Citations

We read with great interest the manuscript by Brassetti et al. recently published in your journal and hope it will encourage discussion and debate around the optimization of the surgical management of patients with penile cancer (PECa) [...]

Abstract Background: Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in OC, immune checkpoint blockade therapy has been ineffective. Recently studies have shown that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity. One target involved in the DDR that has the potential to be of therapeutic value is protein phosphatase 4 (PP4); however, the effect of PP4 deficiency on anti-tumor immunity remain unknown. Results: Our results show that PP4 inhibition combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that PP4 inhibition triggers inflammatory signaling via NF-κB and STAT1 activation resulting in increased expression of pro-inflammatory cytokines including IFNβ1, CCL5, CXCL10, and IL-6. Moreover, conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and NK cells over carboplatin treatment alone. Knockdown of STING in OC cells significantly abrogated the increase in CD8 T cell migration induced by PP4 inhibition. PPP4C or PPP4R3B knockdown resulted in strong induction of NK cell IFN-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of ovarian cancer resulted in significant reduction of tumor growth in vivo. PP4C low tumors had increased infiltration of CD161+ NK cells and CD4+ T cells. Addition of low dose carboplatin treatment in vivo led to increased CD8+ T cell infiltration in PP4C low tumors when compared to the untreated groups. Conclusions: Our work has identified a role for PP4 inhibition in promoting anti-tumor immune activation. These findings provide the rationale for combining PP4 inhibitors with immunotherapy as a new approach in ovarian cancer treatment. Citation Format: Remya Raja, Christopher Wu, Esen Y. Bassoy, Thomas Rubino, Emma Utagawa, Paul Magtibay, Kristina Butler, Marion Curtis. Protein phosphatase 4 inhibition stimulates anti-tumor immunity in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4455.

Abstract Low count (LC) MBL is characterized by a circulating population of clonal B-cells and is a precursor state to CLL. Most individuals with LC MBL do not progress to CLL and never come to clinical attention. An active area of research is to identify factors that distinguish those individuals with MBL who progress from those who do not. Currently, 41 single nucleotide polymorphisms (SNPs) are known to be associated with CLL risk, and a portion of these SNPs have also been found to be associated with MBL risk. We hypothesize that these CLL-susceptibility SNPs associated with both MBL and CLL risk are SNPs associated with developing a B-cell clone; whereas the remaining SNPs found not to be associated with MBL risk are associated with progression of the B-cell clone (i.e., progression from MBL to CLL). To test this hypothesis, we evaluated the association of these 41 SNPs with risk of progression to CLL using a case-control study of 1,911 CLL cases and 1,579 individuals with MBL from Mayo Clinic. Individuals with MBL were identified through screening using 8-color flow cytometry assay. Individuals with LC MBL (defined by having a clonal B-cell population with the percent clonal B-cells out of total B-cell count <85%) who had an immunophenotype consistent with CLL were included as controls. CLL cases were obtained through the Mayo Clinic hematology practice. DNA was genotyped from whole blood. We analyzed the 41 CLL susceptibility SNPs with risk of progression to CLL using logistic regression and estimated odds ratio (OR) and 95% confidence interval (CI), adjusting for sex and age. We evaluated the CLL polygenic risk score (CLL-PRS), comprised of a weighted average of the 41 SNPs, with risk of CLL. Out of the 41 established CLL susceptibility SNPs, 15 were associated with CLL progression from MBL to CLL (all P<0.05). Nine of these 15 significant SNPs were not previously found to be associated with risk of MBL. The most significant SNPs were rs9880772 (OR=1.23, CI: 1.11-1.36, P=6.1 × 10−5), rs888096 (OR=1.25, CI: 1.12-1.38, P=3.0 × 10−5), and rs305065 (OR=1.28, CI: 1.14-1.43, P=1.5 × 10−5). One SNP (rs77551289) had a suggestive association (OR=1.21, P=0.069). The remaining 25 SNPs had no evidence of association with progression (ORs ranging between 0.9-1.1 and all P>0.1). The CLL-PRS was also associated with CLL risk (continuous OR = 1.25, CI: 1.15 - 1.35, P = 6.7 × 10−8). In our study accounting for the presence of B-cell clonal population, we identified 15 out of 41 CLL-susceptibility SNPs to be significantly associated with progression from LC MBL to CLL. The remaining 25 non-significant CLL-SNPs were not associated with progression and may instead be associated with development or initiation of the B-cell clone. These findings suggest that the established CLL susceptibility SNPs may play differing role (i.e., development or progression of the clone) in the etiology of CLL. Citation Format: Raphael Mwangi, Geffen Kleinstern, Sara J. Achenbach, Dennis Robinson, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie G. Waller, Nicholas J. Boddicker, James R. Cerhan, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, Tait Shanafelt, Susan L. Slager. Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5222.

Abstract Background: Colorectal cancer (CRC) incidence has increased in those 50 years or younger compared to older individuals in recent decades. While the intra-tumoral microbiome is a potent facilitator of tumorigenesis, the role of the microbiome in early onset (eo)CRC is unclear. Identification of the intra-tumoral microbiome may enhance prediction of adverse outcomes to improve survival of CRC overall age groups and eoCRC. Methods: We performed targeted sequencing of tumor and matched normal DNA samples from 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted from formalin fixed paraffin embedded (FFPE) or fresh frozen tissue. Normal tissue DNA was extracted from blood or normal surrounding colorectal tissue. We designed probes for the bacterial pathogens enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), and Fusobacterium nucleatum (Fn). We used logistic regression to assess tumor-associated bacteria and clinical parameters, tumor molecular characteristics, and tumor stage adjusted for age, sex, and study and a Cox promotional hazard to assess association with CRC-specific survival, overall and in eoCRC, adjusted for age at diagnosis, sex, mutational burden, and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 and 308 patients, overall and in eoCRC, respectively. Results: The prevalence of different bacteria in tumors differed slightly overall vs. in eoCRC tumors: ETBF (5.2%, 6.3%), pks+ EC (12.9%, 14.8%), and Fn (10.4%, 8.2%). Overall, men had a lower probability of tumors with Fn (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.64-0.99, p=0.04) but higher odds of tumors with pks+ EC (OR=1.38, CI=1.14-1.68, p=0.001), compared to women. The odds of prevalent Fn were higher for proximal vs distal tumors (OR=1.76, CI=1.38-2.26, p<0.001), but there was no association between tumor site and pks+ EC or ETBF. MSI-high tumors were more likely to carry Fn (OR=2.79, CI=2.08-3.75, p<0.001) and less likely to carry pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with MSS tumors. In patients with non-hypermutated tumors, we observed that pks+ EC positive tumors were associated with better CRC-specific survival (HR=0.73, CI=0.57-0.93, p=0.01), whereas ETBF positive tumors (HR=1.99, CI=1,18-3.36,p=0.009) and Fn positive tumors (HR=1.33, CI=1.04-1.72, p=0.03) were associated with poorer survival. In eo-CRC patients, Fn positive status was associated with poorer survival (HR=2.4, CI= 1.056 - 5.492, p=0.026). Conclusions: We showed that the intra-tumoral microbiome differs in distribution across patient populations and patient survival over all age groups and, specifically, in eoCRC. Citation Format: Meredith A. Hullar, Keith R. Curtis, Tabitha Harrison, Yi Lin, Robert Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David A. Drew, Jane Figueiredo, Amy J. French, Tabitha A. Harrison, Mark A. Jenkins, Johannes Melaku, Victor Moreno, Tomotaka Ugai, Shuji Ogino, Conghui Qu, Chenxu Qu, Steven Thibodeau, Syed Zaidi, Amanda I. Phipps, Ulrike Peters, on behalf of GECCO and CCFR investigators. Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3039.

9 Background: The efficacy of neoadjuvant platinum-based chemotherapy (NAPC) in penile squamous cell carcinoma (PSCC) was provided by small clinical trials, leaving clinicians with scant evidence-based guidance. Thus, we aimed to analyze real-world outcomes of patients with PSCC who received NAPC prior to surgical resection. Methods: Patients from 9 tertiary care centers who had undergone NAPC prior surgical resection for PSCC were included with locally advanced (cTany, cN+), non-metastatic (M0) disease. The primary and secondary outcomes were overall survival (OS) and progression-free survival (PFS), Response was determined via best overall response measured with RECIST 1.1 criteria. Results: 167 patients were included, 137 (84%), of which received TIP prior to surgical resection. Ninety-two (55%) patients died during follow up, and the mean follow up for survivors was 57 months. The median OS was 42 months, while the median PFS was 17 months. The table details the response rates to NAPC, the associated survival, and Cox Proportional Hazards Modelling of OS and PFS. Predictors for OS and RFS included lymphovascular invasion, positive number of lymph nodes, extranodal extension, downstaging, best overall response, and ypN status. Figure 1 demonstrates OS outcomes by RECIST best response after chemotherapy, presence of downstaging after NAPC, ypN stage, and presence of extranodal extension. Conclusions: Upfront NAPC is effective in patients with lymph node metastases from penile carcinoma. Unsurprisingly, patients who demonstrated a robust response to therapy had improved survival outcomes compared to those who had not—as measured by RECIST criteria, tumor downstaging, pN staging, and presence of extranodal extension. This study represents the largest conglomeration of multi-institutional penile carcinoma patients treated with NAPC, and provides further supportive data for multimodal therapy for advanced penile cancer patients while prospective clinical trials complete accrual. [Table: see text]

Abstract Adoptive Cell Transfer (ACT) therapies have recently fostered excitement for the treatment of hematogenous cancers. However, efficacy has largely been stunted in the treatment of solid tumors, especially for ovarian cancer, in part due to nutrient restriction within the tumor microenvironment. We recently demonstrated that intracellular delivery of biodegradable microparticles (MPs) made from polymers of alpha-ketoglutarate modulated dendritic cell energy metabolism and immunophenotype, altering regulatory and helper T cell frequencies. To determine if continuous metabolite release from MPs would rescue T cell dysfunction in nutrient-poor, immunosuppressive conditions, we generated MPs of polymerized glycolytic and tricarboxylic acid cycle central-carbon metabolites. Flow cytometric analysis and confocal microscopy indicated that FITC-conjugated MPs were efficiently loaded into the cytoplasm of activated primary murine T cells via membrane adsorption or electroporation. The MPs were retained intracellularly and sustained proliferation of mouse T cells for over five days in vitro. Glycolysis and mitochondrial respiration stress tests using an Agilent Seahorse XFe96 Analyzer demonstrated that primary mouse CD8 T cells utilized the MPs as a metabolic substrate. In particular, MPs increased metabolic function, as indicated by increased spare respiratory capacity, glycolysis, and glycolytic capacity. The results of this study provide preliminary evidence in support of utilizing sustained-releasing particles of metabolic substrates for the metabolic reprogramming of immune cells used in ACT therapies. This strategy has potential to improve or expand the efficacy of existing ACT therapies for solid tumors.

Abstract Background Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in OC, immune checkpoint blockade therapy has been ineffective. Recently studies have shown that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity. One target involved in the DDR that has the potential to be of therapeutic value is protein phosphatase 4 (PP4); however, the effect of PP4 deficiency on anti-tumor immunity remain unknown. Results Our results show that PP4 inhibition combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using a panel of ovarian cancer cells, we show that PP4 inhibition triggers inflammatory signaling via NFκB and STAT1 activation resulting in increased expression of pro-inflammatory cytokines including IFNβ1, CCL5, CXCL10, and IL-6. We further show that PP4 inhibition and carboplatin-induced STAT1 activation is significantly blunted upon STING knockdown in ovarian cancer cells. Moreover, using mouse ID8 ovarian cancer cell line and OT-I cells, we demonstrate that the combination of PP4 inhibition and carboplatin significantly increased CD8 T cell migration over carboplatin treatment alone. In addition, the same treatment resulted in enhanced NK cell-mediated tumor cell killing and T cell killing in an autologous human organoid model. Conclusions Our work has identified a role for PP4 inhibition in promoting anti-tumor immune activation. These findings provide the rationale for combining PP4 inhibitors with immunotherapy as a new approach in ovarian cancer treatment. Supported by DOD W81XWH2110489

Background Increased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian cancer (OC), the frequency of responses to immune checkpoint blockade (ICB) therapy in OC is much lower than other cancer types. Recent studies have highlighted that deficiencies in the DNA damage response (DDR) can drive increased genomic instability and tumor immunogenicity, which leads to enhanced responses to ICB. Protein phosphatase 4 (PP4) is a critical regulator of the DDR; however, its potential role in antitumor immunity is currently unknown. Results Our results show that the PP4 inhibitor, fostriecin, combined with carboplatin leads to increased carboplatin sensitivity, DNA damage, and micronuclei formation. Using multiple OC cell lines, we show that PP4 inhibition or PPP4C knockdown combined with carboplatin triggers inflammatory signaling via Nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 1 (STAT1) activation. This resulted in increased expression of the pro-inflammatory cytokines and chemokines: CCL5 , CXCL10 , and IL-6 . In addition, IFNB1 expression was increased suggesting activation of the type I interferon response. Conditioned media from OC cells treated with the combination of PP4 inhibitor and carboplatin significantly increased migration of both CD8 T cell and natural killer (NK) cells over carboplatin treatment alone. Knockdown of stimulator of interferon genes (STING) in OC cells significantly abrogated the increase in CD8 T-cell migration induced by PP4 inhibition. Co-culture of NK-92 cells and OC cells with PPP4C or PPP4R3B knockdown resulted in strong induction of NK cell interferon-γ, increased degranulation, and increased NK cell-mediated cytotoxicity against OC cells. Stable knockdown of PP4C in a syngeneic, immunocompetent mouse model of OC resulted in significantly reduced tumor growth in vivo . Tumors with PP4C knockdown had increased infiltration of NK cells, NK T cells, and CD4 + T cells. Addition of low dose carboplatin treatment led to increased CD8 + T-cell infiltration in PP4C knockdown tumors as compared with the untreated PP4C knockdown tumors. Conclusions Our work has identified a role for PP4 inhibition in promoting inflammatory signaling and enhanced immune cell effector function. These findings support the further investigation of PP4 inhibitors to enhance chemo-immunotherapy for OC treatment.

Background: Regional anesthesia (“block”) is an important component of upper extremity (UE) surgery pain control. However, little is known about patient experience related to perioperative opioid use. This study assessed patient-reported pain control and satisfaction with UE blocks and evaluated how opioid consumption impacted these outcomes before the block “wore off.” Methods: A postoperative phone survey was administered to patients who underwent outpatient UE surgery at a surgery center for more than 16 months. It assessed pain scores (scale 1-10), satisfaction with block duration (scale 1-5), duration until return of UE function, and opioid consumption. Analyses used Mann-Whitney U tests, Fisher exact tests, and bivariate and multivariable linear and ordered logistic regressions to understand relationships between opioid use and outcomes. Results: A total of 509 patients (61%) completed the survey, and 441 (88%) were satisfied with block duration. Initial and final pain scores were significantly higher in patients who took opioids prior to the block wearing off (6 and 4.5, P = .04 and 3.5 and 2, P = .002, respectively). Although satisfaction with block duration was not different in group comparisons (ie, patients who premedicated vs those who did not), in a multivariable analysis, patients who premedicated with opioids had 78% increased odds of reporting the highest level of satisfaction compared with the lower 4 levels ( P = .03). Conclusions: Upper extremity blocks are associated with high overall patient satisfaction and postsurgical pain control. Premedicating before the block wears off may increase patient satisfaction with block duration even if pain is not notably impacted.

AbstractHeparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.