Post-treatment Biopsy Samples Research Articles

Abstract CT111: Pharmacokinetic and pharmacodynamic evaluation of NXP800, a novel GCN2 activator, in a first in human clinical trial

Abstract Background NXP800 is an oral, small molecule that activates the integrated stress response through activation of the kinase GCN2. The drug causes growth inhibition and regression in various preclinical models, including ARID1A mutated ovarian and endometrial xenograft and cholangiocarcinoma PDX models. Methods We describe results of the dose-defining dose escalation cohort and the toxicity-pharmacokinetics and pharmacodynamic evaluation in a first-in-human phase I trial of NXP800. Dose escalation was guided by a Bayesian modified continual reassessment method that targeted a dose-limiting toxicity DLT probability (DLTp) closest to 30% but <33%. Toxicity was assessed using NCI.CTC.V5. Detailed sampling of blood for pharmacokinetic analysis and blood for mRNA and protein analysis in peripheral blood mononuclear cells was conducted on C1D-3 to -7 (run in prior to continuous dosing) and C2D1. Two schedules (once per day [QD] and twice per day [BID] and a dose range of 50 to 150 mg/day were evaluated. Results Eighteen patients were treated: at baseline, the median age was 65 years (range 42-77), 9 (50%) were female and 14/18 (78%) had an ECOG score of 1. The most common treatment emergent adverse events (more than 20% of patients) were nausea, vomiting, diarrhea, fatigue, decreased appetite, AST increase and thrombocytopenia. All these side effects were grade 1-2 except grade 3 nausea and grade 3 diarrhea reported in one patient each and grade 3 thrombocytopenia in 2 patients. The MTD for the QD schedule was 100 mg (DLTp=26%), no doses in the BID schedule had estimated DLTp of <33%. The average Cmax at 50 mg and 75 mg OD was 190 nM and 384 nM during the run-in and 495 nM and 656 nM in cycle 2 reached by 3 hours. ATF4 protein in PBMCs was elevated in 57% and 80% of patients during run in and Cycle 2 respectively, 6 hours after 50 mg and 75 mg treatment. The mRNA biomarkers in blood most consistently elevated included ATF4 transcriptional targets NUPR1, ULBP1 and TRIB3 at 2 - 24 hours after 50 mg and 75 mg treatment. Further, mRNA levels for these biomarkers were elevated in post-treatment biopsy samples in one of the two patients tested. It is envisaged that NXP800 will need to be dosed continuously and although the protocol defined MTD at 28 days at 100 mg QD, it was decided to take a dose of range of 50 mg and 75 mg OD which caused biomarker modulation, forward for further evaluation. Conclusions A tolerable dose/schedule of continuous dosing of NXP800 has been established with biomarker modulation in blood. Patients will be randomized to two dose levels 50 and 75 mg/day in a QD schedule to evaluate response in a population of ARID1A mutated, platinum-resistant, clear cell ovarian cancer (NCT05226507). Citation Format: Simon Pacey, Ching Leung, Simon Rodney, Ana Filipa Palma DosReis, Ruth Ruddle, Matthew Tall, Karen Swales, Robert te Poele, Paul Workman, Diane Marsolini, Megan Sardone, Enrique Poradosu, Shay Shemesh, Udai Banerji. Pharmacokinetic and pharmacodynamic evaluation of NXP800, a novel GCN2 activator, in a first in human clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT111.

Abstract 5100: Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade

Abstract Background: Why some patients fail or have short lived response to immune checkpoint blockade (ICB) immunotherapy remains largely unknown. While baseline molecular assessments have provided clues to prognostic factors, insights into resistance drivers remains elusive. This is partially due to the difficulty in getting access to post progression samples from patients that were either primary resistant or developed acquired resistance after an initial response to ICB. Thus, the tumor-intrinsic and -extrinsic features that are selected for during progression and potentially drive primary and acquired resistance to immunotherapy remain underexplored. Methods: To compare clinical features and immunogenomic drivers of acquired and primary resistance to ICB across major cancers, we analysed and annotated de-identified patient records in the Tempus real-world database. We built an immuno-oncology cohort consisting of >2500 multimodal (DNA, RNA and clinical outcome data) pre-treatment baseline with >1500 post-treatment tumour biopsy samples from mainly NSCLC, TNBC, HNC and Bladder cancer patients. We used bulk RNA-seq data to estimate activation of the hallmark oncogenic pathways and immune cell composition and used panel DNA-seq data (>500 genes) to quantify mutation selection at the gene and pathway levels using dndscv. Results: Compared to acquired, primary resistant patients tended to have a higher observation of liver lesions at progression. Post-ICB, acquired resistant NSCLC and HNC patients showed a significantly inflamed tumour microenvironment (TME) characterised by higher estimation of infiltration of T cells and myeloid cells and higher activation of interferon gamma (IFNg) signalling as compared to primary resistant patients. In addition, in post-ICB acquired resistance in NSCLC we observed selection for mutations in genes involved in known immunomodulatory pathways, including loss-of-function mutations in B2M. Consistently, acquired resistance patients showed stronger selection for mutations in Hedgehog and Notch pathways as compared to primary resistance patients across NSCLC, HNC and TNBC post-ICB. Conclusions: Acquired and primary ICB resistant patients have distinct clinical and molecular features at progression. Their tumours’ TME is fundamentally different with acquired resistance TMEs being infiltrated with immune cells albeit escaped post progression. In addition, ICB selects mutations that potentially activate pathways such as Notch and Hedgehog. This multi-modal Real-World Data with post therapy biopsies has given insights for patient selection strategies and provides rational into combination treatment options for acquired resistant patients. Citation Format: Mohamed Reda Keddar, Sebastian Carrasco Pro, Kathleen Burke, Ana Camelo Stewart, Mark Cobbold, Ross Stewart, Sajan Khosla, Ben Sidders, Scott Hammond, Douglas C. Palmer, Jonathan Dry, Martin L. Miller. Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5100.

Dynamics and survival associations of T cell receptor clusters in patients with pleural mesothelioma treated with immunotherapy

BackgroundImmune checkpoint inhibitors (ICIs) are now a first-line treatment option for patients with pleural mesothelioma with the recent approval of ipilimumab and nivolumab. Mesothelioma has a low tumor mutation burden...

Phase 2 trial of TRC102 (methoxyamine HCl) with temozolomide (TMZ) in patients with granulosa cell ovarian cancer.

5564 Background: TRC102 is a novel small molecule that binds to reactive aldehyde created in apurinic/apyrimidinic (AP) sites, inhibiting base excision repair (BER), which is implicated as a pathway of resistance to alkylating agents. In preclinical studies, TRC102 demonstrated synergistic anti-tumor activity when TRC102 was combined with the alkylating agent temozolomide (TMZ) and the phase 1 trial of this combination reported 4 patients with partial response, two of which were of granulosa cell ovarian cancer (GCOC) histology. Here we report the phase 2 results of the combination TRC102 and TMZ in the GCOC cohort (NCT01851369). Methods: We conducted an open-label, single center Simon 2-stage trial including patients (pts) with GCOC who received ≥ one line of therapy in the metastatic setting. TRC102 was dosed at 125 mg (100 mg for BSA < 1.6) and TMZ was dosed at 150 mg/m2 orally on day (D) 1-5 in 28-day cycle (C). Restaging CT scans every 2 Cs were evaluated by RECIST v1.1. Mandatory paired biopsies (C1D1 pretreatment and C1D4 3-4 hours after drug administration) and optional blood samples for circulating tumor cells (CTC) were collected at set time points for pharmacodynamic analyses. Results: Nine pts with GCOC (7 adult, 2 juvenile histology) were enrolled as of 12/23/2022. Median age was 53 years (range 21-79) and median prior lines of therapy was 6 (range 3-9). Most common grade (G) 1 and G2 treatment-related adverse events (trAE) were fatigue, myelosuppression, nausea, and vomiting. One pt had G3 trAE of vomiting. No toxicity-related treatment discontinuations and no treatment-related deaths were reported. The median PFS for the 8 evaluable pts was 3.7 months. One pt exited the study after one C per pt’s choice with no restaging available. Four pts had stable disease (SD) as their best response. Of those who had SD, one pt completed 26 Cs prior to progression, one pt completed 11 Cs as of data cut-off but continues on study, two pts completed 6 Cs (one pt went off study for PD and one by pt choice). MGMT analysis was performed for 3 pts, including the pt still on study after 11 Cs with SD. MGMT immunohistochemistry (IHC) of pretreatment biopsies were positive for protein expression, consistent with unmethylated MGMT status. No significant induction in the levels of the DNA damage response markers γH2AX, pNbs1, and RAD51 was detected in 5 evaluable post-treatment biopsy samples as compared to the pre-treatment timepoint. Conclusions: TRC102 combined with TMZ was well-tolerated and demonstrated durable disease control in 4 pts, which is promising in this heavily pretreated GCOC cohort. MGMT analysis suggests that unmethylated MGMT status and protein expression does not preclude response to TRC102/TMZ combination therapy. Analysis of CTCs and biopsy samples are ongoing to further elucidate possible biomarkers of response. Funded by NCI Contract No. HHSN261201500003I. Clinical trial information: NCT01851369 .

Phase 2 Trial of Stereotactic Ablative Radiotherapy for Patients with Primary Renal Cancer

BackgroundMost renal cell carcinomas (RCCs) are localized and managed by active surveillance, surgery, or minimally invasive techniques. Stereotactic ablative radiation (SAbR) may provide an innovative non-invasive alternative although prospective data are limited. ObjectiveTo investigate whether SAbR is effective in the management of primary RCCs. Design, setting, and participantsPatients with biopsy-confirmed radiographically enlarging primary RCC (≤5 cm) were enrolled. SAbR was delivered in either three (12 Gy) or five (8 Gy) fractions. Outcome measurements and statistical analysisThe primary endpoint was local control (LC) defined as a reduction in tumor growth rate (compared with a benchmark of 4 mm/yr on active surveillance) and pathologic evidence of tumor response at 1 yr. Secondary endpoints included LC by the Response Evaluation Criteria in Solid Tumors (RECIST 1.1), safety, and preservation of kidney function. Exploratory tumor cell–enriched spatial protein and gene expression analysis were conducted on pre- and post-treatment biopsy samples. Results and limitationsTarget accrual was reached with the enrollment of 16 ethnically diverse patients. Radiographic LC at 1 yr was observed in 94% of patients (15/16; 95% confidence interval: 70, 100), and this was accompanied by pathologic evidence of tumor response (hyalinization, necrosis, and reduced tumor cellularity) in all patients. By RECIST, 100% of the sites remained without progression at 1 yr. The median pretreatment growth rate was 0.8 cm/yr (interquartile range [IQR]: 0.3, 1.4), and the median post-treatment growth rate was 0.0 cm/yr (IQR: –0.4, 0.1, p < 0.002). Tumor cell viability decreased from 4.6% to 0.7% at 1 yr (p = 0.004). With a median follow-up of 36 mo for censored patients, the disease control rate was 94%. SAbR was well tolerated with no grade ≥2 (acute or late) toxicities. The average glomerular filtration rate declined from a baseline of 65.6 to 55.4 ml/min at 1 yr (p = 0.003). Spatial protein and gene expression analyses were consistent with the induction of cellular senescence by radiation. ConclusionsThis clinical trial adds to the growing body of evidence suggesting that SAbR is effective for primary RCC supporting its evaluation in comparative phase 3 clinical trials. Patient summaryIn this clinical trial, we investigated a noninvasive treatment option of stereotactic radiation therapy for the treatment of primary kidney cancer and found that it was safe and effective.

Abstract 2031: High plex proteomic prognostic marker discovery for patients with pancreatic adenocarcinoma using digital spatial profiling

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) long-term outcomes following surgical resection remain poor, with only 20% of patients surviving 5 years post pancreatectomy. With its immune-privileged nature, starting from the early pre-neoplastic state, it appears to easily evade the antitumor immune response. Although there is rationale for targeting PDAC immune pathways, little benefit has been observed to date. The aim of the study was to interrogate the immune landscape of PDAC utilising the Nanostring GeoMx Digital Spatial Profiler (DSP), enabling high-plex proteomic characterisation whilst maintaining tumor microenvironment (TME) topographical features. We assessed tumor samples from 28 treatment-naive PDAC cases represented in a multiregional FFPE tissue microarray (TMA) for which extensive IHC, molecular, genomic characterisation and clinicopathological follow-up data is available. Multiplex immunofluorescent staining for nuclei, panCK, aSMA and CD3 was used to guide region selection using the GeoMx DSP system in multiple TMA cores. 60 immune markers were quantified simultaneously in different tissue compartments defined by immunofluorescence co-localization including tumor (panCK+), immune stromal (PanCK-) regions. The spatially informed assessment by DSP was validated by both regression and variable prognostication compared with IHC for stromal CD3, CD8 and CD68 in near serial TMA PDAC sections. Unsupervised analysis of proteome data (DSP) in the panCK-negative regions identified an immune poor group associated with shorter OS (13.0 versus 31.1mths, P=0.005). When transcriptomic subtype was considered, the checkpoint inhibitor B7-H3 was significantly upregulated in the squamous subtype tumours versus the classical group (Log2: 1.63, P = 0.001). Patients with high B7-H3 expression, using a median expression cut-off, were associated with shorter OS on multivariate analysis (HR: 4.16, P=0.01) a finding that was validated in an external cohort at the bulk transcriptome level. This pilot scale discovery study shows the potential of the Nanostring DSP technology to identify spatially-informed biomarkers with prognostic relevance in biopsy sized samples from treatment-naive PDAC. A number of relevant candidate immune predictors were identified in a spatial context, which are currently undergoing validation in a larger independent cohort and within the neoadjuvant setting. Future studies will apply this technology to pre and post treatment biopsy samples. Citation Format: Assya Legrini, Holly Leslie, Stephan Dreyer, Joanne Edwards, Andrew Biankin, David Chang, Nigel Jamieson. High plex proteomic prognostic marker discovery for patients with pancreatic adenocarcinoma using digital spatial profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2031.

Histological Characteristics of Port-Wine Stains with Complete Regression After Photodynamic Therapy Treatment: A 7-Year Follow-Up.

Background: Vascular-targeted photodynamic therapy (PDT) is an effective alternative treatment choice for port-wine stains (PWSs). The histological characteristics of PWSs after PDT treatment have not yet been reported. Objective: To investigate the morphological features of PWSs treated by PDT and define the histopathological characteristics of PWS that achieve clinical cure. Methods: Thirteen patients with facial PWSs, who presented with complete regressive PWS lesions after a mean of 4.38 (standard deviation = 4.907) sessions of PDT. Post-treatment biopsy samples were obtained from each patient. The number of blood vessels, vascular diameter, and depth were measured and compared in all samples of PDT-regressive sites, PDT-resistant sites, and normal skin. Results: Within the 7-year follow-up after PDT, there was no recurrence in the regression area of PDT. In the PDT-regressive sites, within 800 μm of the dermal-epidermal junction, the dilated vessels were occluded and remained fissure-like after PDT. Conclusions: When the vascular lesions within 800 μm of the dermal-epidermal junction were closed after PDT, a stable clinical cure (no recurrence) was achieved.

Spatially resolved transcriptomics deconvolutes histological prognostic subgroups in patients with colorectal cancer and synchronous liver metastases.

165 Background: Up to 50% of patients with Colorectal Cancer (CRC) will metastasise to the liver (CRLM). KRAS-mt liver metastases particularly when co-mutated with TP53 are associated with poor prognosis. We have used the Glasgow Microenvironment Score (GMS), a histological score performed on H+E slides utilising immune and stromal components of the microenvironment to robustly stratify outcome in primary CRC. The aim of the current study was firstly to determine the utility of GMS in metastatic CRC and secondly to employ the NanostringTM GeoMx Digital Spatial Profiler (DSP), a state-of-the art analysis platform enabling spatial transcriptomic characterisation while maintaining tumour microenvironment (TME) topographical features, to interrogate the functional biology underlying the GMS. Methods: FFPE specimens from primary and metastatic lesions from 44 patients undergoing synchronous resection of CRLM underwent GMS, IHC and panel genomic assessment. Primary endpoints were recurrence-free survival (RFS) and cancer-specific survival (CSS). In addition to bulk transcriptomic assessment, 4 matched pairs from the cohort were selected for GeoMx analysis: 2 samples were GMS0 (high-immune) and 2 were GMS1 (low-immune) with an equal distribution of KRASmt and wt. After multiplex IF staining (PanCK, CD45, DAPI, αSMA), 48 regions of interest were selected and Cancer Transcriptome Atlas Transcriptomic outputs (2000 genes) were analysed using Pathway enrichment analysis with immune deconvolution of the transcriptome performed. Results: GMS0 (high-immune) was associated with improved RFS (p=0.0048) and CSS (p=0.0012) remaining an independent predictor of survival on multivariate analysis (HR 2.90, 95% C.I 1.18-7.16 P=0.021). GMS0 lesions were enriched for adaptive immune (NES=2.20 p.adj&lt;0.0005) and IL-10 (NES=1.9 p.adj&lt;0.0005) pathways specifically at the invasive edge. In contrast, a poor prognostic KRAS/TP53 lesion demonstrated profound immunosuppression, upregulated NOTCH signalling (NES=2.13 p.adj&lt;0.0005) and neutrophil degranulation (NES=1.99 p.adj&lt;0.0005). Topographical Immune-cell deconvolution demonstrated significantly higher populations of CD4 (p=0.05) and CD8 (p=0.0003) cells in GMS0 leading edges. Conclusions: We have demonstrated that spatial transcriptomic analysis using the Nanostring GeoMx tool can reveal potential novel mechanisms underlying biologically relevant histological and mutational subgroups (KRAS-mt) of CRC, providing potential therapeutic targets requiring further investigation. Future studies will apply this technology to pre and post treatment biopsy samples.

High-plex proteomic prognostic marker discovery for patients with pancreatic cancer adenocarcinoma using digital spatial profiling.

611 Background: The long-term outcomes following surgical resection for Pancreatic Ductal Adenocarcinoma (PDAC) remain poor, with only 20% of patients surviving 5 years after pancreatectomy. With its immune-privileged nature, starting from the early pre-neoplastic state, it appears to escape easily from the antitumor immune response. Despite rationale for targeting immune pathways in PDAC, there has been little benefit observed at this point. The aim of the current study was to interrogate immune landscape of PDAC utilising the Nanostring GeoMx Digital Spatial Profiler (DSP), a state-of-the art analysis platform enabling Hi-plex proteomic characterisation whilst maintaining tumor microenvironment (TME) topographical features. Methods: We assessed Formalin Fixed Paraffin Embedded (FFPE) tumor samples from 28 treatment-naive PDAC cases represented in a multi-regional tissue microarray (TMA) for which extensive IHC, molecular, genomic characterisation and clinicopathological follow-up data is available. Following multiplex IHC staining for DAPI, panCK, aSMA and CD3 to guide region selection, we employed the GeoMx DSP system (NanoString) to select regions within multiple TMA cores. We quantified 60 immune markers simultaneously in multiple tissue compartments defined by immunofluorescence co-localization including (tumor [panCK+ve], immune stroma (PanCK-ve]. Data analysis was performed by a combination of DSP analysis suite and custom R pipeline. Results: The spatially informed variable assessment by DSP was validated by both regression and variable prognostication compared with IHC for stromal CD3, CD8 CD68 in near serial TMA PDAC sections. Unsupervised analysis of DSP proteome data in the panCK-negative regions identified an immune poor group associated with shorter Overall Survival (OS) (13.0 versus 31.1months, P = 0.005). When transcriptomic subtype was considered, the checkpoint inhibitor B7-H3 was significantly upregulated in the squamous subtype tumours versus the classical group (Log2: 1.63, P = 0.001). Patients with high B7-H3 expression, using a median expression cut-off, were associated with shorter OS on multivariate analysis (Hazard Ratio: 4.16, P = 0.01) including lymph node and resection margin status, a finding that was validated in an external cohort at the transcriptome level. Conclusions: This pilot scale discovery study shows the potential of the Nanostring DSP technology in the identification of spatially-informed biomarkers with prognostic relevance in biopsy sized samples from treatment-naive PDAC. We identified a number of relevant candidate immune predictors in spatial context that are currently undergoing validation in larger independent cohorts and the neoadjuvant setting. Future studies will apply this technology to pre- and post-treatment biopsy samples.

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

Safety and efficacy of AMG 714 in adults with coeliac disease exposed to gluten challenge: a phase 2a, randomised, double-blind, placebo-controlled study

Interleukin 15 (IL-15) is implicated in the pathophysiology of coeliac disease. AMG 714 is the first anti-IL-15 monoclonal antibody to be investigated for the treatment of coeliac disease. We aimed to investigate the effects of AMG 714 in patients with coeliac disease who underwent gluten challenge. This randomised, double-blind, placebo-controlled, parallel-group, phase 2a trial was done at three clinical sites in Finland. Inclusion criteria included age 18-80 years, a confirmed diagnosis of coeliac disease, and adherence to a gluten-free diet for at least 12 months before screening. Patients were randomly assigned (1:1:1) to 150 mg AMG 714, 300 mg AMG 714, or placebo using permuted blocks and stratified by study site and sex. Patients and study staff were masked to treatment assignment. Treatments were administered by two subcutaneous injections every 2 weeks for 10 weeks (total six doses). Patients without severe villous atrophy at baseline received a gluten challenge (2-4 g daily) during weeks 2-12. Small bowel biopsy samples were obtained for histological assessments at baseline and week 12. The primary efficacy endpoint was the percentage change from baseline to week 12 in villous height-to-crypt depth (VHCD) ratio. Secondary endpoints were CD3-positive intraepithelial lymphocyte density; clinical symptoms measured by gastrointestinal symptom rating scale (GSRS), coeliac disease GSRS, and Bristol stool form scale (BSFS); and changes in anti-tTG and anti-DGP antibodies from baseline. The primary analysis was done in the per-protocol 1 population of patients who received at least one dose of study drug and who underwent the gluten challenge. Safety analyses were done in all patients who received at least one dose of study drug. This trial is registered at ClinicalTrials.gov, NCT02637141 and EudraCT, 2015-003647-19. Between April 13, 2016, and Nov 22, 2016, 64 patients were enrolled and randomly assigned to either the 150 mg AMG 714 group (n=22), the 300 mg AMG 714 group (n=22), or the placebo group (n=20). Two patients did not start treatment and two did not provide post-treatment biopsy samples. 49 patients underwent the gluten challenge (per-protocol 1 population) and 11 patients did not because of baseline villous atrophy. AMG 714 did not prevent mucosal injury due to gluten challenge. The least square mean difference in the relative change from baseline in VHCD ratio was -2·49% (95% CI -16·82 to 11·83; p=0·73) between 150 mg AMG 714 and placebo and 6·39% (-7·07 to 19·85; p=0·34) between 300 mg AMG 714 and placebo. Neither comparison was statistically significant. The density of CD3-positive intraepithelial lymphocytes increased in all groups, with smaller increases in the 300 mg group (-41·24% [95% CI -79·28 to -3·20] vs placebo, nominal p=0·03) but not the 150 mg group (-14·32% [-54·39 to 25·74], nominal p=0·47). Clinical symptoms were ameliorated with AMG 714 treatment between baseline and week 12, particularly diarrhoea as measured by the BSFS (nominal p=0·01 for 150 mg vs placebo, and nominal p=0·0002 for 300 mg vs placebo). Serum antibody titres for anti-tTG and anti-DGP antibodies increased in all three treatment groups, with no significant difference between AMG 714 and placebo. Treatment-emergent adverse events occurred in 21 (95%) patients in the 150 mg AMG 714 group, 0 (95%) in the 300 mg AMG 714 group, and 19 (100%) in the placebo group. The most common treatment-emergent adverse events were gastrointestinal disorders (17 [77%] participants in the 150 mg AMG 714 group, 16 [76%] in the 300 mg AMG 714 group, and 13 [68%] in the placebo group). Injection site reactions were the most common individual adverse event, reported in eight (36%) patients in the 150 mg AMG 714 group, 11 (52%) in the 300 mg group, and five (26%) in the placebo group. No serious adverse events occurred. The primary endpoint, change in VHCD ratio from baseline after 12 weeks of treatment in patients with coeliac disease undergoing gluten challenge, was not significantly different between placebo and AMG 714 at either 150 mg or 300 mg. Effects on intraepithelial lymphocyte density and symptoms suggest that further research of AMG 714 may be warranted in patients with non-responsive coeliac disease. Celimmune and Amgen.

Genomic Profiling Identifies Outcome-Relevant Mechanisms of Innate and Acquired Resistance to Third-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy in Lung Cancer

PURPOSEThird-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are effective in acquired resistance (AR) to early-generation EGFR TKIs in EGFR-mutant lung cancer. However, efficacy is marked by interindividual heterogeneity. We present the molecular profiles of pretreatment and post-treatment samples from patients treated with third-generation EGFR TKIs and their impact on treatment outcomes.METHODSUsing the databases of two lung cancer networks and two lung cancer centers, we molecularly characterized 124 patients with EGFR p.T790M-positive AR to early-generation EGFR TKIs. In 56 patients, correlative analyses of third-generation EGFR TKI treatment outcomes and molecular characteristics were feasible. In addition, matched post-treatment biopsy samples were collected for 29 patients with progression to third-generation EGFR TKIs.RESULTSCo-occurring genetic aberrations were found in 74.4% of EGFR p.T790-positive samples (n = 124). Mutations in TP53 were the most frequent aberrations detected (44.5%; n = 53) and had no significant impact on third-generation EGFR TKI treatment. Mesenchymal-epithelial transition factor (MET) amplifications were found in 5% of samples (n = 6) and reduced efficacy of third-generation EGFR TKIs significantly (eg, median progression-free survival, 1.0 months; 95% CI, 0.37 to 1.72 v 8.2 months; 95% CI, 1.69 to 14.77 months; P ≤ .001). Genetic changes in the 29 samples with AR to third-generation EGFR TKIs were found in EGFR (eg, p.T790M loss, acquisition of p.C797S or p.G724S) or in other genes (eg, MET amplification, KRAS mutations).CONCLUSIONAdditional genetic aberrations are frequent in EGFR-mutant lung cancer and may mediate innate and AR to third-generation EGFR TKIs. MET amplification was strongly associated with primary treatment failure and was a common mechanism of AR to third-generation EGFR TKIs. Thus, combining EGFR inhibitors with TKIs targeting common mechanisms of resistance may delay AR.

Chemoradiation therapy induces in vivo changes in gene promoter methylation & gene transcript expression in patients with invasive cervical cancer.

Background & objectives:Invasive cervical cancer patients are primarily treated with chemoradiation therapy. The overall and disease-free survival in these patients is variable and depends on the tumoral response apart from the tumour stage. This study was undertaken to assess whether in vivo changes in gene promoter methylation and transcript expression in invasive cervical cancer were induced by chemoradiation. Hence, paired pre- and post-treatment biopsy samples were evaluated for in vivo changes in promoter methylation and transcript expression of 10 genes (ESR1, BRCA1, RASSF1A, MYOD1, MLH1, hTERT, MGMT, DAPK1, BAX and BCL2L1) in response to chemoradiation therapy.Methods:In patients with locally advanced invasive cervical cancer, paired pre- and post-treatment biopsies after 10 Gy chemoradiation were obtained. DNA/RNA was extracted and gene promoter methylation status was evaluated by custom-synthesized methylation PCR arrays, and the corresponding gene transcript expression was determined by absolute quantification method using quantitative reverse transcription PCR.Results:Changes in the gene promoter methylation as well as gene expression following chemoradiation therapy were observed. BAX promoter methylation showed a significant increase (P< 0.01) following treatment. There was a significant increase in the gene transcript expression of BRCA1 (P< 0.01), DAPK1 and ESR1 (P< 0.05), whereas MYOD1 and MLH1 gene transcript expression was significantly decreased (P< 0.05) following treatment.Interpretation & conclusions:The findings of our study show that chemoradiation therapy can induce epigenetic alterations as well as affect gene expression in tissues of invasive cervical cancer which may have implications in determining radiation response.

Dynamic molecular analysis and clinical correlates of tumor evolution within a phase II trial of panitumumab-based therapy in metastatic colorectal cancer

BackgroundMutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution.Materials and methodsArchival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing.ResultsThe rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n = 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, −0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes.ConclusionsThis first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored.ClinicalTrials.gov IdentifierNCT00891930

Abstract CT053: Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study

Abstract Background: CAVATAK, an oncolytic immunotherapy, is a bio-selected oncolytic strain of Coxsackievirus A21 (CVA21). Following intratumoral (IT) injection, CVA21 preferentially infects ICAM-1 expressing tumor cells, resulting in viral replication, cell lysis, and a systemic anti-tumor immune response. The Phase II CALM study investigated the efficacy and safety of IT CVA21 in pts with advanced melanoma with durable responses being observed in both injected and non-injected melanoma metastases, suggesting the generation of significant host anti-tumor responses. Pre-clinical studies in an immune-competent mouse model of melanoma revealed that combinations of intratumoral CAVATAK and anti-PD-1 or anti-CTLA-4 mAbs mediated significantly greater antitumor activity and compared to use of either agent alone. Here we report on an extension study aimed at understanding the impact of CVA21 on immune cell infiltrates and immune checkpoint molecules within treated lesions of advanced melanoma patients. Methods: In the CALM extension study a cohort of 13 advanced melanoma patients received up to 3 × 108 TCID50 CVA21 IT on study days 1,3,5 and 8 and then every three weeks for a further 6 injections. Sequential tumor biopsies of injected lesions (study days 1 and 8) were monitored for evidence of viral-induced changes immune cell infiltrates (Multi-spectral imaging) and immune checkpoint molecules (NanoString® PanCancer Immune profiling panel). Serial serum samples were monitored for viral loads, anti-CVA21 neutralizing antibody (nAb) and levels of immune-inflammatory cytokines. Results: Intratumoral CVA21 administration in a number of cases induced significant increases in immune cell infiltrates within the tumor microenvironment, in particular CD8+ cells (p = 0.044) and increased expression of PD-L1+ cells (p = 0.041) as assessed by multi-spectral imaging. Analysis of a number of pre- and post-treatment biopsy samples by NanoString® digital RNA counting identified notable increases in the levels of immune checkpoint molecules including PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3 and sizable up-regulation of a number of immune modulation elements, including Th1-gene shifts, with increases in interferon-induced genes. Of particular interest was the reconstitution of immune cell infiltrates in a number of CVA21 treated lesions from patients failing previous immune-checkpoint blockade or other immunotherapies. Conclusions: Intratumoral administration of CVA21 can notably influence the dynamics of the tumor micro-environment as evidenced by increases in both immune cell infiltrates and levels of immune-checkpoint genes. Such CVA21 induced immune-changes appear to be beneficial in combination immunotherapy, with preliminary overall response rates in a Phase 1b study CVA21 and anti-CTLA-4 blockade (ipilimumab) in advanced melanoma patients being higher than published response rates for either agent used alone. The use of CVA21 to reconstitute immune cell infiltrates in lesions resistant to immune-checkpoint blockade prior to re-commencing such treatment is an exciting novel therapeutic option. Citation Format: Robert Andtbacka, Brendan Curti, Sigrun Hallmeyer, Zipei Peng, Christopher Paustian, Carlo Bifulco, Bernard Fox, Mark Grose, Darren R. Shafren. Intratumoral coxsackievirus A21 increases immune-cell infiltrates and upregulates immune-checkpoint molecules in the tumor microenvironment of advanced melanoma patients: phase II CALM extension study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT053.

Phase II Trial of Intravenous Administration of Reolysin® (Reovirus Serotype-3-dearing Strain) in Patients with Metastatic Melanoma

Reovirus, a replication competent RNA virus, has preclinical activity against melanoma lines and xenografts. We conducted a phase II trial of reovirus in metastatic melanoma patients. Patients received 3 × 10(10) TCID50 on days 1-5 of each 28 day cycle, administered intravenously. Twenty-one eligible patients were enrolled. Treatment was well tolerated without any dose reductions having to be implemented. Post-treatment biopsy samples were obtained in 15 patients, 13/15 contained adequate tumor for correlative analysis. In two patients, productive reoviral replication (viral antigen coexpression with tubulin) was demonstrated, despite increase in neutralizing antibody titers. There were no objective responses although 75-90% tumor necrosis, consistent with treatment effect, was observed in one patient who had metastatic lesions surgically removed. Median time to progression and survival were 45 days (range 13-96 days) and 165 days (range 15 days-15.8 months) respectively. In conclusion, reovirus treatment was well tolerated in metastatic melanoma patients; viral replication was demonstrated in biopsy samples. Based on preclinical data showing synergy with taxane and platinum compounds, a phase II combination trial in metastatic melanoma patients is ongoing.

Histopathology of de novo autoimmune hepatitis

De novo autoimmune hepatitis (DAIH) is a well-recognized complication of pediatric liver transplantation (LT). The diagnosis is largely based on elevated liver function test results and the development of autoimmune antibodies. The histology of DAIH was first described in 1998. We present detailed histological data from the largest series to date of pretreatment and posttreatment biopsy samples from pediatric LT patients with DAIH. The histological evaluation included first an assessment of the predominant pattern of injury (hepatitis, rejection, or bile duct obstruction). Then, the necroinflammatory activity (interface, lobular, and perivenular), plasma cell density, rejection activity index, and fibrosis were scored. Seventy of 685 pediatric patients (10.2%) who underwent LT developed DAIH according to clinical and biopsy findings. Fifty-one pretreatment biopsy samples and 38 posttreatment biopsy samples were available for a retrospective review. The predominant pattern of injury (hepatitis, rejection, or bile duct obstruction) was determined, and biopsy samples were scored for the necroinflammatory activity (interface, lobular, and perivenular), plasma cell density, rejection activity index, and fibrosis. The most common pattern of injury was lobular hepatitis, which was frequently unaccompanied by interface necroinflammatory activity or prominent plasma cell infiltrates. Seven of the 51 cases had features strongly suggestive of acute rejection. Posttreatment biopsy samples showed a reduction in the degree of necroinflammatory activity and plasma cell infiltrates. In most patients, the degree of fibrosis was stable or had regressed. Because the histological features of DAIH are variable and nonspecific, a high index of suspicion and correlation with autoimmune antibodies are necessary to establish the diagnosis. In the majority of patients with DAIH, treatment appears to yield good clinical outcomes and histological improvements.

Abstract LB-423: Whole genome comparisons of pre- and post- aromatase inhibitor treatment in estrogen receptor positive breast cancer

Abstract Background: Estrogen receptors are over-expressed in around 70% of breast cancer cases. The genetic changes that occur during aromatase inhibitor (AI) treatment are not well understood and may differ depending upon the patient's response phenotype. Methods: We performed whole genome sequencing (WGS) of matched blood, pre-treatment, and post-treatment biopsy samples from 22 estrogen receptor positive breast cancer patients treated with neoadjuvant aromatase inhibitors. For 5 cases, we performed the whole genome sequencing (WGS) on patients’ matched normal, two pre AI-treatment, and two post AI-treatment DNA isolates from biopsy samples. We validated all putative coding and non-coding somatic mutations using deep sequencing. By comparing the validated somatic mutations from pre- and post- AI treatment biopsy samples, we were able to determine the alterations in the tumor genomes. In every case we defined the clonal architecture of each pair of pre-treatment and post-treatment biopsy samples by comparing the variant allele frequencies from thousands of validated somatic mutations. Results: Comparisons of the two pre AI-treatment biopsy samples from the same patient indicates that the variant allele frequencies of mutations showed high concordances in all 5 cases, 0.74 to 0.95 range of correlation coefficient. Only a small percentage of somatic mutations were detected in one pre-treatment sample and not the other (4.65% overall). In comparing the somatic variations between pre-treatment and matched post-treatment biopsy samples in 22 cases, we found that patients with good clinical response to AI treatment retained known driver mutations only in their pre-treatment tumors. Conversely, those patients with poor clinical response presented new driver mutations in their post-treatment samples. Furthermore, the variant allele frequency for most mutated genes decreased in post AI treatment samples for patients with good AI treatment response; on the contrary, the variant allele frequency increased for patients with poor clinical response. Conclusions: From WGS of matched normal, pre-treatment, and post-treatment biopsy samples, we identified new driver genes mutated in patients with poor clinical response, while patients with good clinical response had lost mutated driver genes in their post-treatment biopsy samples. The genetic landscape revealed by WGS of pre-treatment and post-treatment biopsy samples reveals mutational repertoires are remodeled by AI therapy. This finding suggests deep sequencing of AI treated samples will be necessary to reveal the complete complement of mutations present in a patient's tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-423. doi:1538-7445.AM2012-LB-423

Wnt pathway is affected by endocrine therapy in breast carcinomas

Background and aim: Identifying potential predictive biomarkers of response to endocrine therapy would benefit most estrogen receptor-positive (ER+) breast cancer patients. Our aim was to compare the expression of the wingless type (Wnt) pathway-related proteins (adenomatous polyposis coli [APC], E-cadherin, beta-catenin, cyclin D1, and c-myc) in postmenopausal women with ER+ invasive ductal carcinomas (IDC), prior to and after tamoxifen (n= 18) or anastrozole (n= 15) treatment, in a double-blind, placebo-controlled (n= 25), prospective study for 26 days prior to surgery. Methods: Tissue microarray blocks were constructed from pre- and post-treatment biopsy samples. Nuclear immunostaining of c-myc, APC, estrogen and progesterone receptor levels were assessed using the Allred scoring system, and cytoplasmic immunostaining of cyclin D1, beta-catenin and E-cadherin was assessed with the Hercep-Test system. An anova statistical analysis estimated general equations and analysis of variance with a significance level of 0.05. Results: Tamoxifen increased c-myc (P= 0.0061) and APC (P= 0.0452) expression. Anastrozole did not significantly affect expression of any Wnt-related proteins. Conclusions: In post-menopausal ER+ IDC, tamoxifen for 26 days prior to IDC surgery influenced statistically the expression of important cell cycle regulators as APC and c-myc, whereas anastrozole therapy did not interfere with this pathway during the same period. These Wnt related proteins may contribute to selective estrogen receptor modulator resistance.

Short-term anastrozole therapy reduces Ki-67 and progesterone receptor expression in invasive breast cancer: a prospective, placebo-controlled, double-blind trial

The objective of this study was to compare Ki-67, Bcl-2, Bax, Bak, ER, and PgR expression in postmenopausal women with ER-positive invasive breast cancer (IBC) before and after short-term hormone therapy (HT) with either tamoxifen or anastrozole in order to identify a possible biomarker profile associated with hormone resistance. Fifty-eight patients with palpable IBC were assigned to receive neoadjuvant therapy with either anastrozole, placebo, or tamoxifen for 26 days prior to surgery. Tissue microarray blocks were constructed from pre- and post-treatment biopsy samples and used for immunohistochemical analysis. Biomarker (Ki-67, Bcl-2, Bax, Bak, ER, and PgR) levels were assessed semiquantitatively using the Allred score. A statistical analysis was performed using general estimating equations (GEE) and analysis of variance (ANOVA) with a significance level of 0.05. There was a significant reduction in PgR scores from baseline (mean, 4.22) to post-treatment (mean, 1.94) in the anastrozole group, but only a non-significant trend toward an increase in PgR scores was found in the tamoxifen group. There was a significant reduction in Ki-67 scores from baseline (mean, 3.61) to post-treatment (mean, 2.56) in the anastrozole group (P = 0.01), but only a non-significant trend toward a reduction in Ki-67 scores was found in the tamoxifen group. There was a significant reduction in PgR and Ki-67 expression in the group treated with anastrozole. In the present study, the short-term HT was not associated with changes in apoptosis-related protein levels, regardless the type of drug used.