Plasma-derived proteomic biomarkers in human leukocyte antigen-haploidentical or human leukocyte antigen-matched bone marrow transplantation using post-transplantation cyclophosphamide

Recent studies have suggested that plasma-derived proteins may be potential biomarkers relevant for graft-versus-host disease and/or non-relapse mortality occurring after allogeneic blood or marrow transplantation. However, none of these putative biomarkers have been assessed in patients treated either with human leukocyte antigen-haploidentical blood or marrow transplantation or with post-transplantation cyclophosphamide, which has been repeatedly associated with low rates of severe acute graft-versus-host disease, chronic graft-versus-host disease, and non-relapse mortality. We explored whether seven of these plasma-derived proteins, as measured by enzyme-linked immunosorbent assays, were predictive of clinical outcomes in post-transplantation cyclophosphamide-treated patients using plasma samples collected at serial predetermined timepoints from patients treated on prospective clinical studies of human leukocyte antigen-haploidentical (n=58; clinicaltrials.gov Identifier: 00796562) or human leukocyte antigen-matched-related or -unrelated (n=100; clinicaltrials.gov Identifiers: 00134017 and 00809276) T-cell-replete bone marrow transplantation. Day 30 levels of interleukin-2 receptor α, tumor necrosis factor receptor 1, serum STimulation-2 (IL1RL1 gene product), and regenerating islet-derived 3-α all had high areas under the curve of 0.74–0.97 for predicting non-relapse mortality occurrence by 3 months post-transplant in both the human leukocyte antigen-matched and human leukocyte antigen-haploidentical cohorts. In both cohorts, all four of these proteins were also predictive of subsequent non-relapse mortality occurring by 6, 9, or 12 months post-transplant and were significantly associated with non-relapse mortality in univariable analyses. Furthermore, day 30 elevations of interleukin-2 receptor α were associated with grade II–IV and III–IV acute graft-versus-host disease occurring after day 30 in both cohorts. These data confirm that plasma-derived proteins previously assessed in other transplantation platforms appear to retain prognostic and predictive utility in patients treated with post-transplantation cyclophosphamide

Acupuncture for dyspnea in advanced cancer: a randomized, placebo-controlled pilot trial [ISRCTN89462491]

BACKGROUND: Dyspnea, or shortness of breath, is a common symptom in patients with advanced cancer. Pharmacologic management is of proven benefit, but it does not help all patients. Preliminary data suggest that acupuncture can relieve dyspnea in a variety of populations, including cancer patients. We conducted a pilot study (ISRCTN89462491) preparatory to a fully powered randomized, placebo-controlled trial to determine whether acupuncture reduces dyspnea in patients with lung or breast cancer. METHODS: The study sample was comprised of forty-seven patients with lung or breast cancer presenting with dyspnea. Patients receiving symptomatic treatments were not excluded as long as no changes in management were planned during the trial. Patients were randomized to receive a single session of true or placebo acupuncture in addition to their existing dyspnea treatments. Semi-permanent acupuncture "studs" were then inserted: patients applied pressure to these studs twice a day to provide ongoing stimulation to acupuncture points. The subjective sensation of dyspnea was assessed with a 0 – 10 numerical rating scale immediately before and after acupuncture treatment and daily for a week thereafter. RESULTS: All but two of 47 randomized patients provided follow-up data. Dyspnea scores were slightly higher for patients receiving true versus placebo acupuncture, for both the period immediately following acupuncture treatment and for the daily one week follow-up (differences between means of 0.34, 95% C.I. -0.33, 1.02 and 0.56, 95% C.I. -0.39, 1.51). The 95% confidence interval excludes the prespecified minimum clinically significant difference of a 20% greater improvement in dyspnea for patients receiving acupuncture. CONCLUSION: The acupuncture technique used in this trial is unlikely to have effects on dyspnea importantly larger than placebo for patients with advanced cancer

Targeted Next Generation Sequencing of Circulating Tumor DNA in Multiple Myeloma

Introduction: Immunophenotypic and chromosomal analysis among patients with multiple myeloma (MM) has traditionally been performed on plasma cells enriched from bone marrow aspirates. However, due to the patchy nature of MM, a bone marrow aspirate may not accurately represent the total body burden of disease nor does it reflect the true heterogeneity of the cancer cells. Additionally, since it is an invasive procedure, frequent resampling to monitor clonal evolution is infeasible. Next generation sequencing of circulating tumor DNA is a promising new approach that may address these shortcomings. Circulating tumor DNA, either from cell-free DNA or circulating tumor cells, originates from multiple different sites of disease and may be obtained from a single blood sample. This non-invasive approach may enable clinicians to detect early signs of resistance, monitor minimal residual disease, identify therapeutic targets, and provide prognostic information to help guide treatment decisions. Methods: Bone marrow mononuclear cells (BMMC), peripheral blood mononuclear cells (PBMC), and plasma samples were obtained from 45 patients treated at our center at the time of MM diagnosis, remission, or disease relapse. All samples were obtained through an Institutional Review Board approved protocol. DNA was extracted and library preparation for next generation sequencing was performed using unique molecular identifiers (UMI). The incorporation of UMIs allows for the identification and removal of errant base calls introduced during PCR amplification and Illumina sequencing thereby increasing the sensitivity of the assay to detect rare mutations. Target enrichment was performed using a panel of primers specific to exons of genes known to be significantly, recurrently mutated in MM as well as genes with predictive or prognostic value. Illumina sequencing was performed to a depth of 10,000X. Comparison of single nucleotide variants (SNV) and indels between matched BMMC, PBMC and plasma samples was determined. Correlation of clinical outcomes with mutation status and variant allele frequency was performed. Results: While good concordance was found between BMMC, PBMC, and plasma samples among the SNVs and indels detected, distinct variants unique to each tissue compartment were also identified. Indicating the assay may have utility for disease monitoring, variants detected in the blood pre-treatment were no longer detectable in post-treatment remission samples. Additionally, new variants in blood samples not present prior to initiation of therapy were detected at later time points providing evidence for clonal evolution. Finally, driver mutations were found in genes for which known targeted therapy is available or in genes that may theoretically confer drug resistance such as CUL4A that binds to Cereblon, a target of the immunomodulatory drug lenalidomide. Conclusions: While bone marrow biopsy represents the standard of care for diagnosis and disease monitoring in patients with multiple myeloma, the procedure is subject to sampling bias and frequent serial biopsies are impractical. A noninvasive approach using targeted next generation sequencing of circulating tumor cells is an appealing alternative. We demonstrate the feasibility of this technique in MM PBMC and plasma samples and find evidence that the assay may support clinical decision making. No relevant conflicts of interest to declare

Different measures, different outcomes? Survey into the effectiveness of chronic pain clinics in a London tertiary referral center

Savan Shah,1,2 Alexandra C Ho,1,2 Bianca M Kuehler,1 Susan R Childs,1 Glyn Towlerton,1 Ian D Goodall,1 Carsten Bantel1,2 1Pain Medicine, Chelsea and Westminster Hospital, NHS Foundation Trust, 2Section of Anaesthetics, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK Background: Chronic pain clinics aim to improve challenging conditions, and although numerous studies have evaluated specific aspects of therapies and outcomes in this context, data concerning service impact on outcome measures in a general pain population are sparse. In addition, current trends in commissioning increasingly warrant services to provide evidence for their effectiveness. While a plethora of outcome measures, such as pain-intensity or improvement scores, exist for this purpose, it remains surprisingly unclear which one to use. It also remains uncertain what variables predict treatment success. Objectives: This cross-sectional study was conducted to evaluate clinic performance employing different tools (pain scores, pain categories, responder analysis, subjective improvement, satisfaction), and to determine predictors of outcome measures. Patients and methods: Patients attending scheduled clinic follow-up appointments were approached. They were asked to complete the modified short-form Brief Pain Inventory (BPI-SF) that also included assessments for satisfaction and subjective improvement. Comparisons were made with BPI-SF responses that were completed by each patient on admission. Nonparametric tests were employed to evaluate service impact and to determine predictors for outcome. Results: Data of 118 patients were analyzed. There was considerable variation in impact of pain clinics depending on the outcome measure employed. While median pain scores did not differ between admission and follow-up, scores improved individually in 30% of cases, such that more patients had mild pain on follow-up than on admission (relative risk 2.7). Furthermore, while only 41% reported at least moderate subjective improvement after admission to the service, the majority (83%) were satisfied with the service. Positive treatment responses were predicted by “number of painful regions” and “changes in mood”, whereas subjective improvement was predicted by “helpfulness of treatments”. Conclusion: Depending on the outcome measure employed, pain clinics showed varying degrees of impact on patients’ pain experiences. This calls into question the current practice of using nonstandardized outcome reporting for evaluation of service performances. Keywords: Brief Pain Inventory, chronic pain clinics, pain-intensity scores, patient satisfaction, responder analysis, subjective improvemen

T-Cell Receptor Sequencing of Kaposi Sarcoma Tumors to Identify Candidate Tumor-Reactive T Cells

Abstract 60 Background: Development of Kaposi sarcoma (KS) is strongly associated with immune dysfunction in the context of HIV infection, but little is known about T-lymphocyte responses against KS tumor cells or human herpesvirus-8, the viral cause of KS. Increasing evidence suggests that treatment response in KS is attributable in part to an antitumor immune response that is mediated by tumor-infiltrating lymphocytes (TIL). The aim of this work was to identify TIL characteristics that are associated with tumor regression in patients with KS who were treated with antiretroviral therapy and chemotherapy as well as to identify a molecular signature of response. Methods: High-throughput sequencing of the T-cell receptor β chain ( TRB) was used to define the repertoire of T cells that infiltrate up to two pretreatment and two post-treatment KS tumors and matched normal skin obtained from HIV-infected adults with KS who received care at the Uganda Cancer Institute. We compared TRB repertoire in serially collected tumors to identify TRB sequences carried in candidate tumor-reactive T cells. Results: TRB sequencing was performed on KS tumor and matched normal skin samples from 12 HIV-infected adults with KS who collectively demonstrated a range of treatment responses. Unique populations of T cells were identified in pretreatment tumors but not in normal skin in all patients, which suggested the presence of KS-specific T-cell responses. Durable complete response to treatment in one patient was associated with significant expansion of a small number of T-cell clones, one of which carried a TRB sequence that was associated with a public CD8 + Epstein-Barr virus–associated T-cell receptor. Conclusion: Understanding the immune response to KS through cellular and molecular dissection of TIL will provide important insights into KS biology and may ultimately guide new immune-based strategies to stage and treat this often-refractory cancer. Funding: Solid Tumor Translational Research Transformative Team Grant, Fred Hutchinson Cancer Research Center; National Institutes of Health/National Cancer Institute Grant No. K23-CA150931. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST No COIs from the authors

Comparative Analysis of Endemic and Sporadic Burkitt Lymphoma By RNA Sequencing

Abstract Introduction: Burkitt lymphoma (BL) is an aggressive B-lineage non-Hodgkin lymphoma that has traditionally been classified into 3 subtypes: "endemic", "sporadic", and HIV-associated. The highest incidence of BL - prompting the "endemic" classification - is observed in children in sub-Saharan Africa, where the distribution of the disease is closely associated with that of P. falciparum malaria. The tumor cells in most cases of BL from sub-Saharan Africa are infected with Epstein Barr virus (EBV). In contrast, a minority of BL tumors from Europe and North America - which are commonly described as "sporadic" - are EBV positive. Although excellent outcomes have been observed for patients with BL treated with multi-agent chemotherapy in Europe and North America, the outcomes of BL patients in sub-Saharan Africa are suboptimal. We hypothesized that improved understanding of the molecular heterogeneity that exists within BL, particularly the molecular features that distinguish cases from sub-Saharan Africa from those from Europe/North America, will enable more effective treatment strategies that can readily be implemented in low-resources settings. Methods: Tumor biopsies were collected from 19 pediatric patients who presented to the Uganda Cancer Institute with maxillofacial tumors histologically confirmed to be BL. Total RNA was extracted and polyA-selected sequencing libraries were prepared. Paired-end, 50-base pair sequencing was performed on the Illumina HiSeq 2500 platform at a depth of 100 million reads per sample. A publicly available RNA sequencing dataset from 28 BL cases from Europe and North America that were previously analyzed by both microarray (NEJM 2006; 354[23]: 2431-2442) and RNA sequencing (Nature 2012; 490[7418]: 116-120) was analyzed in parallel for comparison. RNA sequencing on the 28 sporadic BL tumors was performed on polyA-selected sequencing libraries with paired-end, 108-base pair sequence reads generated on the Illumina HiSeq 2000 platform. The reads from all 47 BL tumors were aligned to the human and EBV (GenBank ID KC207813.1) genomes using the STAR aligner. Tumors were deemed EBV positive if the ratio of mapped viral reads to human reads exceeded 0.001%, since the EBV genome is 0.005% the size of the human genome. Normalization and differential expression analysis were performed on aligned reads using the DESeq2 R package. Somatic variants were identified by the Genome Analysis Tool Kit. Analysis of alterative splicing was performed with the SGSeq R package. Results: All of the Uganda BL cases but only 70% of the previously published European/North American cases were obtained from children less than 18 years; 61% and 87% were from male patients, respectively. One half of patients in both groups presented with Ann Arbor stage I or II disease, while the remainder presented with higher stage disease. The median survival in the 19 Ugandan patients was less than one year. Sequence reads from 15 (79%) of the Ugandan tumors and 4 (15%) of the European/North American tumors aligned to the EBV genome. Although BL is reported to express only latency type I EBV genes, expression of latency type I, II, and III genes was detected. Among the 4 tumors from Uganda that did not contain EBV-derived reads, 1 was from a child who was HIV-seropositive, while the others were from HIV-seronegative patients. Unsupervised hierarchical clustering and principal component analysis of all annotated genes failed to separate EBV positive and EBV negative tumors. Cluster analysis of the Ugandan tumors revealed no association with clinical variables. Ongoing analyses are being performed to identify associations between differentially expressed genes and somatic mutations and alternative splicing that may distinguish the tumor subtypes. Conclusions: While the terms "endemic" and "sporadic" have traditionally been used to classify BL, this nomenclature is archaic and does not accurately capture the underlying biology of the tumor subtypes. We performed a comparative whole transcriptome analysis of BL tumors from patients in Uganda to a dataset of BL tumors from patients in Europe/North America to identify features that distinguish the two cohorts. While cluster analysis failed to clearly separate tumors by their EBV or clinical status, ongoing analyses are being performed to identify alterations that may contribute to their distinct gene expression profiles. Disclosures No relevant conflicts of interest to declare

Simultaneous Detection of Single Nucleotide Variants and Expression of RNA and Protein in Multiple Myeloma Bone Marrow Aspirates

Introduction: Multiple myeloma (MM) is generally an incurable plasma cell neoplasm that evolves from a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS). Primary genomic events leading to the development of MGUS include hyperdiploidy, translocations involving the immunoglobulin heavy chain genes, and copy number variations. Single-nucleotide variants (SNVs) as well as insertions and deletions are secondary events that trigger progression to active MM. Identifying nucleotide variants that lead to changes in RNA and protein expression is critically important to understanding the biology of the cancer and ultimately identifying therapeutic targets. Analysis of DNA, RNA, and protein has traditionally involved integration of three separate assays and is frequently limited by the availability of sufficient material from MM patient specimens. We used a more efficient approach that simultaneously detects DNA variants, RNA and protein expression in a single sample from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. We evaluated the NanoString nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay performed on 1 x 106cells from bone marrow biopsies obtained from patients with MM. Methods: Bone marrow aspirates from 15 patients diagnosed with MM were obtained through an Institutional Review Board approved protocol. Seven patients had newly diagnosed MM and 8 had relapsed disease. DNA, RNA, and protein were extracted from unsorted, cryopreserved, ficoll-hypaque separated bone marrow mononuclear cells and hybridized with optical barcodes designed to detect 124 DNA somatic variants common to hematologic malignancies as well as 180 mRNA transcripts and 38 total and phospho-proteins. We also used the nCounter PanCancer Pathways Panel Assay to measure gene expression of 770 additional genes from 13 cancer-associated canonical pathways. The hybridized probes in each sample were counted using the nCounter Digital Analyzer and statistical analysis was performed using nSolver™ v4.0 alpha software. Results: The percentage of CD138+ bone marrow plasma cells measured by immunohistochemistry at the time of biopsy varied from 44 to 100% in each sample. One or more DNA variants were detected in 3 pre-treatment and 4 relapse samples. Mutations in KRAS, NRAS, and BRAF were most common and 2 patients were found to have variants in both KRAS and NRAS or KRAS and BRAF . Additionally, variants in DNMT3A and IDH2 were detected in 1 patient each. Since KRAS, NRAS, and BRAF are all members of the mitogen-activated protein kinase (MAPK) signaling pathway, a differential gene expression analysis of both RNA and protein was performed comparing samples with and without mutations in these genes. Findings confirmed that a higher level of expression was detectable in MAPK pathway genes, although this result did not reach statistical significance following correction for replicate testing, potentially the consequence of a small sample size. Conclusions: The Vantage 3D DNA:RNA:Protein Heme Assay is a rapid, sensitive and resource sparing platform for the simultaneous detection of DNA variants, RNA transcripts, and protein expression. We show that this method can feasibly be performed on as few as 1 x 106 cryopreserved bone marrow cells in patients with MM and our findings confirm detection of 3 of the most common MM variants. This technology should have broad application in other hematological malignancies and further evaluation of blood and lymph node biopsies from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) samples is warranted. Demirkan:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment

T Cell Repertoire Evolution after Allogeneic Bone Marrow Transplantation: An Organizational Perspective.

High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-β sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-β sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-β VJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution