bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-cell protein analysis in support of biomarker evaluation for breast cancer immunotherapeutics.
Authors: Plaks Vicki 
Published: February 2018

10 Background: Triple-negative breast cancer (TNBC) is being tested for PD-1/PD-L1 checkpoint inhibitors combination therapy. The relationship between tumor expression of PD-L1 and patient outcomes has been established using immunohistochemistry (IHC) biomarker assays across various malignancies. PD-L1 is expressed in tumor and immune cells within the tumors in TNBC, but only immune-cell PD-L1 is associated with response to anti-PD-L1 (atezolizumab) so differentiating tumor vs. immune PD-L1 expression is essential. The currently available IHC biomarker assays that relay on tissue biopsies struggle to provide clinically meaningful identification of responders vs. non-responders. Poor sensitivity and specificity is partially attributed to tumor heterogeneity that is not well-represented in these biopsies. In addition, PD-L1 expression may vary over time due to disease progression or treatment. Since tissue biopsy collection is often an invasive procedure, it is restricted. Needle biopsies can mitigate some of these issues as a minimally invasive method to probe multiple regions within several cancer lesions across several time points. MILO, a new single cell Western Blot (WB) technology allows quantitative measurements of multiple protein biomarkers within one cell in small samples. Methods: To evaluate the capability of MILO to differentiate between PD-L1 in immune and tumor cell populations, we interrogated BC cell lines as well as peripheral blood mononuclear cells (PBMCs) mixed in known ratios. We used MDA-MB-231 that shows high PD-L1 and low HER2 expression and BT-474, which shows the inverse expression profile for these biomarkers along with PBMCs as PD-L1+ immune cells. Primary breast tumor tissues are currently interrogated to establish utility in needle biopsies. Results: MILO demonstrated heterogeneity at the single-cell level of HER2 and PD-L1 expression in BC tumor cells and enabled differentiation between PD-L1 expression in immune vs. tumor cells using cell-specific markers and low cell numbers. Conclusions: MILO can improve patient selection and prediction of response to immune checkpoint inhibition by promoting the utility of needle biopsies to complement IHC of core biopsies.
Immunotherapy use, outcomes, and toxicities in gastrointestinal malignancies.
Authors: David Rachel Julie , Baran Andrea , Maggiore Ronald John 
Published: February 2018

100 Background: Use of checkpoint inhibitors has expanded over recent years, and the gastrointestinal (GI) oncologic indications for immunotherapy (IO) continue to evolve. IO use in GI malignancies is still in its infancy, and we sought to describe our clinical experience thus far. Methods: We retrospectively identified all patients (pts) with GI malignancies treated with IO at the Wilmot Cancer Institute from December, 2015 through July, 2017. We examined patient and tumor-related clinicopathologic features. Our main outcomes of interest were best response on IO, and frequency/severity of IO toxicity. Fisher’s exact test was used to compare categorical factors between groups of interest. Results: 21 pts received IO for a GI malignancy, all of whom had metastases (mets). 76% of pts were male, and the median age was 62 years (range, 25 – 87). Common diagnoses were colorectal (CRC) (n = 10), gastroesophageal (GE) (n = 5), and hepatocellular cancers (HCC) (n = 4). 76% of pts received pembrolizumab, and 24% of pts received nivolumab. 67% received IO as ≥ 3rd-line therapy. The median time from diagnosis of metastatic disease to initiation of IO was 17.8 mos (range, 3 – 140). The median time on therapy was 2.62 months (95% CI 1.48, 6.52 mos) (range, 0.5 – 11 mos), with 3 pts still responding at the time of the analysis. Pts with HCC had the longest time on IO (8.95 mos), compared to 2.62 mos for GE, 2.39 mos for CRC, and 1.26 mos for all other (p = 0.10). Pts who received IO as 3rd or greater line of therapy had a significantly increased likelihood of non-response or progressive disease/death compared to pts who received IO as 2nd-line (p = 0.02). There was a trend towards non-response to IO in pts with liver mets vs. extrahepatic mets (p = 0.09). 57% experienced grade 3/4 toxicities: the most common were anemia (24%), hyponatremia (14%), and fatigue (14%). No IO-specific grade 3/4 immune-related toxicities were observed. Conclusions: The majority of our pts received IO as 3rd line therapy or beyond, and were significantly less likely to respond to treatment compared to those who received IO earlier on. The presence of liver mets is potentially associated with non-response to IO, and needs further exploration. No unexpected or severe IO-related toxicities were observed.
Expression of endogenous retroviruses and response to immune checkpoint therapy in renal cell cancer.
Authors: Panda Anshuman , De Cubas Aguirre , Beckermann Katy , Riedlinger Gregory , Stein Mark N. , Mayer Tina M. , Mehnert Janice M. , Rathmell Kimryn , Bhanot Gyan , Ganesan Shridar 
Published: February 2018

104 Background: In certain cancers, including renal cell carcinoma (RCC), no clear correlation exists between mutation burden and response to immune checkpoint therapy. To look for other markers of immune activation, we investigated the correlation between expression of endogenous retroviruses (ERV) and evidence of immune checkpoint activation in multiple cancer types. Methods: RNA-seq data of 4,910 tumors of 21 cancers from TCGA was analyzed to identify cancers in which there was correlation between ERV expression and evidence of immune checkpoint activation as shown by increased expression of immune checkpoint genes and evidence of CD8+ T-cell infiltration. Expression of candidate ERVs was measured by quantitative RT-PCR in a set of 20 RCC specimens. Results: In the TCGA clear-cell renal cancer, ER+HER2- breast cancer, and colon cancer datasets showed correlation between expression of a subset of ERVs and markers of local immune checkpoint activation. Using hierarchical clustering, tumors could be classified into 3 groups (high/intermediate/low) based on expression of these ERVs. In all these cancer types, the high ERV expressing group showed evidence of immune activation (robust immune infiltration with high CD8+ T cell fraction) and checkpoint (PD-1, CTLA-4) pathway over-expression. Expression of gene pathways associated with histone modification was significantly correlated with overall ERV expression, suggesting underlying dysregulation of chromatin silencing. Of ERVs analyzed ERV3.2 and ERVK-2 were most consistently associated with markers of immune checkpoint activation in multiple cancer types. For validation, expression of ERVs were measured in tumor sepcimens 20 clear cell renal cancer patients treated with immune checkpoint blockade. Expression of ERV3-2 and ERVK-2, was significantly increased in patients with clinical response to immune checkpoint therapy in this cohort. Conclusions: These data suggest that expression of ERV may be associated with activation of immune checkpoint pathways in renal cell cancer and may predict response to immune checkpoint therapy. Similar associations may also exist in some other solid tumors.
Targeting autophagy and immunotherapy with hydroxychloroquine and interleukin 2 in patients with metastatic renal cell carcinoma (mRCC): A Cytokine Working Group study.
Authors: Appleman Leonard Joseph , Logan Theodore F. , Normolle Daniel Paul , Ernstoff Marc S. , Parikh Rahul Atul , McDermott David F. , Monk Paul , Olencki Thomas , Friedland David , Maranchie Jodi Kathleen , Butterfield Lisa Helene , Liang Xiaoyan , Lotze Michael T. 
Published: February 2018

106 Background: We performed a Phase II study of the combination of the autophagy inhibitor, hydroxychloroquine (HCQ), along with high dose IL-2 in patients with advanced renal cancer. 31 patients were entered on this Cytokine Working Group Study conducted at six member institutions;NCT01550367. This combination in murine models was associated with diminished toxicity and increased efficacy, and, in preliminary studies, diminished high mobility group box 1 (HMGB1) protein, consistent with its established role in serving as a Damage Associated Molecular Pattern (DAMP) molecule and inducer of autophagy. Methods: The Study Design involved initiating oral Hydroxychloroquine 300 mg P.O bid. Aldesleukin (600,000 IU/kg) was administered q8hrs in courses consisting of two cycles separated by 7-14 days and constituting a single course. For patients with stable or responsive disease, additional courses were administered every approximately 85-90 days. Serum, plasma, Paxgene tubes, and peripheral blood mononuclear cells were obtained sequentially prior to therapy initiation and sequentially on D1 and D2 of each cycle following initiation of therapy. Results: 31 patients (9F, 22M) have been registered and 3 confirmed complete responses observed; the current median overall survival has not been reached in the 29pts. The Baseline Karnofsky Score of 100 (17pts), 90 (13pts), and 80(1 pt). The mean age was 57.5 years, range = (45.2, 68.8). 26 patients had a mean of 12.5 doses +/-4.7 (3, 23) with 13 pts receiving a second course and 4, a third. Platelet nadir was diminished from baseline by 26%. Of the 27 patients in the data set, 18 had at least one Grade 4 toxicity at least possibly related to treatment, and 9 patients had at least one Grade 3 adverse event at least possibly related to treatment but no Grade 4 events.Serologic and cellular data and complete clinical data will be submitted with the completed abstract. Conclusions: The combination of high dose aldesleukin and daily oral HCQ was well tolerated. We have concluded this trial and will report mature survival data, toxicity data, and biomarkers/autophagy measures with the final submission. Clinical trial information: NCT01550367.
Clonotypic T-cell responses in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) treated with standard sipuleucel-T (sip-T) compared with patients receiving a booster treatment.
Authors: Zhang Li , Kandadi Harini , Paciorek Alan T , He Tao , Sheikh Nadeem Anwar , Fong Lawrence 
Published: February 2018

107 Background: Sip-T is an FDA-approved autologous cellular immunotherapy for asymptomatic or minimally symptomatic mCRPC. Neoadjuvant sip-T induced activated T-cell infiltration to the prostate tissue (Fong 2014) and broadened the T cell receptor (TCR) repertoire vs control (Sheikh 2016). To test if sip-T induces memory T-cell responses, we compared treatment-induced changes in TCR repertoire of treatment-naïve pts (STRIDE) with those retreated with sip-T (P10-1). Methods: In STRIDE (N=52), pts received sip-T with concurrent or sequential enzalutamide (Petrylak 2015). In P10-1 (N=8), pts previously treated with sip-T in an androgen-dependent setting were retreated with a booster course after a median of 8.6 years (Beer 2013). PBMCs were collected at baseline and during/post–sip-T. Deep sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies). TCR diversity was assessed by Shannon diversity index and clonality. TCR dynamics was evaluated by Morisita’s distance (Zhang 2017). Results: Baseline TCR diversity was similar between the two trials (p=0.590, Shannon diversity index; p=0.700, clonality). Significant decreases in clonality were observed from baseline to wk 4 (p<0.001) and wk 6 (p=0.030) in STRIDE, but not in P10-1, indicating more expansion of T cell clones in STRIDE. Morisita’s distance was significantly higher at wk 2 (p=0.040) and 26 (p=0.013) in STRIDE vs P10-1, indicating a more consistent TCR repertoire in STRIDE across timepoints. The proportion of the clones enriched after sip-T treatment was significantly higher in P10-1 vs STRIDE (wk 6: p=0.007, wk 26: p=0.015, wk 52: p=0.026). The extent of change within the top 100 most abundant TCR sequences (clonal shuffling) was greater and initiated earlier in P10-1 vs STRIDE. Conclusions: Initial sip-T treatment of naïve mCRPC pts programs the TCR repertoire, which is maintained over time. Consistent with immunologic memory, retreatment with sip-T rapidly expands the number of enriched T-cell clones persisting up to 1 year after retreatment, which is characteristic of immunological boosting following successful treatment.
Long-amplicon TCRβ repertoire sequencing to reveal human T-cell receptor variable gene polymorphism: Implications for the prediction and interpretation of immunotherapy outcome.
Authors: Looney Timothy , Glavin Alexander , Pabla Sarabjot , Glenn Sean T. , Miller Lauren , Topacio-Hall Denise , Linch Elizabeth , Pankov Aleksandr , Zheng Jianping , Conroy Jeffrey M. , Morrison Carl , Lowman Geoffrey , Andersen Mark , Hyland Fiona 
Published: February 2018

129 Background: Human T cell antigen receptors play a critical role in protective immune responses but are also implicated in autoimmune disease and immune-mediated adverse events during immunotherapy. The antigen specificity of the T cell receptor is determined in part by the sequence of the CDR and Framework regions encoded by the TCRB variable gene. Previous studies of population sequencing data indicate that current antigen receptor allele databases, such as IMGT, fail to capture a significant portion of human variation. Here we use long-amplicon multiplex sequencing of rearranged TCRB receptors to validate putative novel human variable gene alleles previously recovered from 1000 genomes data. Methods: TCRB rearrangements were amplified from cDNA from 85 Caucasians undergoing treatment for melanoma using AmpliSeq-based multiplex Framework 1 and Constant gene primers to produce ~330bp amplicons. Samples were sequenced using the Ion Torrent S5 530 chip to produce ~1.5M raw reads per sample. Ion Reporter was used for clonotyping and identification of variable gene sequences absent from the IMGT database. Putatively novel sequences were compared to those reported in the Lym1k database of alleles recovered from 1000 genomes data. Results: We identified 15 novel variable gene alleles that are absent from the IMGT database and result in amino acid changes to the CDR or Framework regions of the TCR. Typically, a single individual was found to be heterozygous for a novel variant, though we note two instances where multiple individuals possessed a novel variant. We also identified novel variable gene alleles that are absent from the Lym1k database, potentially due to challenges in inferring receptor alleles from short-read population sequencing studies. Conclusions: We find evidence for significant human diversity in TCRB variable gene alleles beyond what is currently represented in the IMGT database. TCRB sequencing using multiplex Framework 1 and Constant gene targeting primers is ideally suited for studying the role of TCRB polymorphism in autoimmune disease and immune-mediated adverse events during immunotherapy.
Effect of PDL1 signaling blockade on NK cell cytolytic activity towards ovarian cancer.
Authors: Oyer Jeremiah , Gitto Sarah B. , Altomare Deborah , Igarashi Robert , Copik Alicja 
Published: February 2018

13 Background: The PD-1/PDL-1 axis is an immuno-oncology target for abrogating immune suppression. Anti-PD1 targeted mAb therapy has had success in several indications by alleviating suppression of cytolytic T cells. In addition to T cells, NK cells hold promise for adoptive applications for oncology. We developed a nanoparticle based stimulation method for producing large quantities (> 1,000 fold expansion within two weeks) of highly activated NK cells with high anti-tumor cytotoxicity and enhanced cytokine delivery. These NK cells (PM21-NK) release high amounts of IFNγ in response to tumors that stimulates the expression of PDL1 on tumor, which in turn enhances Treg development. Methods: The ability of PM21-NK cells to induce expression of PDL-1 on tumors was tested in vitro and in vivo. Furthermore, the in vivo efficacy of combination treatment of PM21-NK cells and anti-PDL1 was tested in NSG mice models for ovarian cancer. Results: The IFNγ production by PM21-NK cells increased from 1.4% to 9.9% (p < 0.001) upon exposure to SKOV-3 cells. SKOV-3 cells normally do not express PDL1 but PDL1 expression increased with 48 hours exposure to 20 ng/mL IFNγ (0.7% vs 65.1%). In and in vivo experiment, NSG mice intraperitoneally implanted with 1 x 106 SKOV-3 cells were treated with 107 T-cell depleted PM21-NK cells i.p. on days 8 and 13 and 25,000 U IL2 i.p. thrice weekly. Upon euthanization 21 days after tumor seeding, tumors in PM21-NK treated mice had increased PDL1+ tumors vs the untreated group (29.7% vs 14.5%, n = 6, p < 0.0001). Despite T-cell depletion, T-cells made up ~22% of hCD45+ events in perfused tumors, 83% of which were Tregs. In a follow-up survival experiment, combination treatment of anti-PDL1 and NK cells were tested in SKOV-3 implanted NSG mice (as above). Groups of mice were given 107 T-cell depleted PM-NK or vehicle i.p. days 4 and 7 and 25,000 U IL2 3x’s weekly and groups were given 10 mg/kg anti-PDL1 (Atezolizumab) or vehicle ctrl. on day 4 and later 5 mg/kg twice weekly. Untreated and Atezo alone survival were 24 and 23.5 days, respectively. Median survival for PM-NK + Atezo was 48 days compared to 40.5 for PM-NK alone (p = 0.042; n = 8). Conclusions: These data support the use of anti-PDL1 in NK cell therapy regardless of initial tumor PDL1 status.
Use of immune checkpoint inhibitors in a community practice within a vertically integrated health care system.
Authors: Cooper Robert Michael , Yamamoto Christopher , Truong Thach-Giao , Niu Fang , Moon Helen , Spence Michele M. , Chan Jim , Chiu Timothy , Hui Rita L. 
Published: February 2018

130 Background: Four immune checkpoint inhibitors have been FDA approved from 2011 to 2016. Immune checkpoint inhibitors function differently from chemotherapy and have different benchmarks for response as well as different side effect profiles. This report examines how oncologists in two large medical groups have introduced these new treatments into their practices. Kaiser Permanente is a large HMO which insures 8.5 million members in the state of California and has an exclusive contract with 2 large medical groups to care for its members. There are over 200 medical oncologists caring for KP members practice at over 35 medical centers. The care of the KP members has been documented in an EPIC based medical record systems since 2006. Methods: The Pharmacy Research Outcomes Group has conducted a retrospective cohort study looking at patients who initiated Checkpoint inhibitors between March 2011 and September 2016 for this review and followed the patients till December 2016. Electronically available data collected for this review includes patient characteristics such as but not exclusive to: cancer diagnosis, previous therapies, current therapies, age, sex, ethnicity, immune related adverse events (IRAEs) proxy by diagnosis code, duration of immunotherapy, use of systemic steroids, Charlson Co-morbity Index and survival. Results: We reviewed a total of 1760 users of Checkpoint Inhibitors. We show the uptake of this class of medications aligns with approved checkpoint inhibitors and the new indications of these agents. We were able to electronically pull medical information and define IRAEs. Our data closely matches the expected incidence of IRAEs as reported in clinical trials. We were also able to monitor the use of steroids in response to these events. Conclusions: The ability to collect and track this information has allowed the group to better understand patient selection practices as well as management practices in the community practice setting.
Two distinct clinical trajectories of inflammatory thyroiditis evoked by checkpoint blockade in malignant melanoma.
Authors: Olsson-Brown Anna Claire , Lord Rosemary , Sacco Joseph J , Coles Mark Christopher , Wagg Jonathan , Pirmohamed Munir 
Published: February 2018

131 Background: Oncological immune checkpoint inhibitors (CPI) commonly cause immune related adverse events (irAEs). Thyroid irAEs have a significant clinical impact requiring therapeutic intervention and clinical coordination whilst often causing treatment delays. Methods: Retrospective review of all patients treated with CPIs (pembrolizumab, nivolumab +/- ipilimumab) for metastatic melanoma at the Clatterbridge Cancer Centre, UK identified patients experiencing thyroid irAEs. Clinical/biochemical course (T4 and thyroid stimulating hormone (TSH) levels) and autoantibody titers were evaluated to identify characterisable trajectories. Results: From February 2016 to May 2017, 103 patients received anti-PD1 therapy; 90 receiving monotherapy and 13 combination. Thyroid irAEs were seen in 13(14.4%) and 3(23%) patients respectively. Two distinct trajectories were identified: a hyperthyroid phase with subsequent hypothyroidism (HH) and de novo hypothyroidism (DN). There were no outliers. HH was the predominant pattern (75%) and the sole manifestation in combination therapy. In the HH cohort 69% of patients were female. The peak T4 was 39.3pmol/L occurring, on average, 7.5 weeks after CPI initiation. T4 levels declined within 6-21 days and hypothyroidism occurred within 8 weeks. The DN cohort was exclusively female. Thyroid irAE occurred 12 weeks after CPI initiation. Baseline TSH in HH (2.49mU/l) was significantly lower than DN (5.77mU/l); p = 0.0092. Positive thyroid autoantibodies were detected in 5 patients across both groups. One patient (HH) had symptomatic thyroid dysfunction. Corticosteroids conveyed no benefit. All patients had permanent dysfunction requiring long term levothyroxine replacement. All continued with immunotherapy treatment; however notable irAE-related treatment hiatuses occurred. Conclusions: Retrospective review revealed two distinct thyroid irAE trajectories, HH and DN. The trajectories may have biochemical predictors, are rarely associated with symptoms and appear unrelated to thyroid autoantibodies. Trajectory identification will optimise patient outcomes, reduce ineffective steroid use and promote early hormone replacement.