Progression of Site-Specific Recurrence of Pancreatic Cancer and Implications for Treatment.

OBJECTIVE: This study aimed to analyze post-recurrence progression in context of recurrence sites and assess implications for post-recurrence treatment. BACKGROUND: Most patients with resected pancreatic ductal adenocarcinoma (PDAC) recur within two years. Different survival outcomes for location-specific patterns of recurrence are reported, highlighting their prognostic value. However, a lack of understanding of post-recurrence progression and survival remains. METHODS: This retrospective analysis included surgically treated PDAC patients at the NYU-Langone Health (2010-2021). Sites of recurrence were identified at time of diagnosis and further follow-up. Kaplan-Meier curves, log-rank test, and Cox-regression analyses were applied to assess survival outcomes. RESULTS: Recurrence occurred in 57.3% (196/342) patients with a median time to recurrence of 11.3 months (95%CI:12.6 to 16.5). First site of recurrence was local in 43.9% patients, liver in 23.5%, peritoneal in 8.7%, lung in 3.6%, while 20.4% had multiple sites of recurrence. Progression to secondary sites was observed in 11.7%. Only lung involvement was associated with significantly longer survival after recurrence compared to other sites (16.9 months vs. 8.49 months, P=0.003). In local recurrence, 21 (33.3%) patients were alive after one year without progression to secondary sites. This was associated with a CA19-9 of <100U/ml at time of primary diagnosis (P=0.039), nodal negative disease (P=0.023), and well-moderate differentiation (P=0.042) compared to patients with progression. CONCLUSION: Except for lung recurrence, post-recurrence survival after PDAC resection is associated with poor survival. A subset of patients with local-only recurrence do not quickly succumb to systemic spread. This is associated with markers for favorable tumor biology, making them candidates for potential curative re-resections when feasible.

Corrigendum: Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

[This corrects the article DOI: 10.3389/fimmu.2023.1067352.].

POLQ inhibition elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma via cGAS/STING signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination-repair (HR-repair) proteins in 20%-25% of cases. Defects in HR impart a specific vulnerability to poly ADP ribose polymerase inhibitors and platinum-containing chemotherapy in tumor cells. However, not all patients who receive these therapies respond, and many who initially respond ultimately develop resistance. Inactivation of the HR pathway is associated with the overexpression of polymerase theta (Polθ, or POLQ). This key enzyme regulates the microhomology-mediated end-joining (MMEJ) pathway of double-strand break (DSB) repair. Using human and murine HR-deficient PDAC models, we found that POLQ knockdown is synthetically lethal in combination with mutations in HR genes such as BRCA1 and BRCA2 and the DNA damage repair gene ATM. Further, POLQ knockdown enhances cytosolic micronuclei formation and activates signaling of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING), leading to enhanced infiltration of activated CD8+ T cells in BRCA2-deficient PDAC tumors in vivo. Overall, POLQ, a key mediator in the MMEJ pathway, is critical for DSB repair in BRCA2-deficient PDAC. Its inhibition represents a synthetic lethal approach to blocking tumor growth while concurrently activating the cGAS-STING signaling pathway to enhance tumor immune infiltration, highlighting what we believe to be a new role for POLQ in the tumor immune environment.

Tumor infiltrating T cell states and checkpoint inhibitor expression in hepatic and pancreatic malignancies.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103+ tissue resident memory T cells (TRM), CCR7+ central memory T cells, and CD57+ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher TRM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

Single-cell RNA sequencing reveals the effects of chemotherapy on human pancreatic adenocarcinoma and its tumor microenvironment.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

A pan-cancer organoid platform for precision medicine.

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Safety of Combined Yttrium-90 Radioembolization and Immune Checkpoint Inhibitor Immunotherapy for Hepatocellular Carcinoma.

PURPOSE: To investigate the safety of yttrium-90 radioembolization in combination with checkpoint inhibitor immunotherapy for the treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This single-center retrospective study included 26 consecutive patients with HCC who received checkpoint inhibitor immunotherapy within 90 days of radioembolization from April 2015 to May 2018. Patients had preserved liver function (Child-Pugh scores A-B7) and either advanced HCC due to macrovascular invasion or limited extrahepatic disease (21 patients) or aggressive intermediate stage HCC that resulted in earlier incorporation of systemic immunotherapy (5 patients). Clinical documentation, laboratory results, and imaging results at 1- and 3-month follow-up intervals were reviewed to assess treatment-related adverse events and treatment responses. RESULTS: The median follow-up period after radioembolization was 7.8 months (95% confidence interval [CI], 5.6-11.8). There were no early (30-day) mortality or grades 3/4 hepatobiliary or immunotherapy-related toxicities. Delayed grades 3/4 hepatobiliary toxicities (1-3 months) occurred in 2 patients in the setting of HCC disease progression. One patient developed pneumonitis. The median overall survival from first immunotherapy was 17.2 months (95% CI, 10.9-23.4). The median overall survival from first radioembolization was 16.5 months (95% CI, 6.6-26.4). From first radioembolization, time to tumor progression was 5.7 months (95% CI, 4.2-7.2), and progression-free survival was 5.7 months (95% CI, 4.3-7.1). CONCLUSIONS: Radioembolization combined with checkpoint inhibitor immunotherapy in cases of HCC appears to be safe and causes limited treatment-related toxicity. Future prospective studies are needed to identify the optimal combination treatment protocols and evaluate the efficacy of combination therapy.

New Labyrinth Microfluidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

Histotripsy for Non-Invasive Ablation of Hepatocellular Carcinoma (HCC) Tumor in a Subcutaneous Xenograft Murine Model.

Histotripsy fractionates tissue through a mechanical, non-invasive ultrasonic ablation process that precisely controls acoustic cavitation while utilizing real-time ultrasound (US) imaging guidance. This study investigates the potential, feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in a subcutaneous in vivo murine Hepatocellular Carcinoma (HCC) model. Hep3B tumors were generated in the right flanks of 14 NSG and 7 NOD-SCID mice. The mice were grouped as follows: A (acute, NSG with n=9 treatment and n=1 control), B (chronic, NSG with n=2 treatment and n=2 control) and C (chronic NODSCID, with n=6 treatment and n=1 control). Treatment was performed when the tumor diameters reached >5 mm. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were delivered using a custom built 1 MHz therapy transducer attached to a motorized positioner, which scanned the transducer focus to traverse the targeted tumor volume, guided by real-time US imaging. Tumor ablation effectiveness was assessed by obtaining T1, T2 and T2* weighted MR images. Post euthanasia, treated tumor, brain, and lung tissue samples were harvested for histology. Histology of acute group A showed fractionation of targeted region with a sharp boundary separating it from untreated tissue. Groups B and C demonstrated effective tumor volume reduction post treatment on MRI as the homogenate and edema were resorbed within 23 weeks. However, as the tumor was subcutaneous, it was not possible to set adequate treatment margin and since the mice were immune-compromised, residual viable tumor cells eventually developed into tumor regrowth at 3-9 weeks after histotripsy. Groups B and C showed no signs of metastasis in the lung and brain. Our study successfully demonstrated the potential of histotripsy for non-invasive HCC ablation in a subcutaneous murine model. Additional work is ongoing to study the response of histotripsy in immune-competent orthotopic liver tumor models.

Multicenter Study of Staging and Therapeutic Predictors of Hepatocellular Carcinoma Recurrence Following Transplantation.

Orthotopic liver transplantation (OLT) and resection are effective treatments for hepatocellular carcinoma (HCC). However, optimizing OLT and limiting HCC recurrence remains a vexing problem. New HCC Model for End-Stage Liver Disease and allocation algorithms provide greater observation of HCC patients, many while receiving local-regional treatments. Potential benefits of local-regional treatment for limiting HCC recurrence after OLT remain incompletely understood. Therefore, we aimed to define HCC-specific prognostic factors affecting recurrence in a contemporary, multicenter cohort of HCC patients undergoing OLT and specifically whether local-regional therapies limited recurrence. We identified 441 patients undergoing OLT for HCC at 3 major transplant centers from 2008 to 2013. Cox regression was used to analyze covariate-adjusted recurrence and mortality rates after OLT. "Bridging" or "downstaging" therapy was used in 238 (54%) patients with transarterial chemoembolization (TACE) being used in 170 (71%) of treated patients. The survival rate after OLT was 88% and 78% at 1 and 3 years, respectively, with HCC recurrence (28% of deaths) significantly increasing the mortality rate (hazard ratio [HR], 19.87; P < 0.001). Tumor size, not tumor number, either at presentation or on explant independently predicted HCC recurrence (HR, 1.36 and 1.73, respectively; P < 0.05) with a threshold effect noted at 4.0-cm size. Local-regional therapy (TACE) reduced HCC recurrence by 64% when adjusting for presenting tumor size (HR, 0.36; P < 0.05). Explant tumor size and microvascular invasion predicted mortality (HR, 1.19 and 1.51, respectively; P < 0.05) and pathologic response to therapy (TACE or radiofrequency ablation) significantly decreased explant tumor size (0.56-1.62 cm diameter reduction; P < 0.05). In conclusion, HCC tumor size at presentation or explant is the most important predictor for HCC recurrence after OLT. Local-regional therapy to achieve a pathologic response (decreasing tumor size) can limit HCC recurrences after OLT. Liver Transplantation 00 000-000 2018 AASLD.

Individualized Adaptive Stereotactic Body Radiotherapy for Liver Tumors in Patients at High Risk for Liver Damage: A Phase 2 Clinical Trial.

IMPORTANCE: Patients with preexisting liver dysfunction could benefit the most from personalized therapy for liver tumors to balance maximal tumor control and minimal risk of liver failure. We designed an individualized adaptive trial testing the hypothesis that adapting treatment based on change in liver function could optimize the therapeutic index for each patient. OBJECTIVE: To characterize the safety and efficacy of individualized adaptive stereotactic body radiotherapy (SRBT) for liver tumors in patients who have preexisting liver dysfunction. DESIGN, SETTING, AND PARTICIPANTS: From 2010 to 2014, 90 patients with intrahepatic cancer treated with prior liver-directed therapy were enrolled in this large phase 2, single-arm, clinical trial at an academic medical center. All patients had at least 1 year of potential follow-up. INTERVENTIONS: Using indocyanine green retention at 15 minutes (ICGR15) as a direct biomarker of liver function and a Bayesian adaptive model, planned SBRT was individually modified midway through the course of therapy to maintain liver function after the complete course. MAIN OUTCOMES AND MEASURES: The primary outcome was local control; the secondary outcome was safety and overall survival. RESULTS: Patients were 34 to 85 years of age, and 70% (63) were male. Ninety patients (69 [77%] with hepatocellular carcinoma, 4 [4%] with intrahepatic cholangiocarcinoma, and 17 [19%] with metastatic) received treatment to 116 tumors. Sixty-two patients (69%) had cirrhosis, 21 (23%) were Child-Pugh (CP) grade B. The median tumor size was 3 cm; 16 patients (18%) had portal vein involvement. Sixty-two (69%) received all 5 fractions (47 full dose, 15 dose-reduced owing to rising ICGR15). Treatment was well tolerated, with a lower than expected complication rate without adaptation: 6 (7%) experienced a 2-point decline in CP 6 months post-SBRT. The 1- and 2-year local control rates were 99% (95% CI, 97%-100%) and 95% (95% CI, 91%-99%), respectively. CONCLUSIONS AND RELEVANCE: We demonstrated that the treatment strategy of individualized adaptive therapy based on a direct biomarker of liver function can be used to achieve both high rates of local control and a high degree of safety without sacrificing either. Individualized adaptive radiotherapy may represent a new treatment paradigm in which dose is based on individual, rather than population-based, tolerance to treatment. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01522937.

Targeting ADAM17 inhibits human colorectal adenocarcinoma progression and tumor-initiating cell frequency.

ADAM17 (a disintegrin and metalloproteinase 17)/TACE (TNFα converting enzyme) has emerged as a potential therapeutic target in colorectal cancer (CRC) and other cancers, due in part to its role in regulating various tumor cell surface proteins and growth factors and cytokines in the tumor microenvironment. The emergence of MEDI3622, a highly potent and specific antibody-based ADAM17 inhibitor, has allowed testing of the concept that targeting ADAM17 may be an important new therapeutic approach for CRC patients. We demonstrate that MEDI3622 is highly efficacious on tumor growth in multiple human CRC PDX models, resulting in improved survival of animals bearing tumor xenografts. MEDI3622 was further found to impact Notch pathway activity and tumor-initiating cells. The promising preclinical activity seen here supports further clinical investigation of this treatment approach to improve therapeutic outcome for patients diagnosed with metastatic CRC, including patients with KRAS-mutant tumors for whom other therapeutic options are currently limited.

Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial.

BACKGROUND: For patients with advanced hepatocellular carcinoma, sorafenib is the only approved drug worldwide, and outcomes remain poor. We aimed to assess the safety and efficacy of nivolumab, a programmed cell death protein-1 (PD-1) immune checkpoint inhibitor, in patients with advanced hepatocellular carcinoma with or without chronic viral hepatitis. METHODS: We did a phase 1/2, open-label, non-comparative, dose escalation and expansion trial (CheckMate 040) of nivolumab in adults (≥18 years) with histologically confirmed advanced hepatocellular carcinoma with or without hepatitis C or B (HCV or HBV) infection. Previous sorafenib treatment was allowed. A dose-escalation phase was conducted at seven hospitals or academic centres in four countries or territories (USA, Spain, Hong Kong, and Singapore) and a dose-expansion phase was conducted at an additional 39 sites in 11 countries (Canada, UK, Germany, Italy, Japan, South Korea, Taiwan). At screening, eligible patients had Child-Pugh scores of 7 or less (Child-Pugh A or B7) for the dose-escalation phase and 6 or less (Child-Pugh A) for the dose-expansion phase, and an Eastern Cooperative Oncology Group performance status of 1 or less. Patients with HBV infection had to be receiving effective antiviral therapy (viral load <100 IU/mL); antiviral therapy was not required for patients with HCV infection. We excluded patients previously treated with an agent targeting T-cell costimulation or checkpoint pathways. Patients received intravenous nivolumab 0·1-10 mg/kg every 2 weeks in the dose-escalation phase (3+3 design). Nivolumab 3 mg/kg was given every 2 weeks in the dose-expansion phase to patients in four cohorts: sorafenib untreated or intolerant without viral hepatitis, sorafenib progressor without viral hepatitis, HCV infected, and HBV infected. Primary endpoints were safety and tolerability for the escalation phase and objective response rate (Response Evaluation Criteria In Solid Tumors version 1.1) for the expansion phase. This study is registered with ClinicalTrials.gov, number NCT01658878. FINDINGS: Between Nov 26, 2012, and Aug 8, 2016, 262 eligible patients were treated (48 patients in the dose-escalation phase and 214 in the dose-expansion phase). 202 (77%) of 262 patients have completed treatment and follow-up is ongoing. During dose escalation, nivolumab showed a manageable safety profile, including acceptable tolerability. In this phase, 46 (96%) of 48 patients discontinued treatment, 42 (88%) due to disease progression. Incidence of treatment-related adverse events did not seem to be associated with dose and no maximum tolerated dose was reached. 12 (25%) of 48 patients had grade 3/4 treatment-related adverse events. Three (6%) patients had treatment-related serious adverse events (pemphigoid, adrenal insufficiency, liver disorder). 30 (63%) of 48 patients in the dose-escalation phase died (not determined to be related to nivolumab therapy). Nivolumab 3 mg/kg was chosen for dose expansion. The objective response rate was 20% (95% CI 15-26) in patients treated with nivolumab 3 mg/kg in the dose-expansion phase and 15% (95% CI 6-28) in the dose-escalation phase. INTERPRETATION: Nivolumab had a manageable safety profile and no new signals were observed in patients with advanced hepatocellular carcinoma. Durable objective responses show the potential of nivolumab for treatment of advanced hepatocellular carcinoma. FUNDING: Bristol-Myers Squibb.

Non-Invasive Liver Ablation Using Histotripsy: Preclinical Safety Study in an In Vivo Porcine Model.

This study investigates the safety profile for use of histotripsy, a non-invasive ultrasonic ablation method currently being developed for the treatment of liver cancer, for liver ablation in an in vivo porcine model. Histotripsy treatments were applied to the liver and hepatic veins of 22 porcine subjects, with half of the subjects receiving systemic heparinization. Vital signs (heart rate, blood pressure, temperature, electrocardiogram and SpO2) were monitored throughout the procedure and for 1 h post-treatment. Blood was drawn at six points during the experiment to analyze blood gases, liver function and free hemoglobin levels. All treatments were guided and monitored by real-time ultrasound imaging. After treatment, the tissue was harvested for histological analysis. Results indicated that histotripsy generated well-defined lesions inside the liver and around the treated hepatic veins of all subjects in both treatment groups. Vital signs and blood analysis revealed that animals responded well to histotripsy, with all animals surviving the treatment. One animal in the non-heparinized group had a transient increase in pH and decreases in blood pressure, heart rate and PCO2 during the 15-min vessel treatment, with these changes returning to baseline levels soon after the treatment. Overall, the results indicate that histotripsy can safely be performed on the liver without the need for systemic heparinization, even in regions containing large hepatic vessels, supporting its future use for the treatment of liver cancer.

Non-Invasive Ultrasound Liver Ablation Using Histotripsy: Chronic Study in an In Vivo Rodent Model.

Hepatocellular carcinoma, or liver cancer, has the fastest growing incidence among cancers in the United States. Current liver ablation methods are thermal-based and share limitations due to the heat sink effect from the blood flow through the highly vascular liver. Recently, our group has investigated histotripsy as a non-invasive liver cancer ablation method. Histotripsy is a non-thermal ultrasonic ablation method that fractionates tissue through the control of acoustic cavitation. Previous experiments in an in vivo porcine model show that histotripsy can create well-confined lesions in the liver through ribcage obstruction without damaging the overlying ribs and other tissues. Histotripsy can also completely fractionate liver tissue surrounding major vessels while preserving the vessels. In this study, we investigate the long-term effects of histotripsy liver ablation in a rodent model. We hypothesize that the fractionated histotripsy lesion will be resorbed by the liver, resulting in effective tissue healing. To test this hypothesis, the livers of 20 healthy rats were treated with histotripsy using an 8-element 1-MHz histotripsy transducer. Rats were euthanized after 0, 3, 7, 14 and 28 days (n = 4). In vivo and post mortem results showed histotripsy lesions were successfully generated through the intact abdomen in all 20 rats. Magnetic resonance imaging found primarily negative contrast on day 0, positive contrast on day 3 and rapid normalization of signal intensity thereafter (i.e., signal amplitude returned to baseline levels seen in healthy liver tissue). Histologically, lesions were completely fractionated into an acellular homogenate. The lesions had a maximum cross-sectional area of 17.2 ± 1.9 mm(2) and sharp boundaries between the lesion and the healthy surrounding tissue after treatment. As the animals recovered after treatment, the histotripsy tissue homogenate was almost completely replaced by regenerated liver parenchyma, resulting in a small fibrous lesion (<1 mm(2) maximum cross-section) remaining after 28 d. The results of this study suggest that histotripsy has potential as a non-invasive liver ablation method for effective tissue removal.

GM-CSF Mediates Mesenchymal-Epithelial Cross-talk in Pancreatic Cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer-associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. SIGNIFICANCE: The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal-epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886-99. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Sensitivity of KRAS-Mutant Colorectal Cancers to Combination Therapy That Cotargets MEK and CDK4/6.

PURPOSE: The emerging need for rational combination treatment approaches led us to test the concept that cotargeting MEK and CDK4/6 would prove efficacious in KRAS-mutant (KRAS(mt)) colorectal cancers, where upregulated CDK4 and hyperphosphorylated retinoblastoma (RB) typify the vast majority of tumors. EXPERIMENTAL DESIGN: Initial testing was carried out in the HCT-116 tumor model, which is known to harbor a KRAS mutation. Efficacy studies were then performed with five RB(+) patient-derived colorectal xenograft models, genomically diverse with respect to KRAS, BRAF, and PIK3CA mutational status. Tolerance, efficacy, and pharmacodynamic evaluation of target modulation were evaluated in response to daily dosing with either agent alone or concurrent coadministration. RESULTS: Synergy was observed in vitro when HCT-116 cells were treated over a broad range of doses of trametinib and palbociclib. Subsequent in vivo evaluation of this model showed a higher degree of antitumor activity resulting from the combination compared to that achievable with single-agent treatment. Testing of colorectal patient-derived xenograft (PDX) models further showed that combination of trametinib and palbociclib was well tolerated and resulted in objective responses in all KRAS(mt) models tested. Stasis was observed in a KRAS/BRAF wild-type and a BRAF(mt) model. CONCLUSIONS: Combination of trametinib and palbociclib was well tolerated and highly efficacious in all three KRAS-mutant colorectal cancer PDX models tested. Promising preclinical activity seen here supports clinical evaluation of this treatment approach to improve therapeutic outcome for patients with metastatic colorectal cancer.

Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction.

Aerobic glycolysis regulates T cell function. However, whether and how primary cancer alters T cell glycolytic metabolism and affects tumor immunity in cancer patients remains a question. Here we found that ovarian cancers imposed glucose restriction on T cells and dampened their function via maintaining high expression of microRNAs miR-101 and miR-26a, which constrained expression of the methyltransferase EZH2. EZH2 activated the Notch pathway by suppressing Notch repressors Numb and Fbxw7 via trimethylation of histone H3 at Lys27 and, consequently, stimulated T cell polyfunctional cytokine expression and promoted their survival via Bcl-2 signaling. Moreover, small hairpin RNA-mediated knockdown of human EZH2 in T cells elicited poor antitumor immunity. EZH2(+)CD8(+) T cells were associated with improved survival in patients. Together, these data unveil a metabolic target and mechanism of cancer immune evasion.