Post-treatment Specimens Research Articles

Abstract CT185: Phase I dose-escalation study of endoscopic intratumoral injection of OBP-301 (telomelysin) with radiotherapy in esophageal cancer patients unfit for standard treatments

Abstract Background: OBP-301 (telomelysin) is an attenuated type-5 adenovirus with oncolytic potency that contains the human telomerase reverse transcriptase (hTERT) promoter to regulate viral replication. OBP-301 causes selective replication and lysis of a variety of cancer cells, and a phase I study has confirmed the safety and biological activity of OBP-301 alone in patients with advanced solid tumors in the US. We have also completed the preclinical study demonstrating that OBP-301 inhibits the repair of radiation-induced DNA double-strand breaks, leading to radiosensitization. To further determine the feasibility, efficacy, and pharmacokinetics of OBP-301 in combination with radiotherapy, a clinical trial was designed in esophageal cancer patients unfit for standard treatments such as in elderly.Methods: An open-label, phase I dose-escalation study of OBP-301 with radiotherapy (UMIN 000010158) was conducted in 13 histologically confirmed esophageal cancer patients who deemed unfit to receive standard surgery or chemotherapy. Study treatment consisted of intratumoral OBP-301 injections on days 1, 18, and 32 of treatments. Radiation therapy was administered concurrently over 6 weeks, beginning on day 4, to a total of 60 Gy. Virus administration was performed by intratumoral needle injection of the primary tumor through a flexible endoscope. Patients were also monitored for virus distribution and shedding into the body fluids. The primary and secondary end points were incidence of dose-limiting toxicities and objective response rate.Results: Of 13 patients, 7, 3, and 3 patients were treated in the cohorts with 10e10, 10e11, and 10e12 virus particles (vp) of OBP-301, respectively. The patients comprised 10 males and 3 females, with median age of 79.7 years (range, 53 to 92 years). Common grade 1 and 2 toxicities included fever, esophagitis, pneumonitis, anorexia, constipation, and gastroesophageal reflux. All patients developed a transient, self-limited lymphopenia. Distribution studies revealed that viral DNA was detected in 5 out of 6 patents received with10e11 and 10e12 vp of OBP-301, but rarely in the gargle, saliva, and urine. Eight patients had local complete response (CR); all of them exhibited pathologically no viable malignant cells in biopsy specimens, and 3 had partial response. The objective response rate was 84.6%. The clinical CR rate was 80.0% in stage I and 66.7% in stage II/III, respectively. Histopathologic examination in post-treatment specimens showed massive infiltration of CD8+ cells in 3 partially responded tumors.Conclusions: Multiple courses of endoscopic OBP-301 injection with radiotherapy were feasible and provided definite clinical benefits in patients with esophageal cancer, especially who are unfit for standard treatments. Citation Format: Toshiyoshi Fujiwara, Shunsuke Tanabe, Hiroshi Tazawa, Nobuhiko Kanaya, Kazuhiro Noma, Shunsuke Kagawa, Shinji Kuroda, Yasuo Urata, Yasuhiro Shirakawa. Phase I dose-escalation study of endoscopic intratumoral injection of OBP-301 (telomelysin) with radiotherapy in esophageal cancer patients unfit for standard treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT185.

Comparison of Urine Antigen Assays for the Diagnosis of Histoplasma capsulatum Infection.

Urine antigen testing is a rapid sensitive method for detecting active infection with the endemic fungi Histoplasma capsulatum and Blastomyces dermatitidis. Herein, we compared the performance of the MiraVista Diagnostics (MVista) Histoplasma urine antigen assay with the Niche Diagnostics (ND) Histoplasma urine antigen assay for the detection of histoplasmosis and blastomycosis. Two hundred fifty urine samples from 234 patients previously tested by the MVista Histoplasma urine antigen assay as part of routine care were tested by the ND Histoplasma and Blastomyces urine antigen assays. The electronic medical records of all patients whose samples were tested were retrospectively reviewed to identify patients with a clinical diagnosis of and/or treatment for histoplasmosis or blastomycosis, and the diagnostic workup undertaken to support these diagnoses. The MVista and ND Histoplasma urine antigen assays were highly concordant, showing 99% overall agreement (90.5% positive agreement and 99.6% negative agreement). Three specimens collected after antifungal therapy returned discrepant results, with the MVista assay positive in 2 of these and the ND assay positive in 1; in each case, the antigen concentration was near the lower quantification limit. Both Histoplasma assays were positive in all patients with culture-proven blastomycosis (n = 3). The MVista and ND Histoplasma urine antigen assays performed similarly in identifying histoplasmosis cases encountered in routine clinical practice, with discrepancies affecting posttreatment specimens. Given the paucity of Blastomyces-positive samples, further studies are needed to better compare the utility of the MVista and ND Histoplasma urine antigen assays in diagnosing blastomycosis.

Optimization of Window Study Endpoints in Endometrial Cancer.

Pre-surgical window studies rely on the accurate quantification of biomarkers as surrogates of disease response. In endometrial cancer, this has traditionally involved comparing immunohistochemical expression in diagnostic endometrial biopsies with the post-treatment hysterectomy specimen. This strategy is at risk of generating erroneous results if significant hypoxia occurs during surgery or delays in fixation of tissues lead to protein loss. Immunohistochemical expression of commonly studied biomarkers in window studies were compared in pre-operative endometrial biopsies and hysterectomy specimens taken on the same day from 75 women with endometrial cancer enrolled in a clinical trial. Differences in expression were correlated with clinico-pathological variables and tissue handling. Expression of Ki-67, markers of the PI3K-Akt-mTOR, and insulin signaling pathways and hormone receptors was significantly lower in the hysterectomy specimen than the corresponding endometrial biopsy (all p < 0.0001). In contrast, expression of the cancer stem cell markers, CD133 and ALDH, were similar in the two specimens. The extent to which protein expression was lost in the hysterectomy specimen was closely correlated with baseline expression in the endometrial biopsy (all p ≤ 0.001). Bisection of the uterus prior to placement in formalin partially preserved protein expression suggesting prompt fixation is critical. These results call into question findings from earlier endometrial cancer window studies which have relied on the hysterectomy specimen for analysis and suggest a post-intervention endometrial biopsy should be included in trials going forward.

Tumor immune microenvironment (TiME) changes by multiplex IF staining in a pilot study of neoadjuvant talazoparib for early-stage breast cancer patients with a BRCA mutation.

585 Background: We previously reported a median tumor volume loss of 88% (range 30-98%) in 13 patients with early stage BRCA1/2 mutant breast cancer treated on a neoadjuvant trial of the PARP inhibitor talazoparib. The effects of PARP inhibition on immune aspects of the TiME in early-stage breast cancer has not been well described. The goal of this study was to evaluate the TiME in pre and post-treatment core biopsies from enrolled patients. Methods: Eleven paired core biopsies were available for examination. Tumor infiltrating lymphocytes (TILs) were quantified by H&amp;E stained slides by a central pathologist. Specimens were assessed by multiplex immunofluorescence (mIF) using a panel of 6 biomarkers (PD-1, PD-L1, CD3, CD8, CD68 and CK) with the Opal 7-color Kit in LEICA BOND auto stainer, Vectra automated quantitative pathology imaging system and inForm software (PerkinElmer). Results: In the analyzed core biopsies, there was an increase in TILs evaluated by H&amp;E in post-treatment compared to baseline (mean 36 vs 11%). By mIF there was an increase in CD3+ T cell and CD3+CD8+ cytotoxic T cell density in post-treatment samples compared to baseline, summarized in table. PD-L1 expression in tumor cells was rare in the cohort. There was no difference in CD3+PD-1+ or CD3+CD8+ PD-1+ lymphocytes in pre and post-treatment specimens. There was also no differences in macrophages (CD68+). Evaluation of immune phenotype and imaging response will be presented in the final analysis. Conclusions: This is the first study phenotyping the immune response to neoadjuvant talazoparib in BRCA-mutant breast cancer patients. In this small cohort, intratumoral and stromal CD3+ T cells and CD3+CD8+ cytotoxic T cells increased after two months of talazoparib. Clinical trial information: NCT02282345. [Table: see text]

A phase II randomized control trial of erlotinib in combination with celecoxib in patients with operable oral squamous cell carcinoma (OSCC): Erlo-Xib Study.

6054 Background: When combined with COX-2 inhibitors, the EGFR tyrosine kinase inhibitor Erlotinib has shown a better antitumor response in preclinical studies. Since high volume hospitals in many countries usually have a longer waiting period for surgery, neoadjuvant targeted therapy may be helpful in reducing disease progression and downstaging oral squamous cell cancers. Methods: Sixty-four treatment-naïve operable oral cancer patients were randomized into a four-arm window of opportunity study consisting of treatment with erlotinib 150mg daily, celecoxib 200mg twice daily, the combination of both or observation alone (NCT02748707). Since the regular wait period for surgery at our hospital was four to five weeks, we planned a 21-day drug treatment versus observation followed by definitive surgery in the fourth week. MRI scans and biopsies were done before and after drug treatment. Post-operative adjuvant treatments were given as per the standard guidelines used for regular patients. Results: There were 10 females and 54 males with a mean and median age of 44 and 45 years respectively. Taking a 20% reduction in the maximum tumor dimension after drug treatment (assessed clinically and radiologically) as partial response, the combination arm had a 60% partial response and a 25% stable disease. Whereas, 60% in the control arm had disease progression. The ratio of the longest tumor dimension at day 21 versus day 0 (Clinical &amp; MRI assessment) also showed a significant difference between the observation vs erlotinib arms (p &lt; 0.001) using Mann-Whitney Test. Grade II/III rashes was the commonly observed toxicity predominantly in the combination arm. Though not powered for survival analysis, a significant difference (p = 0.048) was observed for two-year overall survival for celecoxib + control (60%) versus erlotinib + combination groups (86%) using Kaplan Meier estimator. Biomarker analysis (transcriptome sequencing and IHC) is being done on pre and post-treatment tumor specimens and the final results will be presented. Conclusion: Preoperative targeted therapy with erlotinib and celecoxib combination can arrest disease progression and downstage tumors with possible impact on survival. The identified biomarkers can further refine a future cohort for effective neoadjuvant targeted therapy in oral cancers. Clinical trial information: CTRI/2012/07/002828.

Safety and immunobiological activity of guadecitabine sequenced with ipilimumab in metastatic melanoma patients: The phase Ib NIBIT-M4 study.

2549 Background: DNA hypomethylating agents show broad immuno-modulatory activity in neoplastic cells, and may improve the effectiveness of cancer immunotherapies. The phase 1b NIBIT-M4 trial investigated a previously unexplored therapeutic strategy using the next-generation DNA hypomethylating agent guadecitabine sequenced with ipilimumab for the treatment of advanced melanoma. Methods: Patients with unresectable Stage III/IV melanoma received escalating doses of guadecitabine 30, 45 or 60 mg/m2 subcutaneously on Days 1–5 every three weeks, and ipilimumab 3 mg/kg intravenously on Day 1 every three weeks, starting one week after guadecitabine, for four cycles. Primary endpoints were the safety, tolerability and maximum tolerated dose of treatment; secondary endpoints included immune-related disease control and objective response. Genome-wide methylation, RNA sequencing, and immunohistochemistry analyses were performed on tumor samples collected at baseline, W4 and W12. (NCT02608437). Results: 19 patients were treated and evaluable for safety and efficacy. The most common treatment-related adverse events of any grade were myelotoxicity (n = 17; 89%) and immune-related adverse events (n = 12; 63%). Grade 3 or 4 myelotoxicity occurred in 15 (79%) patients. There were no dose limiting toxicities. Rates of immune-related disease control and objective response were 8/19 (42%) and 5/19 (26%), respectively. Exploratory analyses of tumour samples (n = 8) showed that median CpG site methylation at Week 4 (74.5%) and Week 12 (75.5%) was significantly lower (p &lt; 0.05) than at baseline (80.3%), with a median of 2454 (Week 4) and 4131 (Week 12) differentially expressed genes identified compared to baseline; among the 136 pathways significantly modulated by treatment, the most frequently activated were immune-related. Tumour immune contexture analysis (n = 11) demonstrated up-regulation of Human Leukocyte Antigen (HLA) class I molecules on melanoma cells, and an increase in CD8+, PD-1+ T cells and in CD20+ B cells in post-treatment tumour core specimens. Conclusions: Sequential guadecitabine and ipilimumab is safe and tolerable in patients with metastatic melanoma, and has promising immunological and anti-tumour activity. Clinical trial information: NCT02608437.

Checkpoint inhibitors assessment in oropharynx cancer (CIAO): Safety and interim results.

6008 Background: Anti-PD-1/PD-L1 are active in metastatic oropharynx squamous cell carcinoma (OPC). Durvalumab (durva) and tremelimumab (tremi) target respectively PD-L1 and CTLA-4, which in combination may be synergistic. Here we report the safety and interim results of durva vs. durva+tremi prior to surgery in a window of opportunity trial in OPC. Methods: Pts were randomized 1:1 to durva 1500 mg or durva 1500 mg + tremi 75 mg IV Q4W x 2 cycles. The primary objective was to quantify pre- and post-treatment differences in CD8+ tumor infiltrating lymphocytes for the two arms. Secondary objectives included safety, toxicity, ORR by RECIST, fraction of patients undergoing surgery at 8 wks, and percentage viable tumor cells in the surgical specimen. Serial pre- and post-treatment blood and tumor specimens were collected for ongoing correlative analyses. Results: 28 pts enrolled: median age 64y, 27 (96%) male, 19 (68%) newly diagnosed, most (63%) at stage IVA (AJCC 7th Ed), 9 (32%) had locoregional recurrence, 24 (86%) p16 positive, and 22 (79%) had ≤ 10 PPY smoking history. Median follow-up was 7.6 months. The most common AEs were fatigue (36%), leukopenia/lymphopenia (25%), transaminitis (25%), and rash (21%). Grade 3 AEs occurred in 4 (14%) pts: 2 elevated lipase, 1 diarrhea, and 1 hepatitis, all were manageable. There were no grade &gt;3 AEs. ORR was 43%: 50% had SD (including 29% tumor shrinkage in 1 pt). Treatment effect in the surgical specimen was observed in 19 (79%) of 24 evaluable pts; 2 pts had major pathologic response (≤ 10% viable tumor) at the primary site. Efficacy was equivalent in both arms. The 2 pts with PD and 1 pt with SD were switched to chemotherapy after durva +/- tremi before resection; interestingly, each achieved a pCR in the primary. Most pts (57%) didn’t receive radiotherapy after surgery. There was a statistically significant association between ORR and treatment effect (p=0.014). The median percentage of viable tumor in the primary was 37.5% in pts with PR, and 82.5% in SD (p=0.003). Conclusions: Durva +/- tremi prior to surgery was well tolerated in OPC pts. Activity is encouraging with treatment effect seen in 79% of pts. The primary endpoint and complete efficacy data will be presented. Clinical trial information: NCT03144778.

Predicting breast cancer response to neoadjuvant chemotherapy based on tumor vascular features in needle biopsies.

In clinical breast cancer intervention, selection of the optimal treatment protocol based on predictive biomarkers remains an elusive goal. Here, we present a modeling tool to predict the likelihood of breast cancer response to neoadjuvant chemotherapy using patient specific tumor vasculature biomarkers. A semi-automated analysis was implemented and performed on 3990 histological images from 48 patients, with 10-208 images analyzed for each patient. We applied a histology-based model to resected primary breast cancer tumors (n = 30), and then evaluated a cohort of patients (n = 18) undergoing neoadjuvant chemotherapy, collecting pre- and post-treatment pathology specimens and MRI data. We found that core biopsy samples can be used with acceptable accuracy (r = 0.76) to determine histological parameters representative of the whole tissue region. Analysis of model histology parameters obtained from tumor vasculature measurements, specifically diffusion distance divided by radius of drug source (L/rb) and blood volume fraction (BVF), provides a statistically significant separation of patients obtaining a pathologic complete response (pCR) from those that do not (Student's t-test; P < 0.05). With this model, it is feasible to evaluate primary breast tumor vasculature biomarkers in a patient specific manner, thereby allowing a precision approach to breast cancer treatment.

Molecular expression characteristics confirm the malignancy concealed by morphological alterations in endometrial cancer after fertility-preserving treatment

Fertility-preserving treatment (FPT) has been widely used for young patients with early stage endometrial cancer (EC). However, the literature on the effectiveness and safety of FPT remains controversial. The aim of this study was to investigate malignant transformation in EC after FPT by immunohistochemistry (IHC). A retrospective analysis of pre- and post-treatment biopsy specimens from 24 patients with grade 1 endometrioid adenocarcinoma (EAC) or complex atypical hyperplasia (CAH) was performed. The expression levels of ARID1A, PTEN, and β-catenin were assessed by IHC. The protein expression levels of ARID1A, PTEN, and β-catenin were not significantly different between pre- and post-treatment specimens. However, there was a significant difference between pre-treatment and normal specimens as well as between post-treatment and normal specimens. The protein expression of β-catenin was significantly increased in patients with progression compared with those without progression after FPT. The morphologic normalization of patients with EC after FPT may not be accompanied by the absence of tumor malignancy, and β-catenin may serve as a biomarker for the response to FPT. These results may contribute to a better understanding of the malignant transformation of EC after FPT and the optimization of treatment strategies for young patients with birth plans.

Increased CD3+ T cells with a low FOXP3+/CD8+ T cell ratio can predict anti-PD-1 therapeutic response in non-small cell lung cancer patients

To determine whether tumor microenvironments affect the clinical response to anti-PD-1 therapy in non-small cell lung cancer, we investigated the expression level of PD-L1 and tumor infiltrating lymphocytes and elucidate their predictive role. Thirty-eight pretreatment and two post-treatment specimens from 36 advanced, treatment-refractory non-small cell lung cancer patients who underwent PD-1 blockade therapy were analyzed. PD-L1 expression by tumor cells and the distribution of CD3, CD8, CD4, FOXP3 and PD-1 positive tumor infiltrating lymphocytes were immunohistochemically assessed and counted using digital image analyzer. CD3+ and CD8+ T cells were distributed more in PD-L1 positive group compared to PD-L1 negative group. Conversely, EGFR mutant group showed fewer CD3+ T cells than EGFR-naïve group. The patients in the clinical benefit group with PD-1 blockade showed a higher number of CD3+, CD8+ T cells and a higher CD8+/CD3+ T cell ratio (p = 0.003, p = 0.001, and p = 0.042) and a lower FOXP3+/CD8+ T cell ratio compared to non-responders (p = 0.001). In multivariate logistic regression analysis, increased CD3+ T cell infiltration and low FOXP3+/CD8+ T cell ratio were found to be independent predictors of clinical benefit with PD-1 blockade (p = 0.014 and p = 0.03, respectively). Using receiver operating characteristic curves, levels of CD3+ T cells and FOXP3+/CD8+ T cell ratio that provide the best distinguishing point between responder versus non-responder to PD-1 blockade were 617.5/mm2 and 25%, respectively (p = 0.007 and p = 0.003). Considering that 1 mm2 is about 5 high power fields (HPF), a good response to the PD-1 blockade can be expected when the number of CD3 T cells is observed to be 120 per HPF and when CD8+ T cells and FOXP3+ T cells are present at a ratio greater than 4:1. Tumor infiltrating lymphocytes might become a promising biomarker as an independent predictive factor of response to PD-1 blockade that may also guide therapeutic decisions.

Muscle invasive bladder cancer (MIBC) demonstrates neoadjuvant cisplatin-based chemotherapy (NAC) related changes in molecular subtype and immune infiltration.

443 Background: Defining the role of MIBC molecular subtypes and immune expression in determining clinical outcomes is an area of active investigation. However, changes in these transcriptomic profiles pre- and post-NAC have not been well characterized. Methods: This retrospective study reviewed 53 pts with MIBC treated with NAC, of whom 12 pts without complete pathological response had both pre- and post-NAC samples of sufficient quality. Post-NAC staging was &gt; = pT2 in 11 pts and pT1 in 1 pt. We performed RNA expression analysis of matched pre-NAC transurethral resection of bladder tumor specimens and post-treatment radical cystectomy primary bladder tumor specimens. We used a customized NanoString panel incorporating previously reported immune signatures (Ayers, JCI 2017; O’Donnell, ASCO 2017) and additional genes to assign basal ( CD14, CD44, PDGFC, KRT14, KRT5) and luminal ( GATA3, PPARG, SHH, CD24, FOXA1, WNT7B, ERBB2) molecular subtypes. Results: We first classified the bladder cancer cohort of The Cancer Genome Atlas into basal and luminal subtypes using the BASE47 signature (Damrauer, PNAS 2014) and the NanoString panel and there was good agreement (Rand Index = 0.72). We then assigned subtypes using the NanoString panel on matched pre- and post-NAC samples and found marked subtype shift (Table). We identified two robust clusters of samples according to immune expression with a 3-fold change of immune expression between them (FDR = 0.0008). We found that 4 pts switched from the low to the high cluster, while 2 switched from the high to the low cluster after NAC (Table). Conclusions: MIBC molecular subtype membership is dynamic and is influenced by NAC. NAC can induce both enhanced and suppressed immune activity. These findings have implications on future studies exploring the predictive value of RNA expression patterns for bladder cancer therapies as well as post-NAC immunotherapy. [Table: see text]

Molecular and imaging correlates of exceptional pathologic response to neoadjuvant ADT plus enzalutamide.

61 Background: AR directed therapies are standard of care for metastatic prostate cancer, and their use for high-risk localized disease may improve survival. Previous trials showed that suppression of AR activity resulting in greater pathologic responses improved survival rates. However, the underlying biology and molecular features of exceptionally responding tumors remain unknown. Methods: Men with locally advanced prostate cancer (median Gleason score 8, median PSA 9.58 ng/dl) received mpMRI and MRI/US-fusion targeted biopsies at baseline. Patients received 6 months of ADT plus enzalutamide, then received a second mpMRI, and underwent radical prostatectomy. RP specimens were whole mounted in the same plane as MRI. The location of each tumor on biopsy was mapped to prostatectomy specimens using pathologic and imaging hallmarks. FFPE sections of baseline and post-treatment tumor were stained, laser capture microdissected, and analyzed using whole exome sequencing. Amongst nonresponding patients (residual tumor burden &gt; 0.05 cc) comparison of pre- and post-treatment tumor specimens identified evolutionary phylogenies over space and time representing intrinsic or clonal selection of pre-existing cells. Amongst responding patients, clonal extinction was confirmed by sequencing and immunostaining of surgical specimens. Results: Gleason score at baseline did not differentiate responding and nonresponding patients. Focal ERG staining in biopsies was absent in 100% of responders and present in 60% of responders. Intraductal carcinoma architecture was also absent in all responding tumors. Focal PTEN loss was observed in all nonresponders at baseline. Responding tumor foci were enriched for deletion of chromosome 6q. Comparison of baseline mpMRI sequences indicated a trend towards lower variance on dynamic contract enhanced (DCE) MR and lower local texture heterogeneity metrics in responding lesions. Conclusions: Response to neoadjuvant intense ADT correlates with distinctive pathologic, molecular, and imaging features that can be observed prior to treatment. Selection of patients based on these parameters may improve overall responses to treatment in subsequent clinical trials. Clinical trial information: NCT02430480.

Abstract P5-12-11: M2 macrophages increase after neoadjuvant HER2 targeted chemotherapy

Abstract Purpose: Recent observations suggest a positive association between the presence of tumor-associated myeloid and lymphoid immune cells and clinical responses to HER2 therapies. The specific immune cell composition of HER2+ tumors and the effects of therapy on the tumor immune microenvironment remains poorly understood; limiting efforts to effectively direct immunomodulatory agents in HER2+ breast cancer. We sought to assess the feasibility of a multiplex immunofluorescence strategy to characterize the immune milieu of HER2+ tumors, pre and post treatment. Methods: We conducted a feasibility study using the Perkin Elmer OPAL multiplex dye chemistry for immunofluorescence of tumor, myeloid, and lymphoid cells in pretreatment biopsy and surgical resection tissues of 11 patients who received neoadjuvant HER2 antibody with chemotherapy. Two panels of up to 6 antibodies were studied including a predominantly myeloid panel targeting CD80, CD68, CD163, CD206, PD-L1, and cytokeratin; and a 'lymphoid/proliferation' panel targeting FoxP3, cytokeratin, Granzyme B, CD4, CD8, and Ki67. For paired samples, analysis of pre/post differences were analyzed using two tailed, t-test. Results: 100% of the pre-treatment biopsy samples yielded high quality immune and tumor cell immunofluorescence profiles for both panels. In contrast, post treatment specimens were more challenging. For the post treatment tissues, ˜25% of specimens failed to yield results in at least one panel. As shown in Table 1, the %CD206+ M2 type macrophage population increased between pre and post treatment (p=0.012). This was reflected in a decrease in the M1:M2 ratio and variability in the ratio in favor of M2 (median 0.34 [IQR = 1.01] to 0.11 [IQR=0.10], p=0.0003). In the lymphoid panel, we observed a non-significant reduction in % FoxP3+CD4 T cells with less variability between patients post treatment and a significant decrease in total CD8+ T cells. Further, there was a significant reduction in %Granzyme B positive T cells (median 6.63 to &amp;lt;1%, p=0.02) and non-significant decrease in proliferating (Ki67+) T cells post treatment. PD-L1 expression was low in both pre and post specimens. MarkerSampleMinMedianMaxIQR%CD206+ Mono/MacPre1.358.7135.8911.05%CD206+ Mono/MacPost25.438.9777.621.54M1/M2 RatioPre0.110.342.141.01M1/M2 RatioPost0.030.110.210.1%Ki67+ T cellsPre0.137.9220.0411.06%Ki67+ T cellsPost04.5621.836.31%PD-L1+ MyeloidPre01.0512.060.95%PD-L1+ MyeloidPost00.554.261.47%Granzyme B T cellsPre2.926.6333.727.2%Granzyme B T cellsPost00.892.481.73%FoxP3+ CD4 +Pre01.6456.0416.44%FoxP3+ CD4 +Post01.166.824.14#Total CD8+ T cellsPre5788,36635,42615,744#Total CD8+ T cellsPost121,3717,9043,658 Conclusions: Multiplex immune profiling is a practical approach to characterize the tumor immune microenvironment in biopsy and post treatment specimens. Neoadjuvant HER2 targeted chemotherapy significantly shifts the myeloid population to an M2 (immunosuppressive) phenotype with evidence for a reduction in the number of Ki67 and Granzyme B+ T cells. These preliminary results suggest immunomodulatory agents that are able to induce or maintain an M1 polarized tumor microenvironment may have utility to enhance long term anti-tumor immunity in HER2 disease. Citation Format: Thompson PA, Uhlik M, Preece C, Gorden K, Harrison B, Graff J, Stopeck A. M2 macrophages increase after neoadjuvant HER2 targeted chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-11.

Abstract P3-11-01: Interaction between molecular subtype and stromal immune infiltration dynamics in breast cancer patients treated with neoadjuvant chemotherapy

Abstract Purpose: High levels of tumor-infiltrating lymphocytes (TILs) before neoadjuvant chemotherapy (NAC) are associated with higher pathological complete response (pCR) rates, and better survival in TNBC and HER2-positive breast cancers (BCs). We investigated the value of changes in TIL levels and final TIL levels after treatment, by evaluating lymphocyte infiltration before and after NAC in a real-life BC cohort. Patients and methods: We assessed stromal TIL levels in 716 pre- and post-treatment matched paired specimens, according to the guidelines of the international TIL working group. Results: Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases of residual disease (33.9% versus 20.3%, p=0.001), in luminal tumors and TNBCs, but not in HER2-positive BCs, (pInteraction =0.001). The association between pre-NAC TIL levels and pCR was non-linear in TNBCs (p=0.005). Mean TIL levels decreased during NAC (pre-NAC TILs: 24.1% versus post-NAC TILs: 13.0%, p&amp;lt;0.001). This decrease was strongly associated with high pCR rates, and TIL level variation was strongly inversely correlated with pre-NAC TIL levels (r=-0.80, p&amp;lt;0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a non-linear manner (p&amp;lt;0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in HER2-positive BCs (HR=1.04, CI [1.02-1.06], p=0.001), but not in luminal tumors or TNBCs (pInteraction =0.04). Conclusion: The associations of pre, post-NAC TIL levels with response to treatment and DFS differ between BC subtypes and may deviate from linearity. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, BC subtype, response to treatment and prognosis. Citation Format: Hamy A-S, Bonsang-Kitzis H, De Croze D, Laas E, Darrigues L, Topciu L, Menet E, Vincent-Salomon A, Lerebours F, Pierga J-Y, Brain E, Feron J-G, Benchimol G, Lam G-T, Laé M, Reyal F. Interaction between molecular subtype and stromal immune infiltration dynamics in breast cancer patients treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-01.

Abstract P3-06-01: Clonal evolution and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy

Abstract Background: Understanding to what extent a breast tumor's genetic composition may change over the course of a few months of neoadjuvant therapy has implications for optimal therapeutic approach. However, genomic changes observed across treatment may result from either treatment-induced clonal evolution or geographically disparate sampling of a heterogeneous tumor. We sought to characterize the geographic heterogeneity in primary breast tumors, and to incorporate this information into analysis of clonal evolution with neoadjuvant therapy. Methods: We assembled the largest cohort to date of multi-region (n=2-3) whole-exome sequenced (WES) or whole-genome sequenced untreated primary breast tumors with matched normal and adequate tumor purity for analysis: four tumors with data generated for this study and five tumors compiled from three previous studies. We also generated the first cohort of multi-region (n=2-6) WES breast tumors post-neoadjuvant HER2-targeted therapy and chemotherapy, sequencing one region from a pre-treatment diagnostic specimen, multiple regions from the post-treatment surgical specimen, and matched normal for five HER2+ breast tumors that did not achieve a pathologic complete response. We used an agent-based model of spatial tumor growth to investigate whether the mutational patterns we observed with treatment were consistent with pre-existing heterogeneity or treatment-induced selection. Results: In untreated primary breast tumors, on average 30% (range 1-70%) of apparently clonal mutations from a single region were absent or rare in a second, spatially disparate region (high-frequency regional, or HFR). Intra-tumor heterogeneity was similar post-treatment (HFR 28%, range 10-54%), and was higher in breast tumors than in previously analyzed colon, brain, lung, and esophageal tumors. Simulation studies confirmed that with high heterogeneity as observed in breast tumors, analysis of one pre-treatment and one post-treatment region could not distinguish treatment-induced clonal evolution from pre-existing heterogeneity; however, obtaining at least two post-treatment regions allowed for detection of clonal shifts with treatment. Analysis of multi-region data revealed that clonal replacement occurred with neoadjuvant therapy in two of the five tumors. Candidate causes of therapeutic resistance included amplifications in CCND1, ERBB4, and MYC in one subclone, and functional protein-altering mutations in ERCC2, SMO, and WT1 in another. Mathematical modeling suggested that these putative resistant subclones comprised 0.02-12.5% of the overall pre-treatment cell population, substantially larger than previous estimates of resistant tumor clone size. Conclusions: WES data from multiple regions of untreated and treated primary breast tumors revealed considerable heterogeneity that remained present throughout treatment with chemotherapy and HER2-targeted therapy, even while major clonal sweeps took place in a minority of tumors. Obtaining at least two samples for analysis from breast tumors post-neoadjuvant therapy may reveal the tumor's evolutionary path and, especially as increasing numbers of molecular and immune therapeutic targets are identified, inform new clinical strategies. Citation Format: Caswell-Jin JL, McNamara K, Reiter JG, Sun R, Hu Z, Ma Z, Suarez CJ, Tilk S, Raghavendra A, Forte V, Chin S-F, Bardwell H, Provenzano E, Caldas C, Lang J, West R, Tripathy D, Press MF, Curtis C. Clonal evolution and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-01.

Clonal replacement and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy

Genomic changes observed across treatment may result from either clonal evolution or geographically disparate sampling of heterogeneous tumors. Here we use computational modeling based on analysis of fifteen primary breast tumors and find that apparent clonal change between two tumor samples can frequently be explained by pre-treatment heterogeneity, such that at least two regions are necessary to detect treatment-induced clonal shifts. To assess for clonal replacement, we devise a summary statistic based on whole-exome sequencing of a pre-treatment biopsy and multi-region sampling of the post-treatment surgical specimen and apply this measure to five breast tumors treated with neoadjuvant HER2-targeted therapy. Two tumors underwent clonal replacement with treatment, and mathematical modeling indicates these two tumors had resistant subclones prior to treatment and rates of resistance-related genomic changes that were substantially larger than previous estimates. Our results provide a needed framework to incorporate primary tumor heterogeneity in investigating the evolution of resistance.

Endoscopic intratumoral injection of OBP-301 (telomelysin) with radiotherapy in esophageal cancer patients unfit for standard treatments.

130 Background: OBP-301 (telomelysin) is an attenuated type-5 adenovirus with oncolytic potency that contains the human telomerase reverse transcriptase (hTERT) promoter to regulate viral replication. OBP-301 causes selective replication and lysis of a variety of cancer cells, and also inhibits the repair of radiation-induced DNA double-strand breaks, leading to radiosensitization. We aimed to assess intratumoral injection of OBP-301 with radiotherapy in esophageal cancer patients unfit for standard treatments. Methods: An open-label, phase I dose-escalation study of OBP-301 with radiotherapy was conducted in 13 histologically confirmed esophageal cancer patients who deemed unfit to receive surgery or chemotherapy. Study treatment consisted of intratumoral OBP-301 injections on days 1, 18, and 32 of treatment. Radiation therapy was administered concurrently over 6 weeks, beginning on day 4, to a total of 60 Gy. Virus administration was performed by intratumoral needle injection of the primary tumor through a flexible endoscope. The primary and secondary end points were incidence of dose-limiting toxicities and objective response rate. Results: Of 13 patients, seven, three, and three patients were treated in the cohorts with 1010, 1011, and 1012 virus particles of OBP-301, respectively. The patients comprised 10 males and 3 females, with median age of 79.7 years (range, 53 to 92 years). Common grade 1 and 2 toxicities included fever, esophagitis, pneumonitis, anorexia, constipation, and gastroesophageal reflux. All patients developed a transient, self-limited lymphopenia. Eight patients had local complete response (CR); all of them exhibited pathologically no viable malignant cells in biopsy specimens, and three had partial response. The objective response rate was 84.6%. The clinical CR rate was 80.0% in stage I and 66.7% in stage II/III, respectively. Histopathologic examination in post-treatment specimens showed massive infiltration of CD8+ cells in three partially responded tumors. Conclusions: Multiple courses of endoscopic OBP-301 injection with radiotherapy were feasible and provided definite clinical benefits in patients with esophageal cancer. Clinical trial information: 000010158.

Immune Profiling of Relapsed or Refractory Multiple Myeloma Patients Treated with Pomalidomide and Low-Dose Dexamethasone in Combination with Daratumumab

Immune Profiling of Relapsed or Refractory Multiple Myeloma Patients Treated with Pomalidomide and Low-Dose Dexamethasone in Combination with Daratumumab

Participation of xCT in melanoma cell proliferation in vitro and tumorigenesis in vivo

Our research group demonstrated that riluzole, an inhibitor of glutamatergic signaling reduced melanoma cell proliferation in vitro and tumor progression in vivo. The underlying mechanisms of riluzole are largely unknown. Microarray analyses on two human melanoma cell lines revealed that riluzole stimulates expression of the cystine-glutamate amino acid antiporter, xCT (SLC7A11). Western immunoblot analysis from cultured human melanoma or normal melanocytic cells showed that xCT was significantly overexpressed in most melanomas, but not normal cells. Studies using human tumor biopsy samples demonstrated that overexpression of xCT was correlated with cancer stage and progression. To further investigate if xCT is involved in melanoma cell growth, we derived several stable clones through transfection of exogenous xCT to melanoma cells that originally showed very low expression of xCT. The elevated xCT expression promoted cell proliferation in vitro and inversely, these melanoma clones showed a dose-dependent decrease in cell proliferation in response to riluzole treatment. Xenograft studies showed that these clones formed very aggressive tumors at a higher rate compared to vector controls. Conversely, treatment of xenograft-bearing animals with riluzole down-regulated xCT expression suggesting that xCT is a molecular target of riluzole. Furthermore, protein lysates from tumor biopsies of patients that participated in a riluzole monotherapy phase II clinical trial showed a reduction in xCT levels in post-treatment specimens from patients with stable disease. Taken together, our results show that xCT may be utilized as a marker to monitor patients undergoing riluzole-based chemotherapies.

Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment.

While treatment with surgery, radiotherapy and/or chemotherapy may prolong life for patients with glioblastoma, recurrence is inevitable. What is still being discovered is how much these treatments and recurrence of disease affect the molecular profiles of these tumors and how these tumors adapt to withstand these treatment pressures. Understanding such changes will uncover pathways used by the tumor to evade destruction and will elucidate new targets for treatment development. Nineteen matched pre-treatment and post-treatment glioblastoma tumors were subjected to gene expression profiling (Fluidigm, TaqMan assays), MGMT promoter methylation analysis (pyrosequencing) and protein expression analysis of the DNA repair pathways, known to be involved in temozolomide resistance (immunohistochemistry). Gene expression profiling to molecularly subtype tumors revealed that 26% of recurrent post-treatment specimens did not match their primary diagnostic specimen subtype. Post-treatment specimens had molecular changes which correlated with known resistance mechanisms including increased expression of APEX1 (p < 0.05) and altered MGMT methylation status. In addition, genes associated with immune suppression, invasion and aggression (GPNMB, CCL5, and KLRC1) and polarization toward an M2 phenotype (CD163 and MSR1) were up-regulated in post-treatment tumors, demonstrating an overall change in the tumor microenvironment favoring aggressive tumor growth and disease recurrence. This was confirmed by in vitro studies that determined that glioma cell migration was enhanced in the presence of M2 polarized macrophage conditioned media. Further, M2 macrophage-modulated migration was markedly enhanced in post-treatment (temozolomide resistant) glioma cells. These findings highlight the ability of glioblastomas to evade not only the toxic onslaught of therapy but also to evade the immune system suggesting that immune-altering therapies may be of value in treating this terrible disease.