Serial Sequencing Research Articles

Agility-based exercise training compared to traditional strength and balance training in older adults: a pilot randomized trial.

BackgroundIn addition to generally high levels of physical activity, multi-component exercise training is recommended for the maintenance of health and fitness in older adults, including the prevention of falls and frailty. This training often encompasses serial sequencing of balance, strength, endurance and other types of exercise. Exercise training featuring integrative training of these components (i.e. agility training) has been proposed, as it more likely reflects real life challenges like stop-and-go patterns, cutting manoeuvers, turns and decision-making. In this study, we compared the efficacy of an agility-based training to the traditional strength and balance training approach with regard to selected risk factors for falls and frailty.MethodsWe trained twenty-seven community-dwelling healthy seniors (16♂; 11♀; age: 69.5 ± 5.3 y; BMI: 26.4 ± 3.7 kg/m2) for 8 weeks in a group setting with 3 sessions per week, each lasting 50 minutes. Participants were randomized into either the agility group (AGI; n = 12), that used the integrative multi-component training, or the traditional strength and balance group (TSB; n = 15). TSB performed balance and strength exercises separately, albeit within the same session. The training of both groups progressively increased in difficulty. Outcomes were static and dynamic balance (single leg eyes open stand, Y-balance test, reactive balance), lower limb (plantar flexion and dorsal extension) and trunk flexion and extension maximum strength and rate of torque development (RTD). In addition, we tested endurance by the six-minute walk test (6MWT). We calculated linear mixed effects models for between-groups comparisons as well as effect sizes (ES) with 95 % confidence intervals.ResultsSmall ES in favor of AGI were found for plantar flexion strength (ES > 0.18[−0.27;0.89]) and RTD (ES > 0.43[−0.19;1.36]) as well as trunk extension RTD (ES = 0.35[−0.05;0.75]). No other parameters showed notable between group differences. Compliance was high in both groups (AGI: 90 ± 8% of sessions; TSB: 91 ± 7% of sessions).DiscussionAgility-based exercise training seems at least as efficacious as traditional strength and balance training in affecting selected physical performance indicators among community-dwelling healthy seniors. In particular, lower limb and trunk extension explosive strength seem to benefit from the agility training.

A randomized study evaluating tailoring of advanced/metastatic colorectal cancer (mCRC) therapy using circulating cell-free tumor DNA (ctDNA) (TACT-D).

TPS277 Background: Identifying non-responders to expensive salvage therapies with modest benefits and substantial treatment related adverse events (TRAEs) (e.g. regorafenib/TAS102 in mCRC) is necessary to maximize benefits and limit toxicities. Serial ctDNA sequencing is reliable for tracking tumor dynamics and appears to predict resistance to therapy earlier than radiographic progression. Methods: TACT-D is a randomized study (N = 100) to validate the ability of changes in ctDNA (ΔctDNA) to predict resistance early and in limiting toxicities. We hypothesize that increase in ctDNA (measured by variant allele fraction) at 2 weeks (wk) into treatment can predict resistance earlier than standard radiographic means [at 8-12 wk] and detecting resistance early can enable prompt change in therapy resulting in reduction of TRAEs. Pts with mCRC eligible for either regorafenib/TAS102 are randomized 2:1 to either standard of care (SOC) or ctDNA arm. On SOC arm, treatment is given as per current paradigm i.e. for 8 wk and then restaging. On ctDNA arm, decision to continue therapy is based on ctDNA change between baseline and 2 weeks [ΔctDNA = ctDNA (C1D15 – C1D1)]. Increase in ctDNA triggers early radiographic staging (4 wk). Treatment is continued for disease stability/regression and discontinued for progression. Study has 2 co-primary endpoints: 1) Association of Δ ctDNA and radiographic progression [62 pts on SOC arm, have 94% power (2-sided α 0.05) to detect difference of 95% vs. 58% in progressive disease between pts with increase vs decrease in ctDNA] and 2) Compare proportion of pts experiencing TRAEs within 4 months between study arms [67 in SOC arm and 33 in ctDNA arm have 82% power (2-sided α 0.05) to detect a 30% decrease in toxicity]. Key secondary endpoints include: patient-reported outcomes (MD Anderson Symptom Inventory and PRO-CTCAE), OS, clinical events of special interest (hospitalizations/ER visits/medical interventions such as blood transfusions/IV hydration), clinical trial referral and cost effectiveness. Study is now actively accruing pts (NCT03844620). Funding: MD Anderson Cancer Center, Houston, TX & Guardant Health Inc., Redwood City, CA. Clinical trial information: NCT03844620.

Applying response surface methodology on the results of serial sequencing batch moving bed reactor

This study introduces the new biological reactor that called serial sequencing batch moving bed reactor. In this research, this reactor was used for treatment of hospital wastewater. Furthermore, three independent variables (retention time, pollutant loading and media filling percentage) were investigated. For the first time, modeling of COD removal percentage (as the dependent variable) in hospital wastewater treatment was investigated by using of response surface methodology. According to the results, the COD removal efficiency increased from 58 to 91%, when pollutant loading decreased from 0.38 to 0.3 kgCOD/kgMLSS. Also, COD removal efficiency increased from 38 to 86% when retention time increased from 16.48 to 58.52 h. According to the results, the media filling percentage had the minimum effect on the COD removal efficiency, so that, COD removal efficiency increased from 63 to 78% with increasing in media filling percentage from 19.77 to 45%. The results revealed that the optimum condition to achieving the highest COD removal efficiency was at the retention time of 46.59 h, pollutant loading of 0.34 kgCOD/kgMLSS and media filling percentage of 42.24%. According to this research results, this reactor have high efficiency in hospital wastewater treatment.

A Phase I Study of Asciminib (ABL001) in Combination with Dasatinib and Prednisone for Untreated BCR-ABL1-Positive ALL in Older Adults

Introduction The BCR-ABL1 gene fusion is the most common molecular aberration in adult ALL and confers an adverse prognosis. The incorporation of ABL1 tyrosine kinase inhibitors (TKIs) into ALL chemotherapy regimens has dramatically improved outcomes for BCR-ABL1+ ALL. Subsequently, reduced-intensity TKI-based regimens have been shown to be as effective and less toxic than intensive TKI-based regimens (Foa et al. Blood 2011; Chalandon et al. Blood 2015). Asciminib (ABL001) is a potent, specific allosteric inhibitor of ABL1 that binds to a site spatially distinct from the catalytic domain. Asciminib was developed for efficacy against BCR-ABL1 mutations conferring resistance to standard TKIs. Preclinical studies demonstrated that the combination of asciminib and TKI leads to sustained disease control in a cell line xenograft model of BCR-ABL1+ CML (Wylie et al. Nature 2017). We also observed that the addition of asciminib to TKI significantly prolongs survival in patient-derived xenograft models of BCR-AB1L+ ALL, even in the context of a pre-existing subclonal T315I mutation (unpublished). An ongoing phase I Novartis study (NCT02081378) has shown safety and efficacy of asciminib alone and in combination with TKIs in relapsed/refractory (R/R) BCR-ABL1+ ALL and CML. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and effective in older adults with BCR-ABL1+ ALL. Methods This is an investigator initiated, phase I study (NCT03595917) of asciminib in combination with dasatinib plus prednisone in BCR-ABL1+ ALL. The study uses a 3+3 dose escalation design with an expansion cohort with the primary objective to determine the MTD or RP2D. Secondary objectives include defining the depth of molecular remissions at early time points and durability of responses. Patients (pts) ≥ 50 years (yrs) with newly diagnosed BCR-ABL+ ALL or CML in blast phase with p190 or p210 transcripts are eligible. Younger pts (18-49 yrs) unfit for intensive chemotherapy, or pts ≥ 18 yrs with ALL R/R to intensive chemotherapy are also eligible (excluded if prior dasatinib or asciminib exposure). Pts are treated with dasatinib 140 mg daily and prednisone 60 mg/m2 days 1-24 (max 120 mg daily, tapered days 25-32) following Foa et al with escalating doses of asciminib once daily administered fasting (DL1: 40 mg; DL2: 80 mg; DL3: 160 mg; DL: 320 mg). Pts receive CNS prophylaxis with intra-thecal chemotherapy. Transplant-eligible pts are recommended to be consolidated with allogeneic transplant as medically appropriate at or after day 85; transplant-ineligible pts may remain on therapy if deriving clinical benefit. Correlative science efforts aim to identify biomarkers of response to combined BCR-ABL1 blockade and molecular signatures enriched in minimal residual disease, based on targeted tumor sequencing and serial single-cell RNA sequencing of leukemia and stromal populations from blood and marrow. Results As of July 31, 2019, 6 adults (3 male/3 female) have enrolled and 3 have completed DL1. All 6 had newly diagnosed, untreated BCR-ABL1+ ALL. The median age at registration was 67 (range 53 - 83) yrs. Three pts treated on DL1 have been evaluated for DLT and discontinued therapy (1 per pt decision after 4 cycles; 2 pts bridged to transplant after 4 cycles). At the time of this report, there has been no induction mortality, no DLTs, and no grade 3 or higher AEs at DL1. At DL1, 1 pt was treated entirely outpatient, the other 2 pts required short hospital stays (4 and 5 days) after treatment initiation. Responses for the 3 pts treated at DL1 are summarized in the table. Pre-treatment tumor genotyping and serial on-treatment, single-cell profiling of tumor and microenvironment fractions from marrow and blood have been performed in all pts to date and have revealed biophysical, transcriptional, and genetic inter-tumor heterogeneity. These data will be used to illuminate disease biology and define predictive biomarkers, pending further maturation of clinical endpoints. Conclusion Dual ABL1 kinase inhibition with asciminib and dasatinib plus prednisone in newly diagnosed BCR-ABL1+ ALL is feasible in older adults with no increase in toxicity. The last pt at DL2 is completing treatment and being evaluated for DLT before planned escalation to DL3. An expansion cohort of 10 pts at the MTD or RP2D is planned to further characterize safety and clinical activity of the combination. Disclosures Stevenson: Celgene: Research Funding. Weinstock:Celgene: Research Funding. DeAngelo:Incyte: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals Inc: Consultancy; Novartis: Consultancy, Research Funding; GlycoMimetics: Research Funding; Abbvie: Research Funding; Shire: Consultancy; Celgene: Consultancy; Takeda Pharmaceuticals: Consultancy; Amgen: Consultancy; Blueprint: Consultancy, Research Funding.

Novel Molecular Pathogenesis and Therapeutic Target in Acute Erythroid Leukemia

Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia (AML) characterized by erythroid predominance and dysplasia, which is morphologically classified into two subtypes: pure erythroid and myeloid/erythroid leukemias (PEL and MEL). Clinically, AEL exhibits aggressive and rapid clinical course, which is considered to be linked to frequent TP53 mutations and complex karyotypes found in this subtype. Although multiple sequencing studies were conducted to explain such phenotypic variety and to search promising therapeutic targets, neither the diagnostic discrimination of PEL and MEL subtypes nor the establishment of standard therapeutic strategy has fully been successful. To clarify genetic characteristics and to identify novel therapeutic targets, we comprehensively characterized the mutation and copy number alteration (CNA) profiles and assessed differential impacts of the frequent driver genetic abnormalities on disease phenotypes and clinical outcomes. We initially analyzed 105 cases with AEL (PEL; n=10, MEL; n=69, and unspecified; n=26). Among these, 9 cases were serially analyzed at 2-6 time points. All samples were obtained according to protocols approved by the ethics board of each participating institution. As control, 907 cases with non-erythroid AML (NEL) distinguished by the low fraction (<50%) of erythroid cells in bone marrow were enrolled. Publicly available information on clinical and genetic findings was collected from previous publications, including The Cancer Genome Atlas and BeatAML datasets. In total, 1,012 cases with AML were enrolled in this study. The genetic profile in PEL and MEL were also compared with that of NEL. Finally, we recognized biological significance of the molecular defects by functional analysis of xenograft model, and showed novel therapeutic possibilities targeting pathogenic pathways. Most frequently observed were mutations in TP53 (39%), STAG2 (20%), and NPM1 (15%), and KMT2A-partial tandem duplication (PTD) (18%) in AEL. On the basis of mutational profiles, consensus clustering divided AEL into 4 subgroups. Group A (n=41, 39%) was defined by TP53 mutations into which all PELs were categorized. In CNA analysis, amplification in 9p, 19p, and 21q were significantly more enriched in PEL than NEL. Intriguingly, amplification of 9p, 19p, and 21q lesions commonly included JAK2, EPOR, and ERG loci, respectively. These genes were affected by amplification or mutations more frequently in AEL than NEL (Figure 1). Group B (n=19, 18%) was enriched for mutations in STAG2, which was significantly more frequent in MEL than in PEL (P<0.05). Compared to STAG2-mutated cases in NEL, those in MEL showed significantly higher frequency of KMT2A-PTD. Group C (n=16, 15%) was defined by NPM1 mutations frequently accompanied by PTPN11 mutations (50% in Group C). This combination was also distinct in MEL and was significantly more frequent than in NPM1-mutated NEL cohort (P<0.05). In Group D (n=29, 28%) without any TP53, STAG2, or NPM1 mutations (triple-negative), RUNX1, ASXL1, BCOR, PHF6, and U2AF1 mutations were enriched compared to triple-negative NEL. In addition, novel loss-of-function USP9X mutations were exclusively identified in Group D AEL (P<0.05, compared to triple-negative NEL), which has been implicated in erythroid leukemogenesis through the TGF beta pathway. Serial sample sequencing analysis revealed that JAK2 mutation positive myeloproliferative neoplasms evolved into PEL. Clinically, patients in Group A showed shorter overall survival time compared to those in any other group. To further search for a novel therapeutic target to the most severe type of AEL, JAK2 inhibitor was administered to xenograft models (from 2 cases) having JAK2 and EPOR amplifications and TP53 mutations. Engrafted cells showed in vitro activation of STAT5 and proliferative capacity enhanced with erythropoietin. In vivo analysis showed that inhibition of JAK/STAT pathway significantly suppressed cell growth and prolonged overall survival. In summary, our findings suggest that AEL is classified into 4 major subgroups having unique genetic and clinical features. PEL and MEL are genetically distinct subtypes, which was also highlighted by comparison to NEL. Frequent involvement of EPOR/JAK/STAT pathway suggests therapeutic indication of JAK inhibition for AEL especially in the most aggressive type of AEL cases with TP53 mutations. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria; Dainippon-Sumitomo: Honoraria; Nippon-Shinyaku: Honoraria; Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria. Takaori-Kondo:Pfizer: Honoraria; Chugai: Research Funding; Janssen: Honoraria; Kyowa Kirin: Research Funding; Takeda: Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Heuser:Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Ogawa:Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

Introducing an applied reactor for treatment of wastewater containing propylene glycol

Propylene glycol (PG), classed as alcohol, has been used in many industrial additives. Leaching PG in aquatic environments would cause a significant decrease in dissolved oxygen, and this is the main reason for treatment of runoffs and wastewater contaminated with this pollutant. Previous researches on PG removal from wastewater indicate that biological methods are more economically suitable. Therefore, a new biological reactor was introduced and used for the treatment of wastewater containing PG. Two main approaches for increasing removal efficiency in presented research are utilizing two serial lab-scale Sequencing Batch Reactors and feed backward connection between these reactors. This novel reactor was named Feed Backward Serial Sequencing Batch Reactor. Moreover, response surface method was used for modeling of PG treatment and investigation of interactions and simultaneous effects of independent parameters. Retention time, influent COD, and flow recirculation percentage were considered as independent variables, where COD removal efficiency was the dependent variable. According to the results, the best COD removal efficiency was 47%, and it was achieved in 3.52 h retention time, 1667.76 mg/L influent COD, and 23.33% flow recirculation percentage. Based on the results of the presented research, PG treatment with the presented reactor is feasible.

P2.04-53 Serial Ultra-Deep Sequencing of ctDNA Reveals the Clonal Evolution in Non-Small Cell Lung Cancer Patients Treated with Pembrolizumab

Immune-therapy with anti-PD1 inhibitors, such as pembrolizumab, is revolutionizing the treatment of non-small cell lung cancers (NSCLC). However, identifying patients for the potential therapeutic response and predicting therapy resistance and early relapse remains a challenge. Between 2016 and 2018, 60 patients were treated with pembrolizumab, among who 12 NSCLC patients had both baseline (before treatment) and serial (on treatment) periodical circulating tumor DNA (ctDNA) samples. Those samples were sequenced on a 329 pan cancer-related gene panel. Analyses of tumor burden, blood tumor mutational burden (bTMB), maximum somatic allele frequency (MSAF), and tumor clonal structure were performed in association with clinical response. Resistance mutations involved in relapse and metastases were further investigated. ctDNA was detected and mutational profiling was performed for each patient. Those with a high baseline bTMBlevel showed significantly improved progression-freesurvival (PFS) after pembrolizumab treatment. Tumor burden and therapeutic response significantly correlated with the MSAF instead of the bTMB. Clone analysis detected tumor progression about 2-4 months ahead of computedtomography (CT) scan. One mutation in gene PTCH1 (Protein patched homolog 1) and two acquired anti-PD1 resistance mutations of gene B2M (β2 microglobulin) were identified in association with distant metastasis. The evolutionary tree of a representative patient was also described. This pilot study showed thatMSAF could be another good indicator of therapeutic response, and clonal analysis could be clinically useful in monitoring clonal dynamics, and detecting resistance and early relapse.

P1.01-46 Response Assessment Using Plasma Cell-Free DNA (cfDNA) – When Is the Optimal Time to Assess Response?

Plasma cfDNA analysis is routine for non-invasive genotyping of advanced NSCLC, however response assessment using plasma cfDNA is not well characterized. We hypothesized that response in cfDNA would be an early process occurring well before routine imaging timepoints. We retrospectively analyzed a total of 48 baseline and serial on-treatment plasma samples collected from 16 patients enrolled across three Experimental Therapeutics Clinical Trials Network (ETCTN) phase I trials of osimertinib combinations in advanced EGFR-mutant NSCLC. For validation, we also retrospectively analyzed a total of 201 baseline and serial on-treatment samples from an institutional cohort of 67 advanced NSCLC patients receiving systemic treatment. Using droplet digital PCR (ddPCR) of key EGFR or KRAS driver mutations, plasma response was defined as any decrease in mutation concentration to below baseline levels. We compared the magnitude of initial (baseline to day 11-30) and subsequent (day 11-30 to day 36-84) plasma response. Finally, we prospectively assessed response using serial amplicon-based plasma next-generation sequencing (NGS) in a pilot cohort of 8 NSCLC patients starting systemic therapy. Of 15 ETCTN patients with any plasma response, best plasma response was seen at the initial response timepoint in 12 patients (80.0%) and ≥90% of the total plasma response was seen at the initial response timepoint in 14 patients (93.3%). In the validation cohort of 61 patients with any plasma response (Figure), best plasma response was seen at the initial response timepoint in 39 patients (63.9%) and ≥90% of the total plasma response was seen at the initial response timepoint in 52 patients (85.2%). Complete plasma responses (-100%) were seen as early as 11 days after initiating therapy. In the prospective clinical cohort, plasma NGS detected genomic alterations and enabled monitoring of changes in mutant allele fraction in all 8 patients. The median turnaround time of the assay was 8 days. Plasma response is an early phenomenon, with the vast majority of plasma response seen within 30 days, and as early as 11 days. These findings suggest that early plasma cfDNA analysis may permit response assessment well before standard imaging timepoints, with potential as an early marker of drug effect. Additional investigation to understand the relationship between early plasma response, radiographic response, and durability of treatment effect is still needed.

Abstract LB-118: Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation

Abstract Background: TRK inhibition is now standard of care for advanced pediatric and adult patients (pts) with TRK fusion solid tumors, regardless of origin. To date, TRK kinase domain mutations are the only known resistance mechanism, and next-generation TRK inhibitors active against these mutations such as LOXO-195 are being developed. We reasoned some pts will develop TRK-independent resistance and hypothesized that these pts will require unique therapeutic approaches. Methods: Paired tumor biopsies and serial cell-free DNA (cfDNA) prospectively collected from pts with TRK fusion-positive cancers treated with first- and next-generation TRK inhibitors before treatment and at progression were sequenced. In parallel, pt-derived and engineered models were analyzed. Results: Alterations involving upstream non-TRK receptor kinases and downstream MAPK pathway members were initially identified in tumors from 3 TRK fusion-positive gastrointestinal (GI) cancer pts who developed resistance to TRK inhibitors. Pt 1 with CTRC-NTRK1 pancreatic cancer developed temporally distinct emergent BRAF V600E and KRAS G12D mutations. Pt 2 with LMNA-NTRK1 colorectal cancer developed temporally distinct KRAS G12A and G12D mutations. Pt 3 with PLEKHA6-NTRK1 cholangiocarcinoma developed focal MET amplification. Phenocopying these clinical observations, pt-derived xenografts and primary cell lines developed BRAF and KRAS mutations following chronic TRK inhibition. Consistently, ectopic expression of these alterations conferred resistance to TRK inhibitors. Given that all 3 index pts had GI cancers, we expanded serial cfDNA sequencing to 5 additional TRK fusion-positive GI disease, identifying 3 with emergent MAPK alterations at progression, bringing the overall frequency of acquired MAPK alterations in GI cancers analyzed to 75% (6/8). To further evaluate whether these emergent alterations induced functional dependence on ERK signaling, pts 1-3 were treated with agents targeting these emergent alterations (dabrafenib + trametinib, LOXO-195 + trametinib, and LOXO-195 + crizotinib, respectively). Pt 1 achieved transient tumor regression, followed by outgrowth of KRAS-mutant disease. Pt 3 achieved a 4.5 months tumor regression. Sequencing at progression in pt 3 identified multiple acquired MET point mutations known to interfere with crizotinib binding. Conclusions: These data suggest that a subset of TRK fusion-positive cancers will develop off-target mechanisms of resistance to TRK inhibition. Relative to other TRK fusion-positive tumors, GI cancers may have a higher propensity for developing these bypass alterations that demonstrate remarkable convergence on ERK signaling. A portion of these mechanisms may be managed with simultaneous targeting of the TRK and MAPK pathways, although additional modeling is required to determine if upfront treatment would confer more durable responses. Citation Format: Emiliano Cocco, Amanda Kulick, Sandra Misale, Rona Yaeger, Pedram Razavi, Helen H. Won, Ryan Ptashkin, Jaclyn F. Hechtman, Eneda Toska, James Cownie, Romel Somwar, Sophie Shifman, Marissa Mattar, S Duygu Selçuklu, Aliaksandra Samoila, Sean Guzman, Brian B. Tuch, Kevin Ebata, Elisa de Stanchina, Rebecca J. Nagy, Richard B. Lanman, Michael F. Berger, Marc Ladanyi, David M. Hyman, Alexander Drilon, Maurizio Scaltriti, Alison M. Schram. Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation [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 LB-118.

COX-2 mediates tumor-stromal prolactin signaling to initiate tumorigenesis

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy.We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture.By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between.After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC.Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO).To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status.As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Phase 1b/2 Combination Study of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Phase 1b/2 Combination Study of APR-246 and Azacitidine (AZA) in Patients with TP53 mutant Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Very Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy for Prevention of Viral Infection after Allogeneic T Cell Depleted Stem Cell Transplantation

▪Background: Reactivation of latent viruses post-allogeneic stem cell transplant (SCT) negatively affects outcomes and increases non-relapse mortality. We have reported over 80% early CMV reactivation rate and significant additional costs in recipients of T cell depleted (TCD) SCT. Ex vivo generated multi-virus specific T cells (MVSTs) are effective as a therapy of active infection, but have not been evaluated as prophylaxis early after transplant. In a Phase I study (NIH 14-H-0182) we transferred MVSTs targeting immunodominant viral proteins of CMV, Epstein-Barr virus (EBV), BK and adenovirus (Ad) immediately post SCT as a novel approach to prevent viral reactivation post-SCT.Methods: All subjects were enrolled in HLA-matched T cell depleted (TCD) transplant protocol (NIH 13-H-0144). MVSTs cells were manufactured from SCT sibling donors by stimulation of elutriated lymphocytes for 14 days with seven overlapping peptide libraries (pepmixes) pulsed onto autologous dendritic cells (DCs) in presence of IL-7, IL-15 and IL-2. MVSTs were infused as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used as follows: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. Three additional subjects received MVST cells manufactured using pepmix-pulsed mononuclear cells as stimulators (Cohort 3A; 1x10e6TNC/kg) under an amended protocol. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT) defined as Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least “probably” or “definitely” related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes of efficacy, immune reconstitution and GVHD biomarkers (ST2, REG3). CDR3 sequencing (ImmunoSEQ) was performed on selected MVST products and peripheral blood samples post MVST and compared to control SCT recipients. GVHD biomarkers were analyzed pre- and post-treatment.Results: Twelve subjects were treated: nine received MVSTs generated using DCs and three subjects using mononuclear cells (cohort 3A). Median time from SCT to MVST administration was 13 days (range D +2 to +52 post-SCT). Median time to MVST for subjects in Cohort 3A was 3 days (range 2-7). There were no immediate infusion-related adverse events or DLT at the highest dose level. De novo grade II-III aGVHD post-MVST infusion was seen in three subjects (one in Cohort 1 and two in Cohort 3A), but GVHD biomarker elevation predated MVST infusion. CMV reactivation post-MVST occurred in 6 out of 12 subjects (50%) vs. 45 out of 52 patients (50% vs 87%) in a historical group of recipients of T cell depleted SCT. In all cases CMV reactivation occurred in the context of high dose steroids and in two subjects MVSTs were derived from CMV seronegative donors with minimal anti-CMV activity. One subject experienced rapidly rising AdV viremia (asymptomatic) and received an additional infusion of MVSTs. We saw self-limiting low level EBV replication in 8 cases and one BK viremia, but no disease. CDR3 sequencing of MVST products and serial peripheral blood from subjects revealed a robust contribution of ex vivo expanded cells to the overall repertoire, in contrast to untreated controls where the repertoire of (sham) MVST cell products generated from the transplant donors did not significantly overlap with the immune repertoire in peripheral blood of TCD-SCT recipients in the early post SCT period. Only at day +180 some convergence of repertoires became visible indicating spontaneous immune reconstitution (Figure). Detailed CDR3 analysis of cytokine-captured CMV pp65 and IE-1 specific CD4+ and CD8+ T cells was performed in a representative subject clearly demonstrating the detrimental effect of high-dose steroids on frequency of anti-viral T cells, precipitating CMV reactivation.Conclusions: This is the first report demonstrating safety and feasibility of using MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen and a minimal risk of aGVHD was observed, as there was no correlation with GVHD biomarkers. As revealed by serial CDR3 sequencing, MVSTs robustly contributed to the T cell repertoire. Our results suggest efficacy of this strategy in reducing viral reactivation. A Phase II study is warranted. DisclosuresNo relevant conflicts of interest to declare.

Genomic Instability and a Preferential Involvement of Ras Pathway in the Myelodysplastic Syndromes Evolution to Secondary Acute Myeloid Leukemia

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are hematological disorders at high risk of progression to acute myeloid leukemia (sAML). Previous high-throughput sequencing studies have provided insight into the mutational dynamics and clonal evolution underlying disease progression. However, large serial sequencing studies are still required to define which type of mutations alone or in combination contribute to leukemic transformation.To assess the mutational profiles and mutational dynamics underlying progression from MDS to sAML, a targeted-deep sequencing (TDS) of 117 MDS/AML related-genes was performed in 110 bone marrow serial samples from 50 MDS/CMML patients who evolved to sAML and 5 patients who did not evolved (controls), at two different time-points: at the time of diagnosis and at sAML progression or after a median of 3 year follow-up, respectively.A total of 269 mutations in 57 different genes were identified at second sampling. At diagnosis, all patients, progressing and not progressing (controls), presented similar number of mutations (p=0.15). Moreover, patients evolving to sAML were then divided by FAB/WHO subtypes at diagnosis (CMML, low-risk and high-risk MDS subgroups) and no differences were observed in the number of mutations (p=0.71) and variant allele frequency (VAF) between each group (p=0.63). It should be noted that mutations in the splicing pathway were significantly more frequent in low-risk MDS patients (89% low-risk MDS vs. 56% high risk MDS, p=0.038). However, after progression, those patients who evolved to sAML displayed a statistically significant increase of mutations (p=0.001) at the leukemic phase, while controls did not at the follow-up sample (p=0.88). This higher number of mutations at second sampling in patients who evolved to sAML, independently of their diagnostic subtype, may be indicative of a higher genomic instability during disease evolution.To study the mutational dynamics and what mutations could be important during disease evolution, the VAFs of mutations detected at both time-points in each patient of transformation cohort were compared. We observed that some mutations identified at the sAML stage (163 mutations) were already present at the MDS stage, at clonal or subclonal levels, and were retained during evolution, for example in genes such as SRSF2 and DNMT3A. However, 106 mutations increased in clonal size or were newly acquired. Interestingly, most of mutations in Ras signaling pathway showed a same pattern: they were not present at time of diagnosis and appeared at sAML. In fact, mutations in this pathway were detected in 25 of 50 patients (50%) included in this cohort and in 22 of them (88%) mutations displayed this dynamic. Therefore, in this study, Ras signaling was the most common pathway involved in the progression from MDS to sAML. Of note, 9 of these patients (18% of the whole cohort) presented, independently of diagnosis, a co-occurring cohesin mutation, that was already present at diagnosis and, in most cases, markedly increased in clonal size at sAML. Thus, the combination of mutations in these two pathways could play an important role during disease evolution.In addition, 22 of 50 patients were treated with a disease-modifying agent (18 azacytidine and 4 lenalidomide) before they progressed to sAML, while the remaining 28 patients received no treatment or supportive care and were considered as non-treated. Thus, we studied the effect of disease-modifying therapy on mutational dynamics in this cohort of patients progressing to sAML. In the treated patients, a higher proportion of newly acquired or increasing mutations at sAML in chromatin modifiers was observed, while in non-treated patients most mutations remained stable (61% vs. 28.6%, p=0.013). By contrast, regarding treatment, no differences were detected in the mutational dynamics of cohesin (p=0.56) or Ras pathway (p=1.00).MDS progression to sAML was characterized by a higher genomic instability, independently of MDS subtypes of patients at diagnosis. Ras signaling was the most frequent affected pathway during disease evolution in this cohort and, interestingly, the co-occurrence of Ras signaling and cohesin mutations could play an important role in the progression. Moreover, mutations in chromatin modifiers genes could be related to the evolution of patients who received disease-modifying treatment before progression to sAML. DisclosuresOlivier:Celgene: Honoraria; Jassen: Honoraria. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract CT068: Phase Ib/II combination study of APR-246 and azacitidine (AZA) in patients with TP53 mutant myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)

Abstract Introduction: TP53 mutant (mTP53) MDS and AML represent a distinct molecular cohort with poor outcomes. Hypomethylating agents (HMA) have emerged as preferred treatment for these patients with CR rate of 20-30% and median OS of 6-12 months. APR-246 is a mutant p53 activator with single agent activity in mTP53 AML. We report initial phase 1b results of APR-246 + AZA in mTP53 MDS/AML. Methods: Eligible pts included HMA naïve mTP53 MDS and oligoblastic AML (≤ 30% blasts) ≥ 18 years of age. Pts received APR-246 in a 3+3 dose escalation design (50, 75, 100 mg/kg lean body weight) IV daily over 4 days in a lead-in phase (days -14 to -10) followed by the same dose of APR-246 (days 1-4) + AZA 75 mg/m2 SC/IV over 7 days (days 4-10 or 4-5 and 8-12) in 28 day cycles. Primary objective was safety with AEs graded by CTCAE v4.03 and DLT assessment over 6 weeks. Secondary endpoints included response by IWG 2006 criteria as well as serial next generation sequencing (NGS) and p53 IHC for evaluation of clonal suppression and remission depth. Results: As of Jan 1, 2017, 9 pts (33% male; median age 65 years (39-73)) have enrolled with 3 pts per cohort. Three pts had AML-MRC and 6 had MDS; all pts had poor risk cytogenetics (11% poor, 89% very poor) and higher risk disease by IPSS-R (22% high, 78% very high). Median BM blasts were 18% (4-30). Seven pts (78%) remain on study: 2 pts in the 50mg/kg cohort discontinued treatment (Tx), 1 pt due to infection during C2 who later died of sepsis unrelated to Tx, and 1 pt electively discontinued in durable marrow CR (mCR) after 5 cycles of therapy. Median time on study is 106 days (14-221). Tx related AEs during the lead-in phase (all G1) included ataxia (n=1), dizziness (n=1), and facial numbness (n=1). AEs occurring in > 1 pt included dizziness (n=3), nausea (n=3), neutropenia (n=3), thrombocytopenia (n=3), infection (n=3), headache (n=2), pain (n=2), weakness (n=2), falls (n=2), facial numbness (n=2), and ataxia (n=2); all G1/G2 except neutropenia/thrombocytopenia (G4). No Tx-related SAEs or DLTs have occurred to date. Five of six pts were response evaluable with 1 pt discontinuing tx prior to 1st disease assessment. ORR by IWG was 100% with 4 CR (80%, 3/3 in DL2) and 1 mCR. All CR patients achieved complete cytogenetic response. One CR patient achieved a mCR and partial cytogenetic response after APR-246 lead-in prior to combination therapy. All CR pts had high p53 positivity by IHC at baseline (55-70%) which normalized on serial assessment (<5%). Serial NGS with a variant allele frequency (VAF) cutoff of 2% was negative in 80% of patients (4/5). The remaining 3 pts entered the study in Dec 17/Jan 18 with no response data available at data cutoff. Conclusions: APR-246 + AZA combination is well tolerated in mTP53 MDS/AML. Responses have been achieved in all pts (80% CR) accompanied by deep molecular remissions. The maximum tolerated dose has not been reached and dose escalation is ongoing. Citation Format: David A. Sallman, Amy DeZern, Kendra Sweet, David P. Steensma, Thomas Cluzeau, Mikkael Sekeres, Guillermo Garcia-Manero, Gail Roboz, Amy McLemore, Kathy McGraw, John Puskas, Ling Zhang, Chirag Bhagat, Armin Graber, Najla H. Al Ali, Eric Padron, Roger Tell, Jeffrey E. Lancet, Pierre Fenaux, Alan F. List, Rami S. Komrokji. Phase Ib/II combination study of APR-246 and azacitidine (AZA) in patients with TP53 mutant myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [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 CT068.

Abstract 3004: Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression

Abstract Myelodysplastic syndromes (MDS) are a collection of clonal diseases with dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. MDS is increasingly being recognized as a molecularly heterogeneous disease with variability in clinical phenotype, prognosis, and response to treatment. The clonal evolution in response to therapy remains a challenge in the management of MDS. However, resolution of complex clonal architectures is difficult with current bulk sequencing approaches. High-throughput single-cell genomic profiling enables the resolution of tumor heterogeneity, and may improve clinical diagnosis and treatment monitoring by allowing for characterization and early identification of resistant subclonal populations. To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cells confined to droplets. The barcodes were then used to reassemble the genetic profiles of cells from next-generation sequencing data. We applied this approach to sequential clinical MDS samples, genotyping the most clinically relevant loci across more than 15,000 individual cells. Additionally, to study effects of subclonal mutations on drug sensitivity, ex vivo functional testing was performed on red blood cell-lysed peripheral blood and/or bone marrow aspirate patient samples. Targeted single-cell sequencing was able to recapitulate bulk sequencing data from both peripheral blood and bone marrow aspirate samples. Furthermore, the single-cell nature of our approach enabled definitive determination of mutational co-occurrence within the same cell. For examples in one sample, bulk sequencing identified mutations in JAK2 and NRAS both at 3% variant allele frequency (VAF), where single-cell analysis suggested that these mutations were mutually exclusive, each defining a distinct subclone. Single-cell sequencing allowed for serial monitoring of clonal evolution, with analysis of sequential samples from this same patient showing increase of NRAS clone from 2.5% to 24.7% at time of disease progression after hypomethylating agent therapy. Furthermore, single-cell analysis was able to identify a distinct subclone characterized by a KRAS mutation (0.4% at initiation of therapy and 6.7% at relapse), missed by serial bulk sequencing. Taken together, our results suggest a greater degree of heterogeneity in MDS samples than suggested with bulk sequencing methods alone, and demonstrate utility of single-cell sequencing for sequential monitoring and identification of resistant clones prior to therapy initiation. We show here that this approach is a feasible, scalable, and effective way to identify and track heterogeneous populations of cells in MDS. Citation Format: Alexey Aleshin, Peter L. Greenberg, Bruno C. Medeiros, Diane Heiser, Marianne Santaguida, Sacha Prashad, Robert Durruthy- Durruthy, Dennis J. Eastburn. Single-cell mutational profiling of clonal evolution in myelodysplastic syndromes (MDS) during therapy and disease progression [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 3004.

Prospective serial sequencing of CTC/cfDNA and NK cell activity in patients undergoing multimodality treatment for triple negative breast cancer.

e24108Background: Patients with early stage triple negative breast cancer (TNBC) are treated with multimodality therapy (chemotherapy, surgery, radiation). Cell-free DNA (cfDNA) and circulating tum...

The formation of the light-sensing compartment of cone photoreceptors coincides with a transcriptional switch.

High-resolution daylight vision is mediated by cone photoreceptors. The molecular program responsible for the formation of their light sensor, the outer segment, is not well understood. We correlated daily changes in ultrastructure and gene expression in postmitotic mouse cones, between birth and eye opening, using serial block-face electron microscopy (EM) and RNA sequencing. Outer segments appeared rapidly at postnatal day six and their appearance coincided with a switch in gene expression. The switch affected over 14% of all expressed genes. Genes that switched off were rich in transcription factors and neurogenic genes. Those that switched on contained genes relevant for cone function. Chromatin rearrangements in enhancer regions occurred before the switch was completed, but not after. We provide a resource comprised of correlated EM, RNAseq, and ATACseq data, showing that the growth of a key compartment of a postmitotic cell involves an extensive switch in gene expression and chromatin accessibility.

Response and progression on midostaurin in advanced systemic mastocytosis: KIT D816V and other molecular markers

AbstractIn advanced systemic mastocytosis (advSM), disease evolution is often triggered by KIT mutations (D816V in >80% of cases) and by additional mutations (eg, in SRSF2, ASXL1, and/or RUNX1 [S/A/Rpos in >60% of cases]). In a recently reported phase 2 study, midostaurin, a multikinase/KIT inhibitor, demonstrated an overall response rate (ORR) of 60% in advSM but biomarkers predictive of response are lacking. We evaluated the impact of molecular markers at baseline and during follow-up in 38 midostaurin-treated advSM patients. The median overall survival (OS) was 30 months (95% confidence interval, 6-54) from start of midostaurin. ORR and OS were significantly different between S/A/Rneg (n = 12) and S/A/Rpos (n = 23) patients (ORR: 75% vs 39%, P = .04; OS: P = .01, HR 4.5 [1.3-16.2]). Depending on the relative reduction of the KIT D816V expressed allele burden (EAB) at month 6, patients were classified as KIT responders (≥25%, n = 17) or KIT nonresponders (<25%, n = 11). In univariate analyses at month 6, reduction of KIT D816V EAB ≥25%, tryptase ≥50%, and alkaline phosphatase ≥50% were significantly associated with improved OS. In multivariate analysis, only KIT D816V EAB reduction ≥25% remained an independent on-treatment marker for improved OS (P = .004, HR 6.8 [1.8-25.3]). Serial next-generation sequencing analysis of 28 genes in 16 patients revealed acquisition of additional mutations or increasing variant allele frequency in K/NRAS, RUNX1, IDH2, or NPM1 associated with progression in 7 patients. In midostaurin-treated advSM patients, the complexity and dynamics of mutational profiles significantly affect response, progression, and prognosis.

Serial genome-wide sequencing of cell-free DNA (cfDNA) by assays used in non-invasive prenatal testing (NIPT) to identify copy number alterations (CNAs) associated with immunotherapy response.

e14533 Background: cfDNA derived from tumor cells can be detected in the plasma of many cancer patients. Noninvasive methods to detect CNAs have been developed to detect chromosomal abnormalities in the setting of prenatal screening. We hypothesized that similar methods can be used to enable quantitative detection of CNAs in cfDNA, as represented by a genome instability number (GIN), in patients with cancer and used to monitor response to immunotherapy. Methods: Peripheral blood for CNA analysis was collected from patients with diverse cancers for whom immunotherapy was planned. Plasma was separated from peripheral blood using centrifugation. cfDNA was collected and subjected to low-coverage, genome-wide sequencing. CNAs were detected using methods developed for noninvasive prenatal testing. GIN was calculated as the cumulative sum of the deviation from baseline for all detected autosomal CNAs. The current analysis focused on patients with a complete or partial response (CR/PR) or progressive disease (PD). Results: Of 48 patients, 11 (23%) attained CR/PR; 17 (35%), PD; 20, stable disease. Twelve of the 28 patients (43%) with CR/PR (5 (42%)) or PD (7 (58%)) had a GIN value consistent with the presence of CNAs at treatment initiation and ≥ 1 additional sample for comparison. Four of the 5 patients with CR/PR had a decrease in their GIN from their baseline while all 7 patients with PD had a sustained increase in their GIN. The mean (standard error of the mean (SEM)) change in GIN from maximum to minimum for responders was –2753.8 (+/-1643); mean (SEM) change in GIN from minimum to maximum for progressors was 1373.76 (+/-576) (p = 0.0221). Interestingly, 3 of the 5 responders had an initial increase in their GIN after 2-3 weeks of treatment followed by a decline, while all 7 progressors had a continuous increase in their GIN. Conclusions: GIN can be used to monitor response to immune checkpoint inhibitors in cancer patients. Serial evaluation shows an initial GIN increase followed by decline in patients who eventually attain CR/PR, but a sustained increase in patients with progression.