Pre-and Post-treatment Samples Research Articles

The heterogeneity and clonal evolution analysis of the advanced prostate cancer with castration resistance

BackgroundNowadays, the incidence rate of advanced and metastatic prostate cancer at the first time of diagnosis grows higher in China yearly. At present, androgen deprivation therapy (ADT) is the primary treatment of advanced prostate cancer. However, after several years of ADT, most patients will ultimately progress to castration-resistant prostate cancer (CRPC). Previous studies mainly focus on Caucasian and very few on East Asian patients.MethodsIn this study, the pre- and post-ADT tumor samples were collected from five Chinese patients with advanced prostate cancer. The whole-exome sequencing, tumor heterogeneity, and clonal evolution pattern were analyzed.ResultsThe results showed that the gene mutation pattern and heterogeneity changed significantly after androgen deprivation therapy. Tumor Mutational Burden (TMB) and Copy Number Alteration (CNA) were substantially reduced in the post-treatment group, but the Mutant-allele tumor heterogeneity (MATH), Socio-Demographic Index (SDI), Intratumor heterogeneity (ITH), and weighted Genome Instability Index (wGII) had no significant difference. According to the clone types and characteristics, the presence of main clones in five pre-and post-treatment samples, the clonal evolution pattern can be further classified into two sub-groups (the Homogeneous origin clonal model or the Heterogeneous origin clonal model). The Progression-free survival (PFS) of the patients with the “Homogeneous origin clonal model” was shorter than the “Heterogeneous origin clonal model”. The longer PFS might relate to MUC7 and MUC5B mutations repaired. ZNF91 mutation might be responsible for resistance to ADT resistance.ConclusionOur findings revealed potential genetic regulators to predict the castration resistance and provide insights into the castration resistance processes in advanced prostate cancer. The crosstalk between clonal evolution patterns and tumor microenvironment may also play a role in castration resistance. A multicenter-research including larger populations with different background are needed to confirm our conclusion in the future.

Exploratory analysis using cfDNA-based fragmentomics to predict disease recurrence in limited disease small cell lung cancer.

8569 Background: The current standard of care (SOC) for limited disease (LD) small cell lung cancer (SCLC) is definitive concurrent chemoradiotherapy (CCRT). Despite the high response rate to SOC, up to 70% of the patients potentially experience disease recurrence and fail to show the prolonged clinical benefit. We investigated the feasibility of cell-free DNA (cfDNA) based genomic alteration and fragmentomic analysis using pre-and post-treatment samples to investigate the predictive value of disease recurrence in LD-SCLC. Methods: The blood sample from fifty LD-SCLC who were treated with definitive CCRT were collected before and after the treatment. Target sequencing, AlphaLiquid scan (IMBdx, Korea), composed of 106 target genes, was conducted using cfDNA and peripheral blood mononuclear cells. Based on the recurrence-free survival (RFS) interval of 12 months, patients were categorized into group with persistent response (PeR, n = 29) and non-PeR (n = 21). Fragmentomics analyses were collected using the proportion of cfDNA fragments sized in P1 (100 – 155 bp) and P2 (160 – 180 bp) ranges. Results: Initial analysis was conducted based on the gene of interest. Patients with RB1 mutation (n = 11) detected in follow-up sample demonstrated significant shorter RFS compared to the RB1 wild type (WT) patients (n = 39) (7.9 mon. vs. NR, P = 0.002). Among the patients who were available for our fragmentomics analysis in follow up samples (n = 19), non-PeR (n = 9) group were significantly high in P1 range ( P = 0.003) and low in P2 range ( P = 0.002) compared to PeR group (n = 10). AUCs using the fragment proportion in P1, proportion in P2, and the fragmentation ratio (proportion in P1 / proportion in P2), were 0.889, 0.911, and 0.889, respectively. The survival analysis using fragmentation ratio showed significantly longer RFS in fragmentation ratio low group compared to the high group (7.6 mon. vs. NR, P = 0.002). Integrating RB1 mutation status with fragmentation ratio, patients with both RB1 WT and fragmentation ratio low (n = 9) showed the most favorable outcomes ( P = 0.006). On the contrary, patients with either RB1 mutated or high in fragmentation ratio (n = 10) showed median RFS of rest than 12 months. Conclusions: In this study, we investigated the clinical feasibility of cfDNA detected mutation and size of the fragment in disease recurrence of LD-SCLC using the sample collected before and after definitive CCRT. It is observed that patients with either RB1 mutation or high fragmentation ratio detected from cfDNA after the completion of CCRT are likely to experience early disease recurrence. Our findings suggest that cfDNA could provide supplementary information in predicting early treatment failure and support the clinical decision in selecting high-risk patients who might need intense monitoring and additional consolidative treatment.

Comprehensive characterization of pre- and post-treatment samples of breast cancer reveal potential mechanisms of chemotherapy resistance.

When locally advanced breast cancer is treated with neoadjuvant chemotherapy, the recurrence risk is significantly higher if no complete pathologic response is achieved. Identification of the underlying resistance mechanisms is essential to select treatments with maximal efficacy and minimal toxicity. Here we employed gene expression profiles derived from 317 HER2-negative treatment-naïve breast cancer biopsies of patients who underwent neoadjuvant chemotherapy, deep whole exome, and RNA-sequencing profiles of 22 matched pre- and post-treatment tumors, and treatment outcome data to identify biomarkers of response and resistance mechanisms. Molecular profiling of treatment-naïve breast cancer samples revealed that expression levels of proliferation, immune response, and extracellular matrix (ECM) organization combined predict response to chemotherapy. Triple negative patients with high proliferation, high immune response and low ECM expression had a significantly better treatment response and survival benefit (HR 0.29, 95% CI 0.10–0.85; p = 0.02), while in ER+ patients the opposite was seen (HR 4.73, 95% CI 1.51–14.8; p = 0.008). The characterization of paired pre-and post-treatment samples revealed that aberrations of known cancer genes were either only present in the pre-treatment sample (CDKN1B) or in the post-treatment sample (TP53, APC, CTNNB1). Proliferation-associated genes were frequently down-regulated in post-treatment ER+ tumors, but not in triple negative tumors. Genes involved in ECM were upregulated in the majority of post-chemotherapy samples. Genomic and transcriptomic differences between pre- and post-chemotherapy samples are common and may reveal potential mechanisms of therapy resistance. Our results show a wide range of distinct, but related mechanisms, with a prominent role for proliferation- and ECM-related genes.

Danofloxacin Treatment Alters the Diversity and Resistome Profile of Gut Microbiota in Calves.

Fluoroquinolones, such as danofloxacin, are used to control bovine respiratory disease complex in beef cattle; however, little is known about their effects on gut microbiota and resistome. The objectives were to evaluate the effect of subcutaneously administered danofloxacin on gut microbiota and resistome, and the composition of Campylobacter in calves. Twenty calves were injected with a single dose of danofloxacin, and ten calves were kept as a control. The effects of danofloxacin on microbiota and the resistome were assessed using 16S rRNA sequencing, quantitative real-time PCR, and metagenomic Hi-C ProxiMeta. Alpha and beta diversities were significantly different (p < 0.05) between pre-and post-treatment samples, and the compositions of several bacterial taxa shifted. The patterns of association between the compositions of Campylobacter and other genera were affected by danofloxacin. Antimicrobial resistance genes (ARGs) conferring resistance to five antibiotics were identified with their respective reservoirs. Following the treatment, some ARGs (e.g., ant9, tet40, tetW) increased in frequencies and host ranges, suggesting initiation of horizontal gene transfer, and new ARGs (aac6, ermF, tetL, tetX) were detected in the post-treatment samples. In conclusion, danofloxacin induced alterations of gut microbiota and selection and enrichment of resistance genes even against antibiotics that are unrelated to danofloxacin.

Microbiome changes in young periodontitis patients treated with adjunctive metronidazole and amoxicillin.

To our knowledge, to date, no studies have comprehensively assessed the changes occurring in the subgingival microbiome of young patients with periodontitis treated by means of mechanical and antibiotic therapy. Thus, this study aimed to use next-generation sequencing to evaluate the subgingival microbial composition of young patients with severe periodontitis treated with scaling and root planing and systemic metronidazole and amoxicillin. Subgingival samples from healthy individuals and shallow and deep sites from periodontitis patients were individually collected at baseline and 90 days post-treatment. The samples were analyzed using 16S rRNA-gene sequencing (MiSeq-Illumina) and QIIME pipeline. Differences between groups for the microbiological data were determined using principal coordinate analysis (PCoA), linear mixed models, and the PERMANOVA test. One hundred samples were collected from 10 periodontitis patients and seven healthy individuals. PCoA analysis revealed significant partitioning between pre-and post-treatment samples. No major differences in the composition of the subgingival microbiota were observed between shallow and deep sites, at baseline or at 90-days post-treatment, and the microbiome of both site categories after treatment moved closer in similarity to that observed in periodontal health. Treatment significantly improved all clinical parameters and reduced the relative abundance of classical periodontal pathogens and of Fretibacterium fastidiosum, Eubacterium saphenum, Porphyromonas endodontalis, Treponema medium, Synergistetes, TM7, and Treponema spp, and increased that of Actinomyces, Rothia, Haemophilus, Corynebacterium, and Streptococci spp. Mechanical treatment associated with metronidazole and amoxicillin promoted a beneficial change in the microbiome of young individuals with severe periodontitis.

Abstract 951: The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients

Abstract Introduction: The phase II neoadjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy (docetaxel, carboplatin) in combination with trastuzumab, trastuzumab and lapatinib or lapatinib alone in HER2+ breast cancer patients. Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor. Trastuzumab is a monoclonal antibody which targets HER2 and is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune effector cells. Studies have shown that tumour infiltrating lymphocyte (TIL) levels and the cytotoxic capacity of circulating immune cells can correlate with response to trastuzumab. Less is known regarding the effect of chemotherapy on the immune response to trastuzumab. This study examines the effects of neo-adjuvant chemotherapy on the phenotype and cytotoxic capacity of peripheral blood mononuclear cells (PBMCs) of HER2+ breast cancer patients. Methods: Matched blood samples were taken pre- and post-neoadjuvant treatment. PBMCs were isolated by density centrifugation and frozen. Direct PBMC-mediated cytotoxicity and trastuzumab-ADCC levels were assessed against the HER2+ breast cancer cell line (SKBR3) and non-MHC class I-restricted leukemic cell line (K562) using a FACS based assay (n=19 matched pre-and post-treatment samples). The immunophenotype of 17 pre-treatment and 13 post-treatment samples was also determined using the DURAClone IM antibody panel (CD16, CD56, CD19, CD14, CD4, CD8, CD3, CD45) on the CytoFLEX platform. Analysis of both data sets was performed using FCS Express and MedCalc. Results: The cytotoxic capacity of PBMCs was reduced following neo-adjuvant chemotherapy. Direct cytotoxicity elicited against the K562 cell line was reduced post-treatment (p=0.04). ADCC elicited against the SKBR3 cell line was also decreased in post-treatment samples (p=0.009). A decrease in cytotoxic capacity was associated with alterations in the immunophenotype of the post-treatment PBMC samples. The proportion of CD56+ NK cells (p=0.001), CD19+ B cells (p=0.001) and CD14+ monocytes (p=0.03) decreased significantly post-treatment. CD56+ NK cells are hypothesised to be the main ADCC effector cell population in peripheral blood. Interestingly, the proportion of CD3+ T cells (p=0.002), including CD4+ (p=0.007) and CD8+ (p=0.035) T cell populations, were increased post-treatment. Conclusion: These results indicate that neo-adjuvant chemotherapy reduces the cytotoxic capacity of circulating immune cells and alters the proportion of adaptive and innate immune cell subsets. These effects warrant further investigation particularly with the emergence of immunotherapies in HER2+ breast cancer. Citation Format: Nicola Gaynor, Alfonso Blanco, Alexandra Canonici, Alexander J. Eustace, Martina McDermott, Barry Moran, Jean M. Fletcher, Norma O'Donovan, John Crown, Denis M. Collins. The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 951.

Abstract P6-03-01: Mapping a personalized chemo-resistome in breast cancer patients by longitudinal transcriptomics

Abstract Background: Understanding resistance mechanisms to chemotherapy is key to improving therapeutic outcomes. Despite the considerable importance of tumor drug resistance to cancer morbidity and mortality, our comprehension of the various molecular mechanisms involved in resistance is limited. The actual response of an individual patient remains a ‘black box’. Previous studies profiling pre-and post-treatment samples were based on population statistics and did not result in a personalized view of resistance. To dissect the individualized emergence of resistance in breast cancer patients we applied longitudinal transcriptomics combined with temporal dynamics analysis approach. Methods: Matched triplets of archived tumor biopsies from pre-treatment, post-treatment and adjacent normal epithelium were collected from 33 individual patients that underwent neo-adjuvant chemotherapy treatment. Full transcriptome analysis was performed by mRNA sequencing. Longitudinal pattern analysis algorithm was developed to follow dynamic expression fluctuations in individual patients. Data analysis incorporated long-term clinical and pathological follow-up information. Pathway enrichment was used to map the resistant pathways and create a “chemo-resistome” map in individual patients by following the rewiring of their molecular pathways through the course of therapy. Results: To identify genes associated with resistance we used longitudinal pattern classification. Each pattern represents a different scenario through tumor progression and treatment stages. We identified 253 genes that their pattern is significantly correlated with pathological response score. Enrichment analysis of these genes pinpointed pathways and functions associated with resistance. We found multiple pathways directly related to the mechanism of action of the administered chemotherapies, such as, pathways involved in microtubule polymerization and DNA repair. Other pathways that emerge involve multi-drug resistance pathways, such as, specific subsets of ABC transporters and pathways related to immune-modulation. Interestingly, we noticed that the mechanisms of resistance are patient-specific. We, therefore, calculated a chemo-resistome map for each patient using the most potent resistant pathway categories. The chemo-resistome maps illustrate the co-existence of several resistance categories in the same patient, whereas some categories exhibit patient- or subtype- specific occurrence. Conclusions: Mapping the complexity of the various resistance pathways in individual patients can provide important insights on the mechanisms underlying tumor cell survival. Depicting an individual road map to resistance by analyzing expression rewiring can offer personalized therapeutic strategies in the future. Citation Format: Maya Dadiani, Gilgi Friedlander, Gili Perry, Nora Balint-Lahat, Anya Pavlovsky, Anjana Shenoy, Iris Barshack, Tamar Geiger, Bella Kaufman, Einav N Gal-Yam. Mapping a personalized chemo-resistome in breast cancer patients by longitudinal transcriptomics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-01.

Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma

Abstract Background: CMP-001 comprises a CpG-A oligodeoxynucleotide packaged within a virus-like particle. It is designed to activate tumor-associated plasmacytoid dendritic cells via TLR9 inducing an interferon-rich tumor microenvironment and anti-tumor CD8+ T cell responses. Materials and Methods: CMP-001-001 is an ongoing phase Ib trial evaluating intratumoral (IT) CMP-001 in combination with pembrolizumab (administered per label) in subjects with advanced melanoma resistant (either did not respond or progressed) on prior anti-PD-1 monotherapy or in combination. During dose escalation, subjects were enrolled to cohorts of ≥ 3 subjects at CMP-001 doses of 1, 3, 5, 7.5, and 10 mg in two dosing schedules (weekly for 7w, followed by q3w; or weekly for 2w, followed by q3w). CMP-001 was administered IT into an accessible lesion(s), and response assessed in all target lesions (injected and non-injected) by RECIST v1.1. Study therapy was continued until progression, toxicity, investigator decision or withdrawal of consent. Baseline and on-therapy serum was collected for cytokine analysis. Immunohistochemical and RNA-Seq analysis was performed on available pre- and post-treatment tumor biopsies. Results: As of December 31, 2017, 68 subjects have been treated (44 in Escalation and 24 in Expansion). Safety data from 63 subjects demonstrated a manageable acute toxicity profile consisting predominately of fever, N/V, headache, hypotension and rigors. Grade 3/4 related AEs reported in ≥1 subject; hypotension (n=7), anemia (n=2), chills (n=2), hypertension (n=2) and fever (n=2). The Objective Response Rates (ORR) across all dose cohorts on weekly (n=40) and q3week schedules (n=13) were 22.5% (9/40; 95 % CI 11-39%) and 7.7%% (1/13; 95% CI 0-36%) respectively. For subjects dosed weekly at 3 and 5 mg, the ORR was 33.3% (6/18 95% CI 13-59%). Of the 10 responders, 1 progressed (w36), 2 withdrew consent (w13, w25), 7 remain on study with 2 subjects maintaining their response though w72. Regression of non-injected tumors occurred in cutaneous, nodal, hepatic, and splenic metastases. CMP-001 induced TLR9 activation with a median 5.9 fold increase in serum CXCL10 (range of 0.9 - 276.3; mean fold increase of 21.8 with SD=48.8; n=39). Immunohistochemical and RNA-Seq analysis of tumor biopsies revealed increases in tumor-infiltrating CD8 T cells (&amp;gt;5 fold), PD-L1 expression (&amp;gt;3 fold increase in H score), and transcriptional signature of inflammation in 2/4 subjects with analyzable pre-and post-treatment samples. Conclusions: CMP-001 in combination with pembrolizumab resulted in objective, durable tumor responses with tolerable toxicities in subjects with advanced melanoma resistant to prior anti-PD-1 therapy. CMP-001 dosing at 5 mg/weekly has been selected for further evaluation in the ongoing dose expansion phase of this study. Citation Format: Mohammed Milhem, Rene Gonzales, Theresa Medina, John M. Kirkwood, Elizabeth Buchbinder, Inderjit Mehmi, Jiaxin Niu, Montaser Shaheen, Ryan Weight, Kim Margolin, Jason Luke, Aaron Morris, David Mauro, Arthur M. Krieg, Antoni Ribas. Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT144.

Effect of intracanal medicaments on matrix metalloproteinase-9 and vasoactive intestinal peptide secretion in periapical lesions of re-treated canals: a randomized controlled clinical study.

The aim of this study was to evaluate the effect of calcium hydroxide (Ca[OH]2) and chlorhexidine (CHX) gel on matrix metalloproteinase-9 (MMP-9) and vasoactive intestinal peptide (VIP) secretion in periapical lesions. A total of 60 patients were randomly divided into two groups that were to receive different medications. Pre-and post-treatment samples were collected from the interstitial fluid of periapical lesions using sterile paper points. VIP and MMP-9 levels were measured by enzyme-linked immunosorbent assay kits, and the data were statistically analyzed. Gender and smoking habits had no effect on the pre- and post-treatment VIP and MMPs levels. Intragroup analyses revealed that in the Ca(OH)2 group, the post-treatment VIP level was found to be significantly higher than the pre-treatment VIP level. In the CHX group, the post-treatment MMP-9 level was significantly higher than the pre-treatment MMP-9 level. According to the results of the present study, the type of the medication affected the amount of periapical VIP and MMP-9 secretion. VIP is a neuropeptide that promotes new bone formation. Thus, intracanal Ca(OH)2 medication may accelerate the repair process of bone tissue.

Abstract PD8-01: CDH1 mutated classic and pleomorphic invasive lobular breast carcinomas differ in genomic signatures and opportunities for targeted and immunotherapies

Abstract Background: Typically defined by negative IHC staining for E-cadherin, classic (CILC) and pleomorphic (PILC) are often combined as a single breast cancer subtype. We queried whether patients with relapsed metastatic disease, mCILC and mPILC, would harbor contrasting genomic alterations (GA)and that molecular information could further differentiate the 2 tumor types and thereby influence therapy selection. Methods: DNA was extracted from 40 µm of FFPE sections of 10,784 invasive breast carcinomas. 454 (4%) CDH1 mutated mILC were selected including 428 classic mCILC (94%) and 26 mPLIC (6%) subtypes. Comprehensive genomic profiling (CGP) was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth &amp;gt;600X for up to 315 cancer-related genes. Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA. Results: mCILC and mPILC patients featured a median age of 63 years (Table). Slide based ER+ status and HER2+ status was significantly different in both groups (P&amp;lt;0.0001). The frequency of base substitutions in ESR1 was significantly higher in mCILC, and this difference was also significantly higher in mCILC metastasis biopsies exposed to hormonal therapy than in pre-treatment primary tumors (P&amp;lt;0.0001). ERBB2 (HER2) GA (amp + non-amp) detected by CGP were higher in mPILC than mCILC in both pre-and post-treatment samples (P&amp;lt;0.0001 for both). The ERBB2 GA frequency was nearly twice as high after hormonal therapy in both mCILC and mPILC. ESR1 and ERBB2 GA were mutually exclusive overall and especially in the mCILC group. PIK3CA GA were the most frequent GA in both mCILC and mPILC. TP53 GA were significantly more frequent in mPILC than mCILC. At 19%, the frequency of TMB &amp;gt; 15 mutations/MB in mPILC was more than twice as frequent than in mCILC (P=0.046). All (100%) of both the CILC and PILC groups were negative for mis-match repair deficiency or MSI high status. mCILC and mPILC patients with post primary therapy associated ESR1 and ERBB2 GA responding to targeted and immunotherapies will be presented. Contrasting Clinical and Genomic Features of CILC and PILC Classic CILC (428 cases)Pleomorphic PILC (26 cases)Median Age6363*ER+98%74%*HER2 IHC/FISH+12 (3%)6 (22%)ESR1 GA Primary Pre-Rx6%0%ESR1 GA Metastatic Post-Rx17%0%ERBB2 GA Primary Pre-Rx7%18%ERBB2 GA Metastatic Post-Rx12%34%Other Significant GAPIK3CA (55%), CCND1 (21%), TP53 (17%), ARID1A, AKT3, MDM4, PTEN (all 11%)PIK3CA (58%), TP53 (30%), AKT1 22%), FGFR4, CCND1, PTEN (all 17%)TMB median (mut/Mb)2.73.6TMB &amp;gt; 15%8%19%*when clinical status available Conclusions: CGP of mCILC and mPILC reveals significant differences in the panorama of GA both in pre-treatment primary and metastatic disease lesions especially in therapy-impacting GA in ESR1 and ERBB2. mCILC is more often driven by ESR1 GA and mPILC by ERBB2 GA. Although both mCILC and mPILC feature subsets of tumors with high TMB, this is more frequent for mPILC likely indicating different potentials for immunotherapies to benefit these patients. Citation Format: Ross JS, Chung J, Elvin JE, Vergilio J-A, Ramkissoon S, Suh J, Severson E, Daniel S, Frampton GM, Fabrizio D, Hartmaier RJ, Albacker LA, Ali SM, Schrock AB, Miller VA, Stephens PJ, Gay LM. CDH1 mutated classic and pleomorphic invasive lobular breast carcinomas differ in genomic signatures and opportunities for targeted and immunotherapies [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-01.

Abstract A50: Immunomonitoring in a phase II/III trial of therapeutic vaccination with CSF-470 plus BCG plus GM-CSF versus IFN-alfa in patients with cutaneous melanoma

Abstract Melanoma is the deadliest form of skin cancer. Once it has metastasized, treatment options are limited and prognosis is bleak. Immunotherapy is one of the current promising treatments for patients (pts) after surgery, but markers for prediction of immune response are still lacking. In a phase II/III randomized clinical trial (CASVAC-0401, NCT01729663), cutaneous melanoma pts with Stages IIB, IIC and III were randomized to receive CSF-470 versus medium-dose IFN-alpha in a 2:1 ratio. The duration of the study is two years, during which the vaccinated pts would receive 13 vaccinations. CSF-470 vaccine is a mini-allograft consisting of four lethally-irradiated allogeneic cutaneous melanoma cell lines, combined with BCG and rhGM-CSF as adjuvants with the goal of stimulating adaptive and innate immunity. Thirty one pts have been included so far in the study; 20 pts have been randomized to the vaccine arm and 11 to the IFN-alfa arm. The DMFS (distant-metastasis-free-survival) is significantly longer for pts in the CSF-470 arm than in those of the IFN-alfa arm (p=0.034, Gehan-Breslow-Wilcoxon Test); besides, quality of life is significantly better in the CSF-470 arm. Looking for parameters involved in the immune system stimulation in vaccinated pts, DTH (Delayed-Type Hypersensitivity) and lymphocyte subsets were periodically evaluated. A significant increase in the DTH response between NED (non evident disease) and PRO (progressed) was observed in vaccinated pts (p=0.0362, unpaired t test with Welch's correction). In order to investigate if the increase in DTH was accompanied by changes in lymphocyte peripheral populations, we investigated changes between pre-and post-treatment samples from 24 enrolled pts, 16 from the vaccine arm and 8 from the IFN-alfa arm. We did not observe significant differences between PRE and POST1 samples (6 months; five vaccinations) in T CD4+, CD8+ or regulatory cells, neither in vaccinated nor in IFN-alfa treated pts. Furthermore, we did not detect any changes in the frequencies of T CD8+ specific clones against MART-1 or gp100 Ags after treatment in HLA*0201 pts. Instead, we observed that only vaccinated pts increased the percentage of Natural Killer (NK) in the POST1 sample (PRE 16.6±8.0% vs POST1 22±12.4% p=0.0106, paired t test). Given these observations, we investigated, using a degranulation assay after challenge with live melanoma cell lines or K562 cells, if NK cells functionality was also augmented. Nevertheless, we did not see significant differences in NK cell activity after vaccination or IFN-alfa treatment. In contrast, we observed significant increases in antibody (Ab) responses against melanoma cells in vaccinated pts sera, that was not observed in IFN-alfa treated pts, as determined by ELISA (vaccine arm: PRE 0.3±0.2 vs POST1 0.8±0.3; p&amp;lt;0.0001, paired t test; IFN-alfa arm: PRE 0.4±0,2 vs POST1 0.4±0,3; p&amp;gt;0.05, paired t test). Anti-melanoma Abs increased both in NED and in PRO vaccinated pts. In conclusion, we suggest that the increase in circulating NK cells that coexist with anti-melanoma Abs could contribute to eliminate circulating tumor cells, thus hampering further metastatic dissemination. Also, since DTH was clearly increased in responding vaccinated pts, we suggest that lymphocyte subsets located in peripheral tissues, which give rise to DTH reactivity, would more closely reflect clinical responses and should receive closer examination than circulating lymphocytes populations. Efforts to understand the impact of CSF-470 in the pts´ immune system are needed to identify the effectors involved in clinical responses. Citation Format: María Betina Pampena, Estrella Mariel Levy, María Paula Blanco, María Marcela Barrio, José Mordoh. Immunomonitoring in a phase II/III trial of therapeutic vaccination with CSF-470 plus BCG plus GM-CSF versus IFN-alfa in patients with cutaneous melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A50.

Effects of narrow-band ultraviolet B on the expressions of thymic stromal lymphopoietin, interleukin-25 and their receptors in peripheral blood of patients with atopic dermatitis

Objective To explore the effects of narrow-band ultraviolet B (NB-UVB) on the serum levels of thymic stromal lymphopoietin (TSLP) and interleukin-25 (IL-25), as well as on the expressions of TSLP receptor (TSLPR) and IL-25 receptor (IL-25R) mRNAs in peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis (AD). Methods A total of 40 patients with AD and 30 healthy volunteers were enrolled in this study. All the patients were treated with NB-UVB at 0.3-2.5 J/cm2 thrice a week for 12 consecutive weeks. Venous blood samples were obtained from these patients before and after the treatment as well as from these healthy controls. Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect serum levels of TSLP and IL-25, and reverse transcription PCR (RT-PCR) to determine the mRNA expression levels of TSLPR and IL-25R in PBMCs from these subjects. The scoring atopic dermatitis (SCORAD) system developed by the European Task Force on Atopic Dermatitis was used to estimate the severity of AD, and visual analogue scale (VAS) to evaluate the degree of itch. Statistical analysis was carried out by the two-independent-sample t-test for intergroup comparisons and paired t-test for comparisons between pre-and post-treatment samples from these patients. Results After the treatment with NB-UVB, the total response rate reached 75% (30/40) in these patients, with a significant decrease in SCORAD score from 55.26±10.88 before the treatment to 20.36±5.12 after the treatment (t=10.29, P 0.05). Conclusions TSLP and IL-25 may play important roles in the development of AD, and NB-UVB may treat AD by downregulating the expressions of them and their receptors. Key words: Dermatitis, atopic; Ultraviolet rays; Thymic stromal lymphopoietin; Interleukin-25

Serum Haptoglobin Concentration to Monitor Recovery from Postpartum Sub-Clinical Endometritis in Murrah Buffaloes

A total of 150 postpartum apparently healthy buffaloes were screened. Out of these 30 buffaloes were found to be positive for sub-clinical endometritis were selected and divided into five groups. The group wise treatment allotted were: Group I (cloprostenol,), Group II (cloprostenol + benzathine cephapirin, single I/U infusion), Group III (100 ig E. coli LPS, single I/U infusion), Group IV (500 mg Oyster glycogen, single I/U infusion) and Group V (0.25% Lugol's iodine 20 ml, single I/U infusion). All the animals were subjected to trans-rectal ultrasonography, endometrial cytology, microbial assay and blood sampling for serum haptoglobin concentration before and after treatment. Total viable bacteria count revealed non-significant (p > 0.05) difference within pre-and post-treatment samples between different treatment groups. Post-treatment total viable bacteria count significantly (p < 0.05) reduced to zero in treatment groups III and IV. Out of 30 pre-treatment uterine lavage samples obtained from all the treatment groups, 25 (83.33%) samples were found positive. E. coli 08 (30.76%) was highly prevalent followed by 07 (26.92%) Staphylococcus spp., 03 (11.53%) Streptococcus spp., 03 (11.53%) Proteus spp., 03 (11.53%) Acinetobacter spp. and 01 (07.69%) Bacillus spp. Serum haptoglobin concentration in different treatment groups ranged from 76.62 ± 1.58 to 85.83 ± 2.12 ig/ml prior to treatment and 26.37 ± 0.86 to 42.57 ± 9.08 ig/ml post-treatment. Significant reduction (p < 0.05) was observed in haptoglobin concentration between pre-and post-treatment values in all the treatment groups. It was concluded that assessment of haptoglobin concentration in sub-clinical endometritic postpartum buffaloes can be used to monitor course of treatment at different points of time.

S3-4: ER Downregulation with Fulvestrant in Combination with pan-PI3K Inhibitor BKM120 Synergizes Against ER+/PI3K-Mutant Breast Cancer Xenografts In Vivo.

Abstract ER+ breast cancers typically respond to aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To discover mechanisms of resistance to AIs, we maintained four ER+, hormone-dependent human breast cancer cell lines in hormone-depleted conditions for several months until estrogen-independent populations emerged (termed long-term estrogen-deprived, LTED). A siRNA kinome screen identified 42 kinases that suppressed hormone-independent MCF-7/LTED cell growth ≥33% (p&amp;lt;0.05). Pathway analysis revealed phosphatidylinositol 3-kinase (PI3K) as a central hub which integrates signaling from, or emits signaling to many of these 42 kinases. Further, treatment with the PI3K/mTOR dual inhibitor BEZ235 or the pan-PI3K inhibitor BKM120 suppressed LTED cell growth, and prevented the emergence of hormone-independent cells. Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are present in about 35% of ER+ breast cancers. We investigated whether PIK3CA mutations modify the tumor response to estrogen deprivation with letrozole in a pre-surgical clinical trial (NCT00651976). Twenty-one patients with stage I-II operable ER+/HER2− breast cancer were treated with letrozole for 10–21 days up to 24 h prior to surgery. Core tumor biopsies were obtained before treatment and at the time of surgical resection. Ki67 immunohistochemistry (IHC) was scored in pre- and post-letrozole specimens. The change in natural log of the Ki67 score between pre-and post-treatment samples was stratified by PIK3CA mutant vs. wildtype (WT) status. PIK3CA-mutant tumors exhibited a statistically lower reduction in Ki67 score compared to tumors with WT PIK3CA (p=0.03). These findings suggest that mutational activation of PI3K attenuates the response to estrogen deprivation. In some LTED cell lines, downregulation of ER with fulvestrant or siRNA inhibited estrogen-independent growth, suggesting that the unliganded ER can maintain proliferation of cells adapted to estrogen deprivation. Prior work suggested that PI3K-ER crosstalk promotes resistance to estrogen deprivation. However, inhibition of PI3K/AKT/TORC1 signaling did not alter ERS167 phosphorylation in LTED cells, and ER downregulation with fulvestrant did not inhibit PI3K/AKT, suggesting that PI3K and ER modulate non-overlapping pathways in cells adapted to estrogen deprivation. Treatment with BKM120 synergized with fulvestrant to inhibit the growth of 6/8 ER+ breast cancer cell lines in estrogen-depleted conditions. Treatment with single-agent fulvestrant or BKM120 in athymic mice devoid of estrogen supplementation partially inhibited the growth of ER+/PI3K-mutant MCF-7 xenografts, while the combination induced near-complete tumor regression. IHC analysis showed increased cleaved caspase-3+ cells in BKM120-treated tumors and decreased Ki67+ cells in fulvestrant-treated tumors, implying that the combination induced tumor regression by both inhibition of tumor cell proliferation and induction of apoptosis. These data suggest that upon progression on an aromatase inhibitor, patients with ER+/PI3K-mutant breast cancer would benefit from a combination of an ER downregulator and a PI3K pathway inhibitor. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-4.

Abstract P4-05-05: Stromal Response to 14-Day Preoperative Therapy in Postmenopausal Oestrogen Receptor Positive Breast Cancer

Abstract Background: Stromal-epithelial interaction is a key factor in tumour progression. Cancer-associated fibroblasts (CAFs) and macrophage infiltration have been associated with early relapse in breast cancer. Bisphosphonates are effective inhibitors of osteoclast activation in metastatic breast cancer but also have a general inhibitory effect on breast cancer progression. In order to monitor a potential tumour stromal response in breast cancer during treatment with an aromatase inhibitor and a bisphosphonate we analysed pre-and post-treatment samples from a neoadjuvant window study and focused on the presence of macrophages and CAFs. Materials and methods: Tissue microarrays (TMAs) from surgical samples and pre-operative core biopsies were immunohistochemically stained for aSMA (CAF marker), CD68 (macrophages) and epithelial proliferation (Ki67). In order to validate if the presence of macrophages and aSMA could be monitored by the TMA approach, we initially analysed a screening cohort of 144 breast cancer samples. We then studied pre-and post-treatment samples from 110 postmenopausal ER-positive invasive breast cancer patients randomised to receive 14 days of preoperative treatment (placebo, Letrozole, or Letrozole plus Zoledronate). Results: In the screening cohort, we observed significant links between aSMA positive fibroblasts and disease recurrence as well as between CD68 positive macrophages and tumour size, grade, lymph node positivity and recurrence. This validated the use of TMAs for stromal analyses and furthersupported a link with key tumour biological events. In both treatment arms, there was a significant drop in absolute Ki67 value compared to placebo (-9.3% Letrozole and -13.1% combination reduction versus 1% increase, P&amp;lt;0.001). Post-treatment CD68 (median 35, range 3 to 117) was significantly linked to a Ki67 drop (p=0.045). Interestingly, this effect was mainly observed in the combination treatment group (p=0.002). aSMA expression was unaffected during treatment in 52%, increased in 35% and decreased in 13% of cases. Patients with aSMA reduction post treatment had a larger Ki67 fall compared to patients with increase or no change in aSMA (p=0.007). Conclusion: Short term treatment response in the epithelial component of cancers was paralleled by specific responses in the tumour stromal component. These novel findings suggest that bisphosphonates and aromatase inhibitors have major effects on tumour stroma in vivo which might augment their inhibitory effect on tumour progression. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-05.

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P<0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.