Digital quantitative tissue image analysis of hypoxia in resected pancreatic ductal adenocarcinomas.

Background: Tumor hypoxia is theorized to contribute to the aggressive biology of pancreatic ductal adenocarcinoma (PDAC). We previously reported that hypoxia correlated with rapid tumor growth and metastasis in patient-derived xenografts. Anticipating a prognostic relevance of hypoxia in patient tumors, we developed protocols for automated semi-quantitative image analysis to provide an objective, observer-independent measure of hypoxia. We further validated this method which can reproducibly estimate pimonidazole-detectable hypoxia in a high-through put manner. Methods: We studied the performance of three automated image analysis platforms in scoring pimonidazole-detectable hypoxia in resected PDAC (n = 10) in a cohort of patients enrolled in PIMO-PANC. Multiple stained tumor sections were analyzed on three independent image-analysis platforms, Aperio Genie (AG), Definiens Tissue Studio (TS), and Definiens Developer (DD), which comprised of a customized rule set. Results: The output from Aperio Genie (AG) had good concordance with manual scoring, but the workflow was resource-intensive and not suited for high-throughput analysis. TS analysis had high levels of variability related to misclassification of cells class, while the customized rule set of DD had a high level of reliability with an intraclass coefficient of more than 85%. Discussion: This work demonstrates the feasibility of developing a robust, high-performance pipeline for an automated, quantitative scoring of pimonidazole-detectable hypoxia in patient tumors.

Pancreatic cancer growth promoted by bone marrow mesenchymal stromal cell-derived IL-6 is reversed predominantly by IL-6 blockade.

Pancreatic cancer is a highly lethal cancer characterized by local invasiveness, early metastasis, recurrence and high resistance to current therapies. Extensive stroma or desmoplasia is a key histological feature of the disease, and interactions between cancer and stromal cells are critical for pancreatic cancer development and progression. Mesenchymal stromal cells [MSCs] exhibit preferential tropism to primary and metastatic tumor sites and may either suppress or support tumor growth. Although MSCs represent a potential source of pancreatic cancer stroma, their contribution to pancreatic tumor growth remains poorly known. Here, we show that bone marrow MSCs significantly contribute to pancreatic cancer growth in vitro and in vivo. Furthermore, MSCs create a pro-carcinogenic microenvironment through the release of key factors mediating growth and angiogenesis, including interleukin (IL)-6, IL-8, vascular endothelial growth factor and activation of STAT3 signaling in tumor cells. IL-6 released by MSCs was largely responsible for the pro-tumorigenic effects of MSCs. Knockdown of IL-6 expression in MSCs by small interfering RNA (siRNA) abolished the MSC growth-promoting effect in vitro, reducing tumor cell proliferation and clonogenic potential. In addition, in a heterotopic nude mouse model of human pancreatic tumor xenografts, blockade of IL-6 with the anti-IL-6 receptor antibody, tocilizumab, or of its downstream effector STAT3 with the small molecule STAT3 inhibitor S3I-201, abrogated MSC-mediated tumor promotion and delayed tumor formation significantly. Our data demonstrate that MSCs promote pancreatic cancer growth, with IL-6 produced by MSCs playing a pivotal role.

Anal Adenocarcinoma: A Rare Entity in Need of Multidisciplinary Management.

BACKGROUND: Anal adenocarcinoma is a rare clinical entity for which the optimal management is not defined. OBJECTIVE: This study aimed to describe the multidisciplinary management and outcomes of patients with anal adenocarcinoma. DESIGN: This is a retrospective cohort study. SETTING: This study was conducted at a quaternary cancer center. PATIENTS: Men and women with anal adenocarcinoma treated between 1995 and 2016 were selected. INTERVENTIONS: Fifty-two patients were treated with either chemoradiotherapy or trimodality therapy including radiation therapy, chemotherapy, and surgical resection. MAIN OUTCOME MEASURES: Local failure, regional failure, and distant metastasis rates were estimated using the cumulative incidence method. The Kaplan-Meier method was used to estimate progression-free survival and overall survival. The multivariable Cox proportional hazards model was used to evaluate the clinical predictors of outcome. RESULTS: There was a higher 5-year rate of local failure in patients treated with chemoradiotherapy compared with trimodality therapy (53% vs 10%; p < 0.01). The 5-year incidence of distant metastases was 29% (trimodality therapy) versus 30% (chemoradiotherapy; p = 0.9); adjuvant chemotherapy did not reduce the incidence of distant metastases (p = 0.8). Five-year overall survival was 73% (trimodality therapy) versus 49.4% (chemoradiotherapy; p = 0.1). On multivariable analysis, factors associated with worse overall survival were treatment with chemoradiotherapy, cT3-4 category disease, and node-positive disease. LIMITATIONS: This study is limited by its small sample size and retrospective nature. CONCLUSIONS: Although treatment may continue to be tailored to individual patients, better outcomes with a trimodality therapy approach were observed. See Video Abstract at http://links.lww.com/DCR/B708.ADENOCARCINOMA ANAL: UNA ENTIDAD POCO FRECUENTE EN NECESIDAD DE UN MANEJO MULTIDISCIPLINARIO. ANTECEDENTES: El adenocarcinoma anal es una entidad clínica poco frecuente por lo que aún no se define el manejo óptimo. OBJETIVO: Describir el manejo multidisciplinario y los resultados de los pacientes con adenocarcinoma anal. DISEO: Estudio de cohorte retrospectivo. ENTORNO CLINICO: Centro de cáncer cuaternario. PACIENTES: Hombres y mujeres con adenocarcinoma anal tratados entre 1995 y 2016. INTERVENCIONES: Cincuenta y dos pacientes fueron tratados con quimiorradioterapia o terapia trimodal que incluyó: radioterapia, quimioterapia y resección quirúrgica. PRINCIPALES MEDIDAS DE VALORACION: Se estimaron las tasas de falla local, falla regional y metástasis a distancia mediante el método de incidencia acumulada. Se utilizó el método de Kaplan-Meier para estimar la supervivencia libre de progresión y la supervivencia global. Los riesgos proporcionales de multivariable Cox se utilizaron para evaluar los predictores clínicos de los resultados. RESULTADOS: Hubo una mayor tasa de falla local a cinco años en pacientes tratados con quimiorradioterapia en comparación con terapia trimodal (53% vs 10%; p < 0,01). La incidencia a cinco años de metástasis a distancia fue del 29% (terapia trimodal) versus 30% (quimiorradioterapia) (p = 0,9); la quimioterapia adyuvante no redujo la incidencia de metástasis a distancia (p = 0,8). La supervivencia global a cinco años fue del 73% (terapia trimodal) versus 49,4% (quimiorradioterapia); p = 0,1. En el análisis multivariable, los factores asociados con una peor supervivencia general fueron el tratamiento con quimiorradioterapia, enfermedad de categoría cT3-4 y enfermedad con ganglios positivos. LIMITACIONES: Este estudio está limitado por su pequeño tamaño de muestra y su naturaleza retrospectiva. CONCLUSIONES: Aunque el tratamiento puede seguir adaptándose a pacientes individuales, se observaron mejores resultados con un enfoque TTM. Conslute Video Resumen en http://links.lww.com/DCR/B708. (Traducción- Dr. Francisco M. Abarca-Rendon).

Patient-derived tumor xenograft and organoid models established from resected pancreatic, duodenal and biliary cancers.

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.

Radioimmunotherapy of human pancreatic cancer xenografts in NOD-scid mice with [64Cu]Cu-NOTA-panitumumab F(ab')2 alone or combined with radiosensitizing gemcitabine and the PARP inhibitor, rucaparib.

INTRODUCTION: Our objective was to determine the feasibility of extending our previously reported PET imaging study of pancreatic cancer (PnCa) with [64Cu]Cu-NOTA-panitumumab F(ab')2 to radioimmunotherapy (RIT) by exploiting the ß-particle and Auger electron emissions of 64Cu (PET theranostic concept). To enhance the effectiveness of [64Cu]Cu-NOTA-panitumumab F(ab')2, we further combined RIT with radiosensitizing gemcitabine (GEM) and the poly(ADP)ribose polymerase inhibitor (PARPi), rucaparib. METHODS: Normal tissue toxicity was assessed in non-tumor-bearing NOD-scid mice injected i.v. with [64Cu]Cu-NOTA-panitumumab F(ab')2 (1.85-9.25 MBq; 10 µg) or [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 (12.95 MBq). Body weight was monitored, and hematopoietic (CBC), liver (ALT) and kidney [creatinine (SCr)] toxicity were assessed. RIT studies were performed in NOD-scid mice with s.c. OCIP23 human PnCa patient-derived xenografts (PDX) administered [64Cu]Cu-NOTA-panitumumab F(ab')2 (3.7 MBq; 10 µg), unlabeled panitumumab F(ab')2 (10 µg) or normal saline every two weeks. Subsequent studies evaluated RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) administered alone or combined with GEM and the PARPi, rucaparib administered on a 14-day treatment cycle for up to 6 cycles in NOD-scid mice with s.c. PANC-1 human PnCa xenografts. The radiation absorbed dose in PANC-1 tumors and normal organs in mice after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) was estimated based on previously reported biodistribution studies of [64Cu]Cu-NOTA-panitumumab F(ab')2. RESULTS: No normal tissue toxicity was observed in non-tumor-bearing NOD-scid mice administered up to 3.7 MBq (10 µg) of [64Cu]Cu-NOTA-panitumumab F(ab')2 but slightly increased ALT was noted at 9.25 MBq. Administration of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 (12.95 MBq; 10 µg) caused some hematopoietic toxicity but no increase in ALT or SCr or decreased body weight. A slight tumor growth delay and increased survival was noted in NOD-scid mice with s.c. OCIP23 PDX treated with [64Cu]Cu-NOTA-panitumumab F(ab')2 (3.7 MBq; 10 µg) or unlabeled panitumumab F(ab')2 (10 µg) compared to normal saline treated mice. RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) combined with GEM + PARPi for up to 6 cycles was most effective for the treatment of PANC-1 tumors. Tumor doubling time increased to 13.3 ± 0.9 days vs. 7.8 ± 3.7 days for RIT alone and 9.3 ± 2.2 days for normal saline treatment. Median survival was significantly longer (P < 0.05) than in mice treated with normal saline (35 days) for RIT + GEM + PARPi (71 days), GEM + PARPi (44 days) and RIT + GEM (43 days) but not for RIT alone (25 days). RIT + GEM + PARPi provided a longer median survival than RIT (P < 0.01), GEM + PARPi (P = 0.01) but not RIT + GEM (P = 0.23). Nonetheless, PANC-1 tumors grew exponentially in all treatment groups. The absorbed dose in PANC-1 tumors after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.85 MBq; 10 µg) was 0.8 Gy, while the dose in normal organs ranged from 0.6-1.2 Gy. CONCLUSIONS: We conclude that RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 did not cause significant normal tissue toxicity but was not effective when administered alone for treatment of PnCa xenografts in NOD-scid mice. Combining RIT with GEM and the PARPi, rucaparib enhanced its effectiveness but tumors continued to grow exponentially. Our results suggest that 64Cu is not feasible for RIT of PnCa due to low tumor absorbed doses. 177Lu which has a higher abundance of moderate energy ß-particle emissions may be more effective than 64Cu. The hematopoietic toxicity of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 may be mediated by binding to mouse EGFR expressed on some hematopoietic stem cells. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Direct extension of PET with 64Cu(Cu)-NOTA-panitumumab F(ab')2 to RIT exploiting the ß-particle and Auger electron emissions of 64Cu is not feasible. Theranostic approaches that combine PET with RIT employing 177Lu may be more promising and should be explored.

Quantifying Reoxygenation in Pancreatic Cancer During Stereotactic Body Radiotherapy.

Hypoxia, the state of low oxygenation that often arises in solid tumours due to their high metabolism and irregular vasculature, is a major contributor to the resistance of tumours to radiation therapy (RT) and other treatments. Conventional RT extends treatment over several weeks or more, and nominally allows time for oxygen levels to increase ("reoxygenation") as cancer cells are killed by RT, mitigating the impact of hypoxia. Recent advances in RT have led to an increase in the use stereotactic body radiotherapy (SBRT), which delivers high doses in five or fewer fractions. For cancers such as pancreatic adenocarcinoma for which hypoxia varies significantly between patients, SBRT might not be optimal, depending on the extent to which reoxygenation occurs during its short duration. We used fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole positron-emission tomography (FAZA-PET) imaging to quantify hypoxia before and after 5-fraction SBRT delivered to patient-derived pancreatic cancer xenografts orthotopically implanted in mice. An imaging technique using only the pre-treatment FAZA-PET scan and repeat dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) scans throughout treatment was able to predict the change in hypoxia. Our results support the further testing of this technique for imaging of reoxygenation in the clinic.

MicroSPECT/CT Imaging of Cell-Line and Patient-Derived EGFR-Positive Tumor Xenografts in Mice with Panitumumab Fab Modified with Hexahistidine Peptides To Enable Labeling with 99mTc(I) Tricarbonyl Complex.

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His6) peptides to panitumumab Fab (PmFab) to enable labeling with [99mTc(H2O)3(CO)3]+ complex and study these radioimmunoconjugates for imaging EGFR-overexpressing tumor xenografts in mice by microSPECT/CT. Fab were reacted with a 10-fold excess of sulfo-SMCC to introduce maleimide functional groups for reaction with the terminal thiol on peptides [CGYGGHHHHHH] that harbored the His6 motif. Modification of Fab with His6 peptides was assessed by SDS-PAGE/Western blot, and the number of His6 peptides introduced was quantified by a radiometric assay incorporating 123I-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His6 in PBS, pH 7.0, with [99mTc(H2O)3(CO)3]+ in a 1.4 MBq/µg ratio. The complex was prepared by adding [99mTcO4]- to an Isolink kit (Paul Scherrer Institute). Immunoreactivity was assessed in a direct (saturation) binding assay using MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Tumor and normal tissue uptake and imaging properties of 99mTc-PmFab-His6 (70 µg; 35-40 MBq) injected i.v. (tail vein) were compared to irrelevant 99mTc-Fab 3913 in NOD/SCID mice engrafted subcutaneously (s.c.) with EGFR-overexpressing MDA-MB-468 or PANC-1 human pancreatic ductal carcinoma (PDCa) cell-line derived xenografts (CLX) at 4 and 24 h post injection (p.i.). In addition, tumor imaging studies were performed with 99mTc-PmFab-His6 in mice with patient-derived tumor xenografts (PDX) of TNBC, PDCa, and head and neck squamous cell carcinoma (HNSCC). Biodistribution studies in nontumor bearing Balb/c mice were performed to project the radiation absorbed doses for imaging studies in humans with 99mTc-PmFab-His6. PmFab was derivatized with 0.80 ± 0.03 His6 peptides. Western blot and SDS-PAGE confirmed the presence of His6 peptides. 99mTc-PmFab-His6 was labeled to high radiochemical purity (≥95%), and the Kd for binding to EGFR on MDA-MB-468 cells was 5.5 ± 0.4 × 10-8 mol/L. Tumor uptake of 99mTc-PmFab-His6 at 24 h p.i. was significantly (P < 0.05) higher than irrelevant 99mTc-Fab 3913 in mice with MDA-MB-468 tumors (14.9 ± 3.1%ID/g vs 3.0 ± 0.9%ID/g) and in mice with PANC-1 tumors (5.6 ± 0.6 vs 0.5 ± 0.1%ID/g). In mice implanted orthotopically in the pancreas with the same PDCa PDX, tumor uptake at 24 h p.i. was 4.2 ± 0.2%ID/g. Locoregional metastases of these PDCa tumors in the peritoneum exhibited slightly and significantly lower uptake than the primary tumors (3.1 ± 0.3 vs 4.2 ± 0.3%ID/g; P = 0.02). In mice implanted with different TNBC or HNSCC PDX, tumor uptake at 24 h p.i. was variable and ranged from 3.7 to 11.4%ID/g and 3.8-14.5%ID/g, respectively. MicroSPECT/CT visualized all CLX and PDX tumor xenografts at 4 and 24 h p.i. Dosimetry estimates revealed that in humans, the whole body dose from administration of 740-1110 MBq of 99mTc-PmFab-His6 would be 2-3 mSv, which is less than for a 99mTc-medronate bone scan (4 mSv).

Mobilization of Leukemic Cells Using Plerixafor as Part of a Myeloablative Preparative Regimen for Patients with Acute Myelogenous Leukemia Undergoing Allografting: Assessment of Safety and Tolerability.

Allogeneic hematopoietic cell transplantation (HCT) is potentially curative for acute myelogenous leukemia (AML); however, a major cause of treatment failure is disease relapse. The purpose of this single-center Phase I study was to determine the safety and tolerability of administration of the CXCR4 inhibitor plerixafor (Mozobil; Sanofi Genzyme) along with myeloablative conditioning in patients with AML undergoing allogeneic HCT. The rationale was that plerixafor may mobilize leukemic stem cells, making them more susceptible to the conditioning chemotherapy (registered at ClinicalTrials.gov; identifier NCT01141543). Three patients were enrolled into each of 4 sequential cohorts (12 patients total). Patients in the first cohort received 1 dose of plerixafor (240 µg/kg s.c.) before the first dose of fludarabine and busulfan, and subsequent cohorts received injections before 2, 3, and 4 days of conditioning chemotherapy. The median age at HCT was 49 years (range, 38 to 58 years). All patients engrafted following HCT, with an absolute neutrophil count ≥.5 × 109/L observed at a median of 14 days (range, 11 to 18 days). Adverse events possibly related to plerixafor were transient and not severe. Main adverse events following the injection were nausea and dizziness in 4 patients (33%) and fatigue in 4 patients (33%). Among the 12 patients, 2 patients (17%) relapsed post-HCT and 6 (50%) were alive at the last follow-up. The median follow-up of survivors was 67 months (range, 53 to 82 months). In conclusion, plerixafor administration is safe and well tolerated when included in a myeloablative conditioning regimen for allogeneic HCT for AML. Further study in a larger cohort is warranted for the investigation of the impact of plerixafor on post-allogeneic HCT outcomes.

Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers Complexed to 177Lu.

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCPs) that present 13 DOTA chelators to complex the ß-emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu (0.3-1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for γ-H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured, and absorbed doses to the cell nucleus were calculated. Normal tissue toxicity was assessed in non tumor-bearing NRG mice by monitoring body weight, complete blood cell counts (CBC), serum alanine aminotransferase (ALT), and creatinine (Cr) after i.v. injection of 6 MBq (10 µg) of panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 µg) of panitumumab-MCP-177Lu. Control mice received nonspecific human IgG-MCP-177Lu (6 MBq; 10 µg), unlabeled panitumumab (10 µg), or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177Lu. Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177Lu (6 MBq; 10 µg) to NRG mice caused no change in body weight, CBC, or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline-treated mice (TGI = 5.8 ± 0.5; P < 0.01). Unlabeled panitumumab had no effect on tumor growth (TGI = 6.0 ± 1.6; P > 0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

Comparison of Practice Guidelines, BRCAPRO, and Genetic Counselor Estimates to Identify Germline BRCA1 and BRCA2 Mutations in Pancreatic Cancer.

Germline BRCA1 and BRCA2 (BRCA) mutation carriers with pancreatic ductal adenocarcinoma (PDAC) may benefit from precision therapies and their relatives should undergo tailored cancer prevention. In this study, we compared strategies to identify BRCA carriers with PDAC. Incident cases of PDAC were prospectively recruited for BRCA sequencing. Probands were evaluated using the National Comprehensive Cancer Network (NCCN) and the Ontario Ministry of Health and Long-Term Care (MOHLTC) guidelines. The probability of each proband carrying a mutation was estimated by surveying genetic counselors and using BRCAPRO. BRCA mutations were detected in 22/484 (4.5%) probands. 152/484 (31.2%) and 16/484 (3.3%) probands met the NCCN and MOHLTC guidelines, respectively. The NCCN guidelines had higher sensitivity than the MOHLTC guidelines (0.864 versus 0.227, P < 0.001) but lower specificity (0.712 versus 0.976, P < 0.001). One hundred and nineteen genetic counselors completed the survey. Discrimination was similar between genetic counselors and BRCAPRO (area-under-the-curve: 0.755 and 0.775, respectively, P = 0.702). Genetic counselors generally overestimated (P = 0.008), whereas BRCAPRO severely underestimated (P < 0.001), the probability that each proband carried a mutation. Our results indicate that the NCCN guidelines and genetic counselors accurately identify BRCA mutations in PDAC, while the MOHLTC guidelines and BRCAPRO should be updated to account for the association between BRCA and PDAC.

Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111In and 177Lu-An EGFR-Targeted Theranostic for Pancreatic Cancer.

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average 13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing 111In or 177Lu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb), panitumumab. Because panitumumab-MCP may be dual-labeled with 111In and 177Lu for SPECT, or radioimmunotherapy (RIT) exploiting the Auger electrons or ß-particle emissions, respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC) group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by measurement of the chromophoric bis-aryl hydrazone bond formed (ε350 nm = 24 500 M-1 cm-1) to achieve two MCPs/panitumumab. Labeling of panitumumab-MCP with 111In or 177Lu demonstrated that masses as small as 0.1 µg were labeled to >90% labeling efficiency (L.E.) and a specific activity (SA) of >70 MBq/µg. Panitumumab-DOTA incorporating two DOTA per mAb was labeled with 111In or 177Lu to a maximum SA of 65 MBq/µg and 46 MBq/µg, respectively. Panitumumab-MCP-177Lu exhibited saturable binding to EGFR-overexpressing MDA-MB-468 human breast cancer cells. The Kd for binding of panitumumab-MCP-177Lu to EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-177Lu (1.0 ± 0.4 nmol/L). 111In and 177Lu were stably complexed to panitumumab-MCP. Panitumumab-MCP-111In exhibited similar whole body retention (55-60%) as panitumumab-DOTA-111In in NOD-scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine and feces. The uptake of panitumumab-MCP-111In in most normal tissues in NOD-scid mice with EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not significantly different than panitumumab-DOTA-111In, except for the liver which was 3-fold greater for panitumumab-MCP-111In. Tumor uptake of panitumumab-MCP-111In (6.9 ± 1.3%ID/g) was not significantly different than panitumumab-DOTA-11In (6.6 ± 3.3%ID/g). Tumor uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In were reduced by preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumor uptake of nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-111In. An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumor uptake via the enhanced permeability and retention (EPR) effect. Tumor uptake of panitumumab-DOTA-111In was 3.5-fold significantly higher than IgG-DOTA-111In. PANC-1 tumors were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-111In or panitumumab-DOTA-111In. Tumors were not visualized with preadministration of excess panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude that linking panitumumab to an MCP enabled higher SA labeling with 111In and 177Lu than DOTA-conjugated panitumumab, with preserved EGFR binding in vitro and comparable tumor localization in vivo in mice with s.c. PANC-1 human PnCa xenografts. Normal tissue distribution was similar except for the liver which was higher for the polymer radioimmunoconjugates.

Impact of tissue transport on PET hypoxia quantification in pancreatic tumours.

BACKGROUND: The clinical impact of hypoxia in solid tumours is indisputable and yet questions about the sensitivity of hypoxia-PET imaging have impeded its uptake into routine clinical practice. Notably, the binding rate of hypoxia-sensitive PET tracers is slow, comparable to the rate of diffusive equilibration in some tissue types, including mucinous and necrotic tissue. This means that tracer uptake on the scale of a PET imaging voxel-large enough to include such tissue and hypoxic cells-can be as much determined by tissue transport properties as it is by hypoxia. Dynamic PET imaging of 20 patients with pancreatic ductal adenocarcinoma was used to assess the impact of transport on surrogate metrics of hypoxia: the tumour-to-blood ratio [TBR(t)] at time t post-tracer injection and the trapping rate k 3 inferred from a two-tissue compartment model. Transport quantities obtained from this model included the vascular influx and efflux rate coefficients, k 1 and k 2, and the distribution volume v d ≡k 1/(k 2+k 3). RESULTS: Correlations between voxel- and whole tumour-scale k 3 and TBR values were weak to modest: the population average of the Pearson correlation coefficients (r) between voxel-scale k 3 and TBR (1 h) [TBR(2 h)] values was 0.10 [0.01] in the 20 patients, while the correlation between tumour-scale k 3 and TBR(2 h) values was 0.58. Using Patlak's formula to correct uptake for the distribution volume, correlations became strong (r=0.80[0.52] and r=0.93, respectively). The distribution volume was substantially below unity for a large fraction of tumours studied, with v d ranging from 0.68 to 1 (population average, 0.85). Surprisingly, k 3 values were strongly correlated with v d in all patients. A model was proposed to explain this in which k 3 is a combination of the hypoxia-sensitive tracer binding rate k b and the rate k eq of equilibration in slow-equilibrating regions occupying a volume fraction 1-v d of the imaged tissue. This model was used to calculate the proposed hypoxia surrogate marker k b. CONCLUSIONS: Hypoxia-sensitive PET tracers are slow to reach diffusive equilibrium in a substantial fraction of pancreatic tumours, confounding quantification of hypoxia using both static (TBR) and dynamic (k 3) PET imaging. TBR is reduced by distribution volume effects and k 3 is enhanced by slow equilibration. We proposed a novel model to quantify tissue transport properties and hypoxia-sensitive tracer binding in order to improve the sensitivity of hypoxia-PET imaging.

Imaging Mass Cytometry.

Imaging Mass Cytometry (IMC) is an expansion of mass cytometry, but rather than analyzing single cells in suspension, it uses laser ablation to generate plumes of particles that are carried to the mass cytometer by a stream of inert gas. Images reconstructed from tissue sections scanned by IMC have a resolution comparable to light microscopy, with the high content of mass cytometry enabled through the use of isotopically labeled probes and ICP-MS detection. Importantly, IMC can be performed on paraffin-embedded tissue sections, so can be applied to the retrospective analysis of patient cohorts whose outcome is known, and eventually to personalized medicine. Since the original description in 2014, IMC has evolved rapidly into a commercial instrument of unprecedented power for the analysis of histological sections. In this Review, we discuss the underlying principles of this new technology, and outline emerging applications of IMC in the analysis of normal and pathological tissues. © 2017 International Society for Advancement of Cytometry.

Activity of the novel polo-like kinase 4 inhibitor CFI-400945 in pancreatic cancer patient-derived xenografts.

BACKGROUND: Polo-like kinase 4 (PLK4) plays a key role in centriole replication. Hence PLK4 inhibition disrupts mitosis, and offers a novel approach to treating chromosomally unstable cancers, including pancreatic cancer. CFI-400945 is a first in class small molecule PLK4 inhibitor, currently undergoing early phase clinical trials. RESULTS: Treatment with CFI-400945 significantly reduced tumor growth and increased survival in four out of the six models tested. Consistent with PLK4 inhibition, we observed reduced expression of the proliferation marker Ki-67 associated with an increase in nuclear diameter during treatment with CFI-400945. Additionally, treatment with CFI-400945 resulted in a significant reduction of tumor-initiating cells. DISCUSSION: These results support the further investigation of PLK4 as a drug target in pancreatic cancer. METHODS: Sensitivity to CFI-400945 was tested in a series of six patient-derived pancreatic cancer xenografts, selected to represent the range of growth characteristics, genetic features, and hypoxia found in pancreatic cancer patients.

Effects of Combined Treatment with Ionizing Radiation and the PARP Inhibitor Olaparib in BRCA Mutant and Wild Type Patient-Derived Pancreatic Cancer Xenografts.

BACKGROUND: The BRCA2 gene product plays an important role in DNA double strand break repair. Therefore, we asked whether radiation sensitivity of pancreatic cancers developing in individuals with germline BRCA2 mutations can be enhanced by agents that inhibit poly (ADP-ribose) polymerase (PARP). METHODS: We compared the sensitivity of two patient-derived pancreatic cancer xenografts, expressing a truncated or wild type BRCA 2, to ionizing radiation alone or in combination with olaparib (AZD-2281). Animals were treated with either a single dose of 12Gy, 7 days of olaparib or 7 days of olaparib followed by a single dose of 12Gy. Response was assessed by tumour growth delay and the activation of damage response pathways. RESULTS: The BRCA2 mutated and wild type tumours showed similar radiation sensitivity, and treatment with olaparib did not further sensitize either model when compared to IR alone. CONCLUSIONS: While PARP inhibition has been shown to be effective in BRCA-mutated breast and ovarian cancers, it is less well established in pancreatic cancer patients. Our results show no radiosensitization in a germline BRCA 2 mutant and suggest that combining PARP inhibition and IR may not be beneficial in BRCA 2 related pancreatic tumors.

Biodistribution of cisplatin revealed by imaging mass cytometry identifies extensive collagen binding in tumor and normal tissues.

Imaging mass cytometry was used for direct visualization of platinum localization in tissue sections from tumor and normal tissues of cisplatin-treated mice bearing pancreas cancer patient-derived xenografts. This recently-developed technology enabled simultaneous detection of multiple markers to define cell lineage, DNA damage response, cell proliferation and functional state, providing a highly detailed view of drug incorporation in tumor and normal tissues at the cellular level. A striking and unanticipated finding was the extensive binding of platinum to collagen fibers in both tumor and normal mouse tissues. Time course experiments indicated the slow release of stroma-bound platinum, although it is currently unclear if released platinum retains biological activity. Imaging mass cytometry offers a unique window into the in vivo effects of platinum compounds, and it is likely that this can be extended into the clinic in order to optimize the use of this important class of agent.

Quantifying hypoxia in human cancers using static PET imaging.

Compared to FDG, the signal of 18F-labelled hypoxia-sensitive tracers in tumours is low. This means that in addition to the presence of hypoxic cells, transport properties contribute significantly to the uptake signal in static PET images. This sensitivity to transport must be minimized in order for static PET to provide a reliable standard for hypoxia quantification. A dynamic compartmental model based on a reaction-diffusion formalism was developed to interpret tracer pharmacokinetics and applied to static images of FAZA in twenty patients with pancreatic cancer. We use our model to identify tumour properties-well-perfused without substantial necrosis or partitioning-for which static PET images can reliably quantify hypoxia. Normalizing the measured activity in a tumour voxel by the value in blood leads to a reduction in the sensitivity to variations in 'inter-corporal' transport properties-blood volume and clearance rate-as well as imaging study protocols. Normalization thus enhances the correlation between static PET images and the FAZA binding rate K 3, a quantity which quantifies hypoxia in a biologically significant way. The ratio of FAZA uptake in spinal muscle and blood can vary substantially across patients due to long muscle equilibration times. Normalized static PET images of hypoxia-sensitive tracers can reliably quantify hypoxia for homogeneously well-perfused tumours with minimal tissue partitioning. The ideal normalizing reference tissue is blood, either drawn from the patient before PET scanning or imaged using PET. If blood is not available, uniform, homogeneously well-perfused muscle can be used. For tumours that are not homogeneously well-perfused or for which partitioning is significant, only an analysis of dynamic PET scans can reliably quantify hypoxia.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP- 1) actively binds TGFß1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFß1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFß1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFß canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFß1-induced EndMT and up-regulated TGFß signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFß1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Porphysome nanoparticles for enhanced photothermal therapy in a patient-derived orthotopic pancreas xenograft cancer model: a pilot study.

Local disease control is a major challenge in pancreatic cancer treatment, because surgical resection of the primary tumor is only possible in a minority of patients and radiotherapy cannot be delivered in curative doses. Despite the promise of photothermal therapy (PTT) for focal ablation of pancreatic tumors, this approach remains underinvestigated. Using photothermal sensitizers in combination with laser light irradiation for PTT can result in more efficient conversion of light energy to heat and improved spatial confinement of thermal destruction to the tumor. Porphysomes are self-assembled nanoparticles composed mainly of pyropheophorbide-conjugated phospholipids, enabling the packing of ∼80,000 porphyrin photosensitizers per particle. The high-density porphyrin loading imparts enhanced photonic properties and enables high-payload tumor delivery. A patient-derived orthotopic pancreas xenograft model was used to evaluate the feasibility of porphysome-enhanced PTT for pancreatic cancer. Biodistribution and tumor accumulation were evaluated using fluorescence intensity measurements from homogenized tissues and imaging of excised organs. Tumor surface temperature was recorded using IR optical imaging during light irradiation to monitor treatment progress. Histological analyses were conducted to determine the extent of PTT thermal damage. These studies may provide insight into the influence of heat-sink effect on thermal therapy dosimetry for well-perfused pancreatic tumors.

A phase I trial of the aurora kinase inhibitor, ENMD-2076, in patients with relapsed or refractory acute myeloid leukemia or chronic myelomonocytic leukemia.

ENMD-2076 is a novel, orally-active molecule that inhibits Aurora A kinase, as well as c-Kit, FLT3 and VEGFR2. A phase I study was conducted to determine the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D) and toxicities of ENMD-2076 in patients with acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Patients received escalating doses of ENMD-2076 administered orally daily [225 mg (n = 7), 375 mg (n = 6), 325 mg (n = 9), or 275 mg (n = 5)]. Twenty-seven patients were treated (26 AML; 1 CMML-2). The most common non-hematological toxicities of any grade, regardless of association with drug, were fatigue, diarrhea, dysphonia, dyspnea, hypertension, constipation, and abdominal pain. Dose-limiting toxicities (DLTs) consisted of grade 3 fatigue, grade 3 typhilitis, grade 3 syncope and grade 3 QTc prolongation). Of the 16 evaluable patients, one patient achieved a complete remission with incomplete count recovery (CRi), three experienced a morphologic leukemia-free state (MLFS) with a major hematologic improvement in platelets (HI-P), and 5 other patients had a reduction in marrow blast percentage (i.e. 11-65 %). The RP2D in this patient population is 225 mg orally once daily.