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.
•Over 30% of patients with unexplained cytopenias who do not meet diagnostic criteria for MDS carry MDS-associated somatic mutations.•Clonal cytopenias of undetermined significance are more common than MDS and show comparable variant allele frequencies and blood counts.
•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.
Predicting response to ICI therapy among patients with renal cell carcinoma (RCC) has been uniquely challenging. We analyzed patient characteristics and clinical correlates from a retrospective ...single-site cohort of advanced RCC patients receiving anti-PD-1/PD-L1 monotherapy (N = 97), as well as molecular parameters in a subset of patients, including multiplexed immunofluorescence (mIF), whole exome sequencing (WES), T cell receptor (TCR) sequencing, and RNA sequencing (RNA-seq). Clinical factors such as the development of immune-related adverse events (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.05-5.91) and immunological prognostic parameters, including a higher percentage of circulating lymphocytes (23.4% vs. 17.4%,
= 0.0015) and a lower percentage of circulating neutrophils (61.8% vs. 68.5%,
= 0.0045), correlated with response. Previously identified gene expression signatures representing pathways of angiogenesis, myeloid inflammation, T effector presence, and clear cell signatures also correlated with response. High PD-L1 expression (>10% cells) as well as low TCR diversity (≤644 clonotypes) were associated with improved progression-free survival (PFS). We corroborate previously published findings and provide preliminary evidence of T cell clonality impacting the outcome of RCC patients. To further biomarker development in RCC, future studies will benefit from integrated analysis of multiple molecular platforms and prospective validation.
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.
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.
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.
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.
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.
Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (
= 24) and validation (
= 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.
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 (
= 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 (
= 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36;
= 0.0004) and overall survival (HR = 0.39;
= 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 (
= 0.000004). In contrast, PD-L1 expression was not predictive of survival.
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.
.
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.
Abstract
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.
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