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Introduction: PD-1 inhibitors have shown limited efficacy in glioblastoma due to microenvironment immunosuppression and low tumor mutational burden. In GBM, PD-L1 expression is not a predictive marker for response to PD-1 or PD-L1 inhibitors. Multiplex immunostaining panel technology allows for detailed analyses of tumor microenvironment cells and their interaction. Our study to evaluates the feasibility and predictive value of quantitative spatial profiling in GBM.Methods: Pre-treatment tumor tissue was collected retrospectively from 27 patients at Columbia University Irving Medical Center with primary glioblastoma who were diagnosed within the past three years, had surgery here, and were either treated with SOC therapy (n= 8) or PD-1 inhibitors at recurrence (n= 19). Multiplex immunofluorescence panels included 1) CD11b/IDO1/HLADR/GFAP, 2) PD1/PD-L1/GFAP, and 3) CD4/CD8/CD25/FoxP3/Ki67/GFAP. Results: Multiplex immunofluorescence panels did not show any correlation with outcomes in patients treated with SOC therapy. Among the 19 patients treated with PD-1 inhibitors, those with more HLA-DR positive cells had worse outcomes (p= 0.02). PD-L1 expression on tumor cells was not predictive of outcomes. There was a correlation trend between PD-1/PD-L1 interaction score (p= 0.08) and outcomes. PTEN loss was correlated with higher Ki67 expression in both tumor cells (p= 0.05) and non-tumors cells (p= 0.03); however, this was not found in Ki67 in CD4+ cells, CD8+ cells, or CD4+CD8+ cells combined. Tumor-associated macrophages, myeloid-derived suppressor cells, CD8+ cells, and CD4+ cells were not significant predictive markers for outcome. Conclusion: Quantitative spatial profiling by multiplex immunofluorescence is feasible in FFPE glioblastoma tissue. More refined and extensive quantitative and spatial microenvironment analyses may allow for the development of biomarkers for immunotherapy in GBM.

Background: Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. Methods: We assessed multiple immunologic biomarkers in a series of 99 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E-based TILs analysis, standard immunohistochemistry (IHC; CD8, CD20, CD56, FOXP3, LAG-3, B7-H4, HLA-A) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK, PD1, CD3). Association among parameters were assessed (up to n=98) including clinical outcome after surgery (n=94). Results: As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS (p=0.0093 and p=0.0376, respectively). H&E-scored TILs were highly correlated to CD3, CD4 and CD8 positive T cells (Spearman r range 0.34-0.4850; all p Conclusions: We have found a paradoxical association of GZMB+ CD8+ T cells with a negative prognosis in NAC-treated TNBC. We hypothesize that while these T cells are poised for cytotoxic activity, they remain restricted through sub-localization outside the tumor core, downregulation of HLA-A on tumor cells preventing interaction with the T cell receptor, and upregulation of B7H4 expression, which has been shown to inhibit cytotoxic T cell activity. Patients with high CD8+GZMB+ tumors in the post-NAC setting may benefit from adjuvant immunotherapy, particularly in combination with therapies that enhance MHC-I (e.g. HLA-A) antigen presentation. Citation Format: Paula I Gonzalez-Ericsson, Violeta Sanchez, Roberto Salgado, Jennifer Bordeaux, Ju Y Kim, Christine Vaupel, Henry Gomez, Melinda E Sanders, Justin M Balko. The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-07.

Background Approximately 10% of patients with SCLC develop a paraneoplastic syndrome (PNS). Neurologic PNS are thought to improve prognosis, which we hypothesized is related to increased tumor-infiltrating lymphocytes and immune recognition. Methods We queried 2,512,042 medical records from a single institution to identify patients who have SCLC with and without PNS and performed manual, retrospective chart review. We then performed multiplexed fluorescence immunohistochemistry and automated quantitative analysis (AQUA Technology) on tumors to assess CD3, CD4, and CD8 T cell infiltrates and programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interactions. T cell infiltrates and PD-1/PD-L1 interaction scores were compared among patients with neurologic PNS, endocrinologic PNS, and a control group without PNS. Clinical outcomes were analyzed using the Kaplan-Meier method and Cox proportional hazards models. Results We evaluated 145 SCLC patients: 55 with PNS (25 neurologic and 30 endocrinologic) and 90 controls. Patients with neurologic PNS experienced improved overall survival compared to patients with endocrinologic PNS and controls (median overall survival of 24 months versus 12 months versus 13 months, respectively). Of the 145 patients, we identified tumor tissue from 34 patients that was adequate for AQUA analysis. Among 37 specimens from these 34 patients, patients with neurologic PNS had increased T cell infiltrates (p = 0.033) and PD-1/PD-L1 interaction (p = 0.014) compared to tumors from patients with endocrinologic PNS or controls. Conclusions Tumor tissue from patients with SCLC with neurologic PNS showed increased tumor-infiltrating lymphocytes and PD-1/PD-L1 interaction consistent with an inflamed tumor microenvironment.

BACKGROUND PD-1 inhibitors have shown limited efficacy in glioblastoma (GBM) due to microenvironment immunosuppression and low tumor mutational burden. In GBM, PD-L1 expression is not a predictive marker for response to PD-1 or PD-L1 inhibitors. Multiplex immunostaining panel technology allows for detailed analyses of tumor microenvironment cells and their interaction. METHODS Pre-treatment tumor tissue was collected retrospectively from 27 patients in our neurooncology database at Columbia University Irving Medical Center with primary glioblastoma who were diagnosed within the past three years, had surgery here, and were either treated with SOC therapy (n= 8) or PD-1 inhibitors at recurrence (n= 19). Multiplex immunofluorescence was done for 1) CD11b/IDO1/HLADR/GFAP, 2) PD1/PD-L1/GFAP, and 3) CD4/CD8/CD25/FoxP3/Ki67/GFAP. RESULTS Multiplex immunofluorescence panels did not show any correlation with outcomes in patients treated with SOC therapy (non-immunotherapy). Among the 19 patients treated with PD-1 inhibitors, those with more HLA-DR positive cells had worse outcomes (p= 0.02). PD-L1 expression on tumor cells was not predictive of outcomes. There was a correlation trend between PD-1/PD-L1 interaction score (p= 0.08), which measures density of PD-1-positive cells in proximity to PD-L1-positive cells, and outcomes. PTEN loss was correlated with higher Ki67 expression in both tumor cells (p= 0.05) and non-tumors cells (p= 0.03); however, this relationship was not found when looking at Ki67 in CD4+ cells, CD8+ cells, or CD4+CD8+ cells combined. This assay allowed us to evaluate tumor- associated macrophages, myeloid-derived suppressor cells, CD8+ lymphocytes, and CD4+ T regulatory cells; however, none of these were significant predictive markers for outcome. CONCLUSION Quantitative spatial profiling by multiplex immunofluorescence is feasible in FFPE glioblastoma tissue. More refined and extensive quantitative and spatial microenvironment analyses may allow for the development of biomarkers for immunotherapy in GBM.

Triple-negative breast cancers (TNBCs) are heterogeneous and aggressive, with high mortality rates. TNBCs frequently respond to chemotherapy, yet many patients develop chemoresistance. The molecular basis and roles for tumor cell–stromal crosstalk in establishing chemoresistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient–derived xenografts (PDXs) established before and after the development of chemoresistance. Interestingly, the chemoresistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemoresistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibited increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemoresistant model exhibited increased immunosuppression, including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemoresistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established Ras/MAPK-associated gene expression signatures correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T cell–recruiting chemokines (CXCL9/10/11) across patients with TNBC, even in the absence of KRAS mutations. MEK inhibition induced tumor suppression in mice while reversing metabolic and immunosuppressive phenotypes, including chemokine production and gMDSC tumor recruitment in the chemoresistant KRAS-mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemoresistance., Longitudinally-derived TNBC xenografts identify acquired KRAS mutation/MEK activation as a driving feature of CXCL1/2-mediated MDSC recruitment.

Programmed cell death protein 1/programmed cell death protein ligand1 (PD-1/PD-L1) interaction is an important immune checkpoint targeted by anti-PD-1/PD-L1 immunotherapies. However, the observed prognostic significance of PD-1/PD-L1 expression in diffuse large B-cell lymphoma treated with the standard of care has been inconsistent and even contradictory. To clarify the prognostic role of PD-1/PD-L1 expression and interaction in diffuse large B-cell lymphoma, in this study we used 3-marker fluorescent multiplex immunohistochemistry and Automated Quantitative Analysis Technology to assess the CD3 +, PD-L1 +, and PD-1 +CD3 + expression in diagnostic samples and PD-1/PD-L1 interaction as indicated by presence of PD-1 +CD3 + cells in the vicinity of PD-L1 + cells, analyzed their prognostic effects in 414 patients with de novo diffuse large B-cell lymphoma, and examined whether PD-1/PD-L1 interaction is required for the prognostic role of PD-1 +/PD-L1 + expression. We found that low T-cell tissue cellularity, tissue PD-L1 + expression (irrespective of cell types), PD-1 +CD3 + expression, and PD-1/PD-L1 interaction showed hierarchical adverse prognostic effects in the study cohort. PD-1/PD-L1 interaction showed higher sensitivity and specificity than PD-1 + and PD-L1 + expression in predicting inferior prognosis in patients with high CD3 + tissue cellularity (“hot”/inflammatory tumors). However, both PD-1 + and PD-L1 + expression showed adverse prognostic effects independent of PD-1/PD-L1 interaction, and PD-1/PD-L1 interaction showed favorable prognostic effect in PD-L1 + patients without high CD3 + tissue cellularity. Macrophage function and tumor-cell MYC expression may contribute to the PD-1-independent adverse prognostic effect of PD-L1 + expression. In summary, low T-cell tissue cellularity has unfavorable prognostic impact in diffuse large B-cell lymphoma, and tissue PD-L1 + expression and T-cell-derived PD-1 + expression have significant adverse impact only in patients with high T-cell infiltration. PD-1/PD-L1 interaction in tissue is essential but not always responsible for the inhibitory effect of PD-L1 +/PD-1 + expression. These results suggest the benefit of PD-1/PD-L1 blockade therapies only in patients with sufficient T-cell infiltration, and the potential of immunofluorescent assays and Automated Quantitative Analysis in the clinical assessment of PD-1/PD-L1 expression and interaction.

Highlights • Neoadjuvant ipilimumab and IFNα were evaluated in regionally advanced melanoma. • Immune cellular profiling investigated tumor immune susceptibility and resistance. • Higher levels of peripheral Th1 cell subsets predicted favorable clinical outcomes. • Higher levels of peripheral Th2 cells was associated with poor prognosis. • Significant reductions in peripheral T-reg and MDSC were seen in responders., Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.

e15036 Background: Patients with locally/regionally advanced melanoma were treated on a clinical trial with a neoadjuvant combination of ipilimumab (ipi) and high dose IFNα2b (HDI) (Tarhini et al, JITC 2018). In this study, immune cell composition in peripheral blood samples collected at various time points was measured to determine any correlation with clinical outcomes and investigate the immune modulating effect of the combination therapy. Methods: Patients were randomized to neoadjuvant ipi at 3 mg/kg or 10 mg/kg, both given in combination with HDI. Tumor radiologic responses were designated as complete (CR), partial (PR), stable disease (SD) or disease progression (PD). Pathologic complete response (pCR) was defined as absence of viable tumor on histologic assessment. Peripheral blood mononuclear cells (PBMC) from treated patients (N = 28) were tested at baseline (before initiating ipi-HDI), then at 6-weeks, 3-months and 12-months (following neoadjuvant ipi-HDI). High complexity (14-color) flow cytometry analysis was performed to detect key immunological biomarkers including myeloid derived suppressor cells (MDSCs), B cells, regulatory T cells (Tregs), PD-1 and TIM3 expression on T-cells, and differentiation of T-cells into Th1, Th2 or Th17 phenotype at different time points during systemic immunotherapy. Statistical significance was determined using R-package employing Kruskal’s test. Results: Lower levels of peripheral Tregs (p = 0.02), MDSCs (p = 0.05), and CD4 effector memory cells (p = 0.04) at 3-months post treatment correlated with radiologic response. In addition, lower change from baseline at 3 months in CD4/CD8 ratio (p = 0.04), levels of Tregs (p = 0.01) and CD4 effector memory cells (p = 0.02) was associated with radiologic response. Patients exhibiting pCR had significantly lower Tregs (p = 0.04) at 6-months post treatment and significantly higher CD8 central memory cells at both 3 months (p = 0.04) and 12 month time-points (p = 0.01) as compared to patients without pCR. Finally, patients without pCR had significantly lower change from baseline in CD19 B cells at 6 months (p = 0.01) and 12 months (p = 0.04) as compared to patients with pCR. Conclusions: Our data demonstrates that the levels of immunosuppressive cells including Tregs and MDSCs in periphery are negatively associated with response. Higher levels of CD8 memory cells and B cells on-treatment are associated with clinical benefit.

Purpose: PD-1/L1 axis–directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti–PD-1 response. Experimental Design: Pretreatment tumor biopsies from 166 patients treated with anti–PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (n = 24) and validation (n = 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti–PD-1 treatment outcomes. Results: In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti–PD-1 response (P = 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond (P = 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36; P = 0.0004) and overall survival (HR = 0.39; P = 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers (P = 0.000004). In contrast, PD-L1 expression was not predictive of survival. Conclusions: Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy. Clin Cancer Res; 24(21); 5250–60. ©2018 AACR.

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes between tumor and stromal cells involved in developing chemo-resistance are under-explored. Here we report studies of paired TNBC patient-derived xenografts (PDX) established before and after chemo-resistance. Despite significant genetic similarities, the chemo-resistant PDX model harbored a rare constitutively-active KRASQ61R mutation which was not present in the chemo-naive PDX. Further analysis demonstrated that the chemo-resistant KRAS-mutant model exhibited altered gene expression changes including increased expression of CXCR2-ligands CXCL1 and CXCL2, which are responsible for recruiting immune cells to tumors. These expression patterns were largely inhibited in vivo by MEK inhibitor (MEKi) treatment. Moreover, in breast cancer cell lines, CXCL1, CXCL2, and granulocyte macrophage-colony stimulating factor (CSF2, stimulates granulocyte and macrophage differentiation from hematopoietic precursor cells, including immunosuppressive myeloid cells) transcripts were also downregulated by MEKi. Notably, chemo-resistant KRAS-mutant tumors harbored increased Gr1+ and Arginase-1+ cells, consistent with recruitment of immunosuppressive M2-like macrophages and/or myeloid-derived suppressor cells (MDSCs), which was inhibited by MEKi. Further experiments demonstrate that CD45+CD11b+Ly6G+ MDSC accumulation in tumors can be inhibited by MEKi treatment alone, or by CXCR2 inhibition, suggesting that the effects of MEK inhibition on MDSC recruitment are CXCL1/2-dependent. Confirming the translational relevance of these findings, in >200 murine and >1000 human breast tumors, Ras/MAPK transcriptional activity correlated with myeloid-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11), even in the absence of activating KRAS mutations. The association with Ras/MAPK activity was also confirmed using immunofluorescence to quantify MHC-II-low myeloid cells in 80 post-chemotherapy TNBC tumors. Importantly, MEKi and chemotherapy combination treatment reversed immunosuppressive cell accumulation and metabolic phenotypes exemplified by altered optical redox ratios (NAD(P)H/FAD) in the chemo-resistant KRAS mutant tumors, resulting in tumor growth suppression in mice. MEKi treatment also reduced redox ratios in 3D cultures of breast cancer cell lines further suggesting that MEK inhibition targets multiple oncogenic processes in breast cancer. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistant disease. Citation Format: Derek A. Franklin, Joe T. Sharick, Paula I. Ericsson-Gonzalez, Violeta Sanchez, Phillip T. Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko. MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC [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 1511.

Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but the composition of TILs has not been explored in granularity, and how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. We assessed multiple immunologic biomarkers in a series of 100 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E TILs analysis, standard immunohistochemistry (IHC; HLA-A, CD4, CD8, LAG-3) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK). TILs and IHC were scored by clinical research pathologists and IF was scored by AQUA® analysis. Where multiple markers were multiplexed together, subset analyses was performed. Association among parameters were assessed (n=83) including clinical outcome after surgery (n=79). As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS. H&E-scored TILs were correlated to both CD4 and CD8 as scored by IHC or IF (Pearson’s r range 0.41-0.55; all p Citation Format: Caroline Nebhan, Paula I. Gonzalez-Ericsson, Roberto Salgado, Jennifer Bordeaux, Ju Young Kim, Christine Vaupel, Henry Gomez, Justin Micah Balko. Molecular characterization of residual triple-negative breast cancers after neoadjuvant chemotherapy identifies immune composition and features associated with clinical outcome [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 4050.

Background Ever increasing knowledge of the interplay between immune composition and a novel drugs’ treatment efficacy or the lack thereof leads to continual advancements in immunotherapies. Given the exponential increase in combination therapies, tools to determine the most promising regimens are critical. Immunotherapeutic agents either reverse T-cell inhibitory pathways (e.g., PD-1) or activate the immune system to induce cytotoxic T-cell activation and proliferation through agonistic stimulation (e.g., Cytokines). These treatment strategies have proven to be efficacious by enhancing the balance between immune-effector and suppressive/regulatory cells that can directly affect the outcome of immunotherapy. To concurrently monitor multiple immune cell subsets responsible for either immuno-suppression or activation, we developed a high-complexity (13-color) flow cytometry panel to reliably enumerate activated T-cells, Tregs (A & B subsets) and Bregs for clinical trial application. Methods The panel includes CD3, CD4, CD8, CD19, FoxP3, CD25, CD127, CD45RA, HLA-DR, CD38, CD24, and CD27 to monitor total Tregs, Treg-subsets, Bregs and T-cell activation. After robust analytical method validation following industry guidelines for exploratory biomarker assessments, we confirmed the clinical utility of this panel in stage III or IV melanoma patients (N= 14) enrolled in a randomized phase II clinical trial receiving high-dose of IL-2 with or without Afliberecept (VEGF inhibitor). Results We observed up-to 20-fold increase in activated CD8+ T-cells following treatment. Interestingly, while no discernible change was observed in the naive Treg-A cells (CD45RAhiFoxp3lo), a remarkable 5-fold expansion of effector Treg-B cells (CD45RAloFoxp3hi) was observed following treatment. Additionally, frequencies of Bregs (CD24hiCD38hi) remained unchanged after treatment. Conclusions In summary, we demonstrated the utility of a powerful high complexity clinical flow cytometry tool for precisely monitoring immune cell subsets involved in tumor escape such as Tregs and Bregs or those involved in tumor killing like CD8+ cytotoxic T-cells. More notably, this tool may be utilized to deprioritize agents that cause immunosuppression and focus on combination agents that lead to additive immune activation. Citation Format: Jennifer Tsau, Michael Abadier, Brittney Atzmiller, Christine Vaupel, Naveen Dakappagari, Ghanashyam Sarikonda, Ahmad Tarhini. Application of high complexity flow cytometry for Monitoring activated T-cells, T and B regulatory subsets in combination immunotherapy trials, melanoma case study [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 4043.

Background Among patients with small cell lung cancer (SCLC), approximately 20% develop a paraneoplastic syndrome (PNS). Neurologic PNS are immune-mediated phenomena predicated on host recognition of an onconeural antigen, while endocrinologic PNS are attributed to ectopic tumor secretion of normal hormones. The presence of neurologic PNS improves prognosis and endocrinologic PNS worsens prognosis in patients with SCLC. We hypothesized that tumors from patients with neurologic PNS may have increased TILs and PD-1/PD-L1 expression compared to tumors from patients with endocrinologic PNS and this improved immune recognition accounts for prognostic differences. Methods We searched electronic medical record text stored in the Vanderbilt University Medical Center (VUMC) clinical data warehouse to identify SCLC patients with and without a PNS. We obtained clinical information through manual, retrospective chart review using an IRB-approved protocol. Overall survival (OS) and progression free survival (PFS) were compared using a log rank test. Archived formalin fixed, paraffin embedded samples were obtained from the Vanderbilt University Pathology Tissue Repository. We performed multiplexed fluorescence immunohistochemistry combined with automated quantitative analysis (AQUA® Technoloy; Navigate BioPharma Services, Inc.) to assess PD-1, PD-L1, CD4, and CD8 expression. A PD-1/PD-L1 interaction score was calculated by measuring the total area of PD-1 positive cells within the proximity of PD-L1 positive cells. This area was then divided by the total area of all non-tumor nucleated cells in the image and multiplied by a factor of 10,000. CD4, CD8, and PD-1/PD-L1 interaction scores were compared using a two sample t-test or Wilcoxon Rank Sum test. Results A total of 145 SCLC patients were identified, 55 with a PNS (25 neurologic and 30 endocrinologic) and 90 control patients. Patients with neurologic PNS exhibited significantly improved OS and PFS compared to patients with endocrinologic PNS and control patients (median OS 24mo, 95% CI 16.4mo-not reached (NR), vs 12mo, 95% CI 8.3mo-15.5mo, vs 13mo, 95% CI 12.2mo-16mo; median PFS 14mo, 95% CI 9.3mo-NR vs 6mo, 95% CI 4.6mo-9.5mo, vs 7mo, 95% CI 6.6mo-8.1mo, respectively). Tumors from patients with neurologic PNS (n=9) had statistically significantly higher PD-1/PD-L1 interaction scores (p=0.02), and increased CD4 (p=0.01) and CD8 (p=0.003) T cell infiltrates compared to tumors from patients with endocrinologic PNS (n=11). Conclusion Our study of tumor tissue from patients with SCLC and PNS demonstrated a statistically significant increase in immune modulation markers' expression in patients with neurologic PNS. Tumor immunomodulation may be the driver of the improved prognosis that has been observed in ours and other retrospective cohorts of patients with SCLC and neurologic PNS. Citation Format: Wade T. Iams, Eileen Shiuan, Catherine B. Meador, Marc Roth, Jennifer Bordeaux, Christine Vaupel, Lucy L. Wang, Joseph T. Schneider, Jeremy L. Warner, Zhiguo Zhao, Christine M. Lovly. Clinical outcomes and differential tumor immune microenvironment in patients with small cell lung cancer and paraneoplastic syndromes [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 1025.

12059Background: Adjuvant chemotherapy (ACT) for ES-NSCLC provides only a modest improvement in survival and is associated with serious adverse effects. Thus, identifying subgroups of ES-NSCLC pati...

12058Background: Although patients with high tumor programmed death ligand-1(PD-L1) expression benefit from PD-1/PD-L1 axis inhibitors, many studies have highlighted that a fraction of patients res...

Although next-generation sequencing has allowed for the detection of somatic mutations in myelodysplastic syndromes (MDS), the clinical relevance of variant allele frequency (VAF) for the majority of mutations is unknown. We profiled TP53 and 20 additional genes in our training set of 219 patients with MDS or secondary acute myeloid leukemia with findings confirmed in a validation cohort. When parsed by VAF, TP53 VAF predicted for complex cytogenetics in both the training (P = 0.001) and validation set (P 40% had a median overall survival (OS) of 124 days versus an OS that was not reached in patients with VAF 40% was an independent covariate (HR, 1.61; P

9517 Background: Although PD-1/L1 axis directed therapies induce durable responses in some mm patients (pts), biomarkers of response remain elusive. We hypothesized that quantifying key immune suppression mechanisms within the tumor microenvironment would provide superior predictors of response to anti-PD-1 compared with single marker assessment. Methods: Pre-treatment tumor biopsies from 124 mm pts treated with anti-PD-1 at 7 academic centers were fluorescently stained with multiple immune markers in discovery (n = 24) and validation (n = 100) cohorts. Selected biomarker signatures, PD-1/PD-L1 interaction score (proportion of PD-1+ cells co-localized with PD-L1) and IDO1/HLA-DR co-expression were evaluated for anti-PD-1 treatment response and survival. Slides were imaged using Vectra; biomarker positive cells and their co-localization were objectively quantified in pathologist-selected regions using novel Automated Quantitative Analysis (AQUA) algorithms. Results: In the discovery cohort, high levels of PD-1/PD-L1 interaction score and/or IDO1/HLA-DR coexpression was strongly positively associated with response to anti-PD-1 (p = 0.0005). In contrast, other individual biomarkers (PD-1, PD-L1, CD8) were not associated with response or survival (p > 0.10). This finding was replicated in the validation cohort: pts with high PD-1/PD-L1 and/or IDO1/HLA-DR were more likely to respond to treatment (p = 0.009). These pts also experienced a three-fold increase in progression free survival (hazards ratio (HR) = 0.33; p = 0.003) and overall survival (HR = 0.34; p = 0.004). Multivariate analyses revealed that these findings were independent of BRAF mutation, stage, LDH and prior therapy. In the combined cohort (n = 124), 80% of responding pts had higher levels of PD-1/PD-L1 interaction scores and/or IDO1/HLA-DR. In contrast, PD-L1 expression alone (≥1% or ≥50%) was not predictive of PFS or OS (p > 0.1). Conclusions: This novel multiplexed method profiling key tumor-immune suppression pathways identified mm pts likely to respond to anti-PD-1 therapy. This method could help stratify patients for PD-1 monotherapy and be useful in guiding future clinical trials.

Background Hypomethylating agent (HMA) therapy represents the standard of care for patients with higher risk myelodysplastic syndromes although only 50% of patients respond to treatment. Recent evidence from molecular profiling through next-generation sequencing (NGS) in myeloid diseases has been conflicting as to the value of somatic mutations as a biomarker for response to HMA. TET2 mutations, predominantly in the absence of ASXL1 mutations (or variant allele frequency (VAF) < 10%), have been shown to predict HMA response in MDS and chronic myelomonocytic leukemia (CMML) (Itzykson et al., 2011; Bejar et al., 2014 (80% decitabine)) while DNMT3A mutations predict response to frontline HMA treatment in acute myeloid leukemia (AML) (Coombs et al., 2016). Therefore, our goal was to identify molecular predictors of response and outcomes to azacitidine in myeloid malignancies. Patients and Methods Genetically profiled higher-risk MDS, CMML and oligoblastic AML (20-30% blasts) cases were retrospectively identified from the Moffitt Cancer Center MDS database. We evaluated gene mutations associated with DNA methylation (TET2, DNMT3A, IDH1, IDH2, and WT1) and up to 19 additional genes. NGS was performed prior to the initiation of HMA in all patients. The lower limit of VAF detection was set at 5% and the minimum depth of coverage at each position was 500X. Clinical variables and outcomes of MDS patients were characterized at the time of sample procurement. Fisher's exact and t-tests were used for comparative analyses. Kaplan-Meier curves were used to estimate overall survival and analyzed from the date of mutation identification. Multivariate Cox regression models were created to adjust for clinical characteristics. Results From May 2013 to February 2016, a total of 77 patients with NGS for somatic mutations prior to HMA therapy were identified with a median age of 70 years and male predominance (66%). Of the cohort, 97% of patients (n=75) were treated with azacitidine with 17% of patients (n=13) proceeding to allogeneic hematopoietic stem cell transplant (AHSCT). A total of 86% of patients (n=66) had at least one pathogenic mutation. Mutations in DNA methylation occurred in 43% of patients (n=33) while TET2 mutation without clonal ASXL1 mutations occurred in 13% of patients (n=10). At a median follow up 17 months, the median OS of the entire cohort was 12.5 months. Patients with a DNA methylation mutation had a median OS that was not reached (NR) vs a median OS of 11.5 months in wildtype (WT) patients (HR 0.38, 95% CI 0.21 to 0.75; P = 0.005), which remained significant when censoring patients at time of AHSCT (P = 0.002). TP53 mutant (MT) patients (n=14, 18% of cohort) had a median OS of 7.9 months vs 15.4 months in WT patients (HR 3.69, 95% CI 3.04 to 28.8; P = 0.0001). The presence of DNA methylation or TP53 mutation in comparison to wildtype patients significantly stratified prognosis in azacitidine treated patients (Figure 1A, P = 0.0001). In multivariable analysis incorporating age and revised international prognostic scoring system (IPSS-R) category, DNA methylation (HR 0.45, 95% CI 0.21 to 0.96; P = 0.04) and TP53 (HR 2.34, 95% CI 1.04 to 5.26; P = 0.04) mutation status remained predictive for survival. Notably, response rates in DNA methylation mutant patients were similar to WT patients (40% versus 42%) with no difference in treatment duration. However, the overall response rate of TET2 MT/ASXL1 WT patients was 70% versus 36% in the rest of the cohort (P =0.08) and 0% in TET2 MT/ASXL1 MT patients (0/6, P =0.01) with a significantly longer duration of treatment in the TET2 MT/ASXL1 WT cohort (median number of cycles 7.5 versus 4; P = 0.002). Additionally, TET2 MT/ASXL1 WT patients had longer survival (median OS NR vs 12.2 months; P = 0.047). When censoring for transplant, the impact of TET2 MT/ASXL1 WT genoptype was significantly strengthened with 70% of patients alive past 15 months (median OS NR vs 9.9 months; HR 0.24, 95% CI 0.19 to 0.75; P = 0.007; Figure 1B). Conclusion In patients with myeloid malignancies, molecular profiling via NGS can predict outcomes to azacitidine therapy. Patients with mutations of DNA methylation have improved OS whereas OS is poor in TP53 MT patients. Most importantly, TET2 MT/ASXL1 WT identifies a genotypic subgroup with particularly good outcomes when treated with HMA without AHSCT and potentially challenges the early timing of AHSCT for higher risk patients and this molecular profile. Figure 1 Figure 1. Disclosures Padron: Novartis: Honoraria; Incyte: Research Funding; CTI: Honoraria, Research Funding; KALOBIOS: Research Funding. Vaupel:Genoptix, a Novartis Company: Employment. Hall:Genoptix, a Novartis Company: Employment. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Boehringer-Ingelheim: Research Funding; Incyte: Consultancy.

While PD-1/L1 axis-targeted therapies have shown promising clinical responses, their use in combination therapies is associated with both benefits and safety concerns. Response rates for single-agent anti-PD-1 therapies are significantly higher in biomarker positive patients, therefore there is a need to utilize predictive diagnostics to enhance benefit-risk profiles and guide treatment decisions. To address this, we developed a novel quantitative multiplexed immunohistochemistry assay that provides objective quantitation of PD-L1 positive cells, but more importantly assesses interactions with immune cells (PD-1+ or CD8+) in formalin-fixed paraffin embedded (FFPE) clinical specimens. The clinical validity of this assay was verified in a small series of melanoma patients treated with anti-PD1 targeted therapies/agents. FFPE melanoma tissues from patients who received anti-PD-1 therapy were fluorescently stained with a combination of anti-PD-1, PD-L1, and S100 antibodies plus DAPI or a combination of anti-CD8, PD-L1, and S100 antibodies plus DAPI. Each slide was then imaged on Vectra platform and the frequencies of biomarker positive cells (PD-L1, PD-1, and CD8) and their interaction scores were objectively evaluated using proprietary Automated Quantitative Analysis (AQUA®) algorithms. Analytical sensitivity, precision, and accuracy were established using standardized PD-L1 and PD-1 tissue control arrays composed of cell lines and lymphoid organs, while range of biomarker expression was verified on archived melanoma clinical specimens (n = 30), including samples taken from melanoma patients prior to anti-PD-1 therapies (n = 21). Frequencies of PD-L1 positive cells could be accurately quantified within 1% to 100% range in predefined control cell line mixtures. PD-L1 and PD-1 measurements were highly reproducible (R2 = 0.98 and 0.97, respectively). A broad range of PD-L1 and PD-1 expression and interaction scores were observed in archival clinical specimens (n = 53). In a cohort of 21 advanced melanoma patients treated with nivolumab (n = 5) or pembrolizumab (n = 16), the PD-1/L1 interaction score was found to reliably distinguish responders from non-responders (p = 0.03) while PD-L1 alone (p = 0.15) or CD-8 alone (p = 0.23) did not. Additionally, patients exhibiting higher PD-1/L1 interaction scores had superior response rates (78% vs. 17%, p = 0.03). Responders experienced significantly longer median progression-free survival (177 vs. 85 days, p = 0.014), and fewer deaths (22% vs 58%) compared with patients having lower PD-1/L1 interaction scores. In terms of diagnostic utility, the PD-1/L1 multiplex test showed superior predictive power (78% Positive Predictive Value, 83% Negative Predictive Value) compared with PD-L1 expression alone. Additional studies are underway to fully establish diagnostic utility and aid in treatment guidance. Citation Format: Jennifer Bordeaux, Douglas Johnson, Jeffrey Sosman, Ju Young Kim, Christine Vaupel, Bashar Dabbas, Justin Cates, Jeff Hall, Jelveh Lameh, Shabnam Tangri, Naveen Dakappagari. Novel quantitative multiplexed PD-1/PD-L1 immunohistochemistry test provides superior prediction of treatment response in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 853.

Background Molecular profiling through next-generation sequencing (NGS) in myeloid diseases has greatly improved prognostic discrimination. In myelodysplastic syndromes (MDS), mutations of TP53, EZH2, ETV6, RUNX1 and ASXL1 were shown in a multivariate model to predict for inferior survival although ASXL1 mutations were least predictive (HR 1.38, P=.049; Bejar et al., 2011). In contrast, mutation analysis of two large chronic myelomonocytic leukemia (CMML) cohorts both identified ASXL1 mutation as the only mutation to be independently predictive of inferior survival in multivariable analysis when including only nonsense and frameshift mutations (Itzykson et al., 2013; Patnaik et al., 2014). Therefore, our goal was to investigate the relative impact of ASXL1 mutations in MDS and CMML. Patients and Methods NGS profiled MDS and CMML cases were retrospectively identified from the Moffitt Cancer Center MDS database. We genotyped ASXL1 and up to 20 additional genes in our cohort. The lower limit of detection was set at 5% mutant allele reads and the minimum depth of coverage was 500X. Clinical variables and outcomes of MDS patients were characterized at the time of sample procurement. Fisher's exact and t-tests were used for comparative analyses. Kaplan-Meier estimates were used to estimate overall survival and analyzed from the date of mutation identification. Multivariate Cox regression models were created to adjust for clinical characteristics. Results From May 2013 to October 2014, 248 patients with NGS for somatic mutations were identified in our database. The baseline characteristics of the MDS and CMML cohorts stratified by ASXL1 mutation status are shown in Table 1. In this cohort, 89% of patients (n=220) had at least one pathogenic mutation. ASXL1 frameshift or nonsense mutations occurred in 22% of patients (n=54) with increased frequency in the CMML cohort (50% versus 18%, P=0.0002). An additional 7% of patients (n=17) had a missense mutation of ASXL1 and were predominantly restricted to the MDS subgroup (16/17). The most common type of ASXL1 mutation were frameshift mutations in the MDS and CMML cohorts, 51% and 69% respectively, with the c.1934dupG; p.G646Wfs*12 variant observed in 33% of CMML patients (n=5) and 21% of MDS patients (n=8). ASXL1 mutant patients had similar survival to wild-type patients with median OS of 15.3 versus 13.7 months, respectively (P = 0.80). Exclusion of missense mutations had no effect on the prognostic impact of ASXL1 mutation (P=0.78). In the MDS cohort, there was again no difference in OS (median OS 14.3 versus 13.3 months; P=0.47) and type of ASXL1 mutation had no effect (P = 0.85). In the CMML cohort (n=30), median OS was 8.8 months in ASXL1 mutant patients and not reached in wild-type patients (HR 3.0, 95% CI 0.87 to 10.37, P=0.08). In comparison to wild-type patients, class of ASXL1 mutation was predictive of survival in the CMML cohort (P=0.008). CMML patients with frameshift mutations of ASXL1 had shorter OS in comparison to nonsense mutations although this did not reach significance (P=0.21). In multivariable analysis incorporating age and IPSS, ASXL1 mutation status was predictive for inferior survival among CMML patients (HR 2.5, 95% CI 1.08 to 5.85; P=0.03). Conclusion In MDS patients, neither ASXL1 mutation status nor exclusion of missense mutations was predictive for inferior survival, while ASXL1 mutant CMML patients had inferior OS, particularly with frameshift mutations. Together, these results highlight biological differences between MDS and CMML and support collaborative efforts in mutational profiling of these patients to improve prognostication. Table 1.Baseline Characteristics by ASXL1 Mutation Status (Frameshift and Nonsense)MDS CohortCMML CohortASXL1 MTASXL1 WTASXL1 MTASXL1 WTn=39n=179n=15n=15Median age72 (54-91)71 (36-100)73 (48-88)72 (57-94)Male29 (74%)109 (61%)13 (87%)9 (60%)Female10 (26%)70 (39%)2 (13%)6 (40%)Median Hemoglobin99.410.111.1Median Platelets64827976Median ANC1.321.395.394.23Median Monocyte count0.230.241.912.46Median BM Blast %4445IPSSlow5 (15%)39 (25%)5 (33%)6 (40%)intermediate 117 (50%)50 (33%)7 (47%)6 (40%)intermediate 23 (9%)36 (24%)2 (13%)2 (13%)high9 (26%)28 (18%)1 (7%)1 (7%) Disclosures Vaupel: Genoptix: Employment; Novartis: Employment, Equity Ownership. Lancet:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Pfizer: Consultancy; Kalo-Bios: Consultancy; Boehringer-Ingelheim: Consultancy; Seattle Genetics: Consultancy. Hall:Novartis: Employment, Equity Ownership; Genoptix: Employment. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Incyte: Consultancy; Celgene: Consultancy, Research Funding; Pharmacylics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

7091 Background: The clinical implementation of next generation sequencing (NGS) has allowed for the quantitative detection of clinically significant somatic mutations in myeloid malignancies. Howe...

Background: Establishing the diagnosis of myelodysplastic syndromes requires excluding benign causes of cytopenias and is based largely on morphologic criteria subject to substantial interobserver variability. Clonal hematopoiesis defined by the presence of a typical karyotype abnormality can serve as presumptive evidence of MDS in the absence of other diagnostic criteria, however these lesions are absent in the majority of cases. Somatic mutations are much more common in patients who meet the diagnostic criteria for MDS. However, the frequency of clonal somatic mutations in patients with meaningful cytopenias who lack morphologic evidence of MDS is not known and could help identify those at increased risk of progressive disease. Methods: We conducted a prospective study enrolling patients with unexplained cytopenias, suspected of having MDS into one of three groups (positive for MDS, equivocal, or negative) based on traditional diagnostic methods of morphology, cytogenetics and flow cytometry. We conducted massively parallel amplicon-based sequencing using the Illumina MiSeq platform on bone marrow samples from consenting patients to examine the mutation status of 21 genes implicated in MDS (SF3B1, SRSF2, U2AF1, ZRSR2, TET2, IDH1, IDH2, DNMT3A, EZH2, ASXL1, SETBP1, TP53, PHF6, RUNX1, ETV6, CBL, NRAS, KIT, JAK2, MPL, NPM1). The minimum coverage was 500x, and only variants with minor allele fractions >5% were reported. Results: Eleven community oncology practices in the US enrolled 145 patients with cytopenias suspected of having MDS. Of these, 86 have been sequenced to date. The ages of the sequenced patients ranged from 31 to 97 years with an average age of 71. Bone marrow material from these 86 patients were examined by two hematopathologists and enrolled into three arms based on their findings; confirmed MDS (n=8), equivocal evidence of MDS (n=12), and non-MDS (n=66). We identified clinically significant MDS-associated somatic variants in all three categories. Variants were found in 5 of 8 confirmed MDS, in 9 of 12 with equivocal MDS, and in 16 of the 66 non-MDS patients. The finding of clinically significant variants in 16 of 66 (24%) in the non-MDS group was unexpected and included 6 patients with mutations in the TET2 gene,6 patients with mutations in the TP53 gene, as well as patients with mutations in RUNX1 (n=2), DNMT3A, SETBP1, ASXL1 and ZRSR2. There were no differences in age or blood counts between patients with mutations and those without them within the non-MDS group. Nor were there any significant differences in these measures between diagnostic groups. Discussion: Somatically acquired variants in the non-MDS group may be the result of early, low grade MDS without sufficient observable pathological characteristics, may indicate the presence of another disease affecting hematopoietic development, or may be incidental age-related somatic mutations. Our study may underestimate the fraction of patients with such mutations as genes not sequenced here may provide evidence of clonal hematopoiesis in more patients. Surprisingly, the number of patients without clear evidence of MDS greatly outnumbered those with a firm morphologic diagnosis in this prospective study. The large fraction of these patients with somatic mutations suggests that clonal cytopenias may be much more common than estimates based on the prevalence of MDS would indicate. Additional follow up may allow us to determine how these patients evolve clinically over time. Sequencing of the remaining 59 patients and additional analyses are ongoing and will include the association of somatic variants with age, type and severity of cytopenia, and features of the bone marrow morphology. All 145 patients, including 103 in the arm without evidence of MDS will be included in the study when presented at the ASH meeting. Conclusion: Somatic mutations in genes typically associated with MDS can be found in a substantial fraction of patients with little or no morphologic evidence of the disease. Identification of clonal cytopenias may help exclude benign alternative diagnoses and impact how these patients are followed clinically over time. Disclosures Hall: Genoptix Medical Laboratory: Employment. Al Hafidh:Genoptix Medical Laboratory: Employment. Balmert:Genoptix Medical Laboratory: Employment. Dabbas:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Vaupel:Genoptix Medical Laboratory: Employment. El Hader:Genoptix Medical Laboratory: Employment. McGinniss:Genoptix Medical Laboratory: Employment. Beruti:Genoptix Medical Laboratory: Employment. Bejar:Genoptix Medical Laboratory: Consultancy, Honoraria, Licensed IP, no royalties Patents & Royalties, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.