neoplasia/cervical carcinoma development and invasiveness and the normalization of tumor vessel functions. These findings may lead to new therapeutic indications of HIV-PI to treat cervical carcinoma and other tumors in either HIV-infected or uninfected patients.

CSPG4-Specific CAR.CIK Lymphocytes as a Novel Therapy for the Treatment of Multiple Soft-Tissue Sarcoma Histotypes.

PURPOSE: No effective therapy is available for unresectable soft-tissue sarcomas (STS). This unmet clinical need prompted us to test whether chondroitin sulfate proteoglycan 4 (CSPG4)-specific chimeric antigen receptor (CAR)-redirected cytokine-induced killer lymphocytes (CAR.CIK) are effective in eliminating tumor cells derived from multiple STS histotypes in vitro and in immunodeficient mice. EXPERIMENTAL DESIGN: The experimental platform included patient-derived CAR.CIK and cell lines established from multiple STS histotypes. CAR.CIK were transduced with a retroviral vector encoding second-generation CSPG4-specific CAR (CSPG4-CAR) with 4-1BB costimulation. The functional activity of CSPG4-CAR.CIK was explored in vitro, in two- and three-dimensional STS cultures, and in three in vivo STS xenograft models. RESULTS: CSPG4-CAR.CIK were efficiently generated from patients with STS. CSPG4 was highly expressed in multiple STS histotypes by in silico analysis and on all 16 STS cell lines tested by flow cytometry. CSPG4-CAR.CIK displayed superior in vitro cytolytic activity against multiple STS histotypes as compared with paired unmodified control CIK. CSPG4-CAR.CIK also showed strong antitumor activity against STS spheroids; this effect was associated with tumor recruitment, infiltration, and matrix penetration. CSPG4-CAR.CIK significantly delayed or reversed tumor growth in vivo in three STS xenograft models (leiomyosarcoma, undifferentiated pleomorphic sarcoma, and fibrosarcoma). Tumor growth inhibition persisted for up to 2 weeks following the last administration of CSPG4-CAR.CIK. CONCLUSIONS: This study has shown that CSPG4-CAR.CIK effectively targets multiple STS histotypes in vitro and in immunodeficient mice. These results provide a strong rationale to translate the novel strategy we have developed into a clinical setting.

Cancer of Unknown Primary (CUP): genetic evidence for a novel nosological entity? A case report.

Cancer of unknown primary (CUP) is an obscure disease characterized by multiple metastases in the absence of a primary tumor. No consensus has been reached whether CUPs are simply generated from cancers that cannot be detected or whether they are the manifestation of a still unknown nosological entity. Here, we report the complete expression and genetic analysis of multiple synchronous metastases harvested at warm autopsy of a patient with CUP. The expression profiles were remarkably similar and astonishingly singular. The whole exome analysis yielded a high number of mutations present in all metastases (fully shared), additional mutations (partially shared) accumulated one after another in a series, and few private mutations were unique to each metastasis. Surprisingly, the phylogenetic trajectory linking CUP metastases was atypical, depicting a common "stream", sprouting a series of linear "brooks", at variance from the extensive branched evolution observed in metastases from most cancers of known origin. The distinctive genetic and evolutionary features depicted suggest that CUP is a novel nosological entity.

Improved Outcome Prediction for Appendiceal Pseudomyxoma Peritonei by Integration of Cancer Cell and Stromal Transcriptional Profiles.

In recent years, cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have substantially improved the clinical outcome of pseudomyxoma peritonei (PMP) originating from mucinous appendiceal cancer. However, current histopathological grading of appendiceal PMP frequently fails in predicting disease outcome. We recently observed that the integration of cancer cell transcriptional traits with those of cancer-associated fibroblasts (CAFs) improves prognostic prediction for tumors of the large intestine. We therefore generated global expression profiles on a consecutive series of 24 PMP patients treated with CRS plus HIPEC. Multiple lesions were profiled for nine patients. We then used expression data to stratify the samples by a previously published "high-risk appendiceal cancer" (HRAC) signature and by a CAF signature that we previously developed for colorectal cancer, or by a combination of both. The prognostic value of the HRAC signature was confirmed in our cohort and further improved by integration of the CAF signature. Classification of cases profiled for multiple lesions revealed the existence of outlier samples and highlighted the need of profiling multiple PMP lesions to select representative samples for optimal performances. The integrated predictor was subsequently validated in an independent PMP cohort. These results provide new insights into PMP biology, revealing a previously unrecognized prognostic role of the stromal component and supporting integration of standard pathological grade with the HRAC and CAF transcriptional signatures to better predict disease outcome.

Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC) with Oxaliplatin, Cisplatin, and Doxorubicin in Patients with Peritoneal Carcinomatosis: An Open-Label, Single-Arm, Phase II Clinical Trial.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is an innovative approach for treating peritoneal carcinomatosis that applies chemotherapeutic drugs into the peritoneal cavity as an under-pressure airflow. It improves local bioavailability of cytostatic drugs as compared to conventional intraperitoneal chemotherapy. The aim of this study is to prove feasibility, efficacy and safety of this new treatment. Patients included in the analysis underwent at least two single port PIPAC procedures; drugs used were Oxaliplatin for colorectal cancers and Cisplatin + Doxorubicin for ovarian, gastric, and primary peritoneal cancers. The primary endpoint was the Disease Control Rate according to the RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Secondary significant endpoints were overall and progression free survival, tumor regression on histology, and quality of life. Safety and tolerability were assessed according to the Common Terminology Criteria for Adverse Events 4. Sixty-three patients were enrolled in this trial. Forty patients (100 PIPAC) were eligible for analysis. Twenty patients were undergoing systemic chemotherapy. Fourteen patients reported an objective response (35%). Median overall survival was 18.1 months; median progression-free survival was 7.4 months. Minor morbidity was observed in seven procedures. Grade 3 complications occurred in two patients, and grade 4 in one patient submitted to reoperation. Single-port PIPAC is feasible, safe, and easy to perform. The combined treatment based on systemic chemotherapy and PIPAC does not induce significant hepatic and renal toxicity and can be considered a valid therapeutic option in patients with advanced peritoneal disease. Further studies on the use of PIPAC alone, possibly with different drug dosages, may define the real effectiveness of the procedure.

Cold Formalin Fixation Guarantees DNA Integrity in Formalin Fixed Paraffin Embedded Tissues: Premises for a Better Quality of Diagnostic and Experimental Pathology With a Specific Impact on Breast Cancer.

Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (stdFFPE) and at 4°C (coldFFPE), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study, n = 21), cohort B (collected at time of the study, n = 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples (mean values: 0.69 vs. 0.36, p < 0.0001) and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.

"Metastatic Cancer of Unknown Primary" or "Primary Metastatic Cancer"?

Cancer of unknown primary (CUP) is an umbrella term used to classify a heterogeneous group of metastatic cancers based on the absence of an identifiable primary tumor. Clinically, CUPs are characterized by a set of distinct features comprising early metastatic dissemination in an atypical pattern, an aggressive clinical course, poor response to empiric chemotherapy and, consequently, a short life expectancy. Two opposing strategies to change the dismal prognosis for the better are pursued. On the one hand, following the traditional tissue-gnostic approach, more and more sophisticated tissue-of-origin (TOO) classifier assays are employed to push identification of the putative primary to its limits with the clear intent of allowing tumor-site specific treatment. However, robust evidence supporting its routine clinical use is still lacking, notably with two recent randomized clinical trials failing to show a patient benefit of TOO-prediction based site-specific treatment over empiric chemotherapy in CUP. On the other hand, with regards to a tissue-agnostic strategy, precision medicine approaches targeting actionable genomic alterations have already transformed the treatment for many known tumor types. Yet, an unmet need remains for well-designed clinical trials to scrutinize its potential role in CUP beyond anecdotal case reports. In the absence of practice changing results, we believe that the emphasis on finding the presumed unknown primary tumor at all costs, implicit in the term CUP, has biased recent research in the field. Focusing on the distinct clinical features shared by all CUPs, we advocate adopting the term primary metastatic cancer (PMC) to denominate a distinct cancer entity instead. In our view, PMC should be considered the archetype of metastatic disease and as such, despite accounting for a mere 2-3% of malignancies, unraveling the mechanisms at play goes beyond improving the prognosis of patients with PMC and promises to greatly enhance our understanding of the metastatic process and carcinogenesis across all cancer types.

Cytokine Induced Killer cells are effective against sarcoma cancer stem cells spared by chemotherapy and target therapy.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

Trabectedin and olaparib in patients with advanced and non-resectable bone and soft-tissue sarcomas (TOMAS): an open-label, phase 1b study from the Italian Sarcoma Group.

BACKGROUND: Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. METHODS: We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675-1·3 mg/m2 every 3 weeks; olaparib 100-300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. FINDINGS: Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2-6; range 1-17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5-23). Considering all dose levels, the most common grade 3-4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6-27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. INTERPRETATION: Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. FUNDING: Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

BRAF-inhibitors can exert control of disease in BRAF T599I mutated melanoma: a case report.

BRAF signaling is involved in melanoma growth in more than half of metastatic patients. In the last few years, new drugs that block this pathway have significantly improved the outcomes of patients with metastatic melanoma. Ninety percent of BRAF mutations involve exon 15, and the most frequent, V600E, results from the amino acid change from valine (V) to glutamic acid (E). BRAF inhibitor treatments have shown a notable overall response rate and improvements in progression-free and overall survival. Rare BRAF mutations of codon 599 have been also described in a few patients with papillary thyroid cancer and melanoma. Nowadays, no evidence is available in the literature, describing the role of target therapies as treatment in patients with this specific codon mutation. We describe the case of a young woman with metastatic melanoma with a particular BRAF mutation, T599I, who has benefited from treatment with a BRAF inhibitor, vemurafenib.