Search

Supplementary Figure S1. Computed Tomographic (CT) findings in patient CGLU116. Supplementary Figure S2. CT findings in patient CGLU127. Supplementary Figure S3. CT findings in patient CGLU161. Supplementary Figure S4. Tumor burden kinetics. Supplementary Figure S5. Neoantigen-specific TCR expansion in stimulated T cell cultures for patient CGLU127. Supplementary Figure S6. Neoantigen-specific TCR expansion in stimulated T cell cultures for patient CGLU161. Supplementary Figure S7. Loss of heterozygosity analyses for patient CGLU116. Supplementary Figure S8. Loss of heterozygosity analyses for patient CGLU117. Supplementary Figure S9. Loss of heterozygosity analyses for patient CGLU127. Supplementary Figure S10. Loss of heterozygosity analyses for patient CGLU161. Supplementary Figure S11. TCR clonality analyses for patients CGLU127 and CGLU161. Supplementary Figure S12. TCR clonality analyses for a responder and a non-responder to PD-1 blockade. Supplementary Figure S13. PD-L1 expression in responsive and resistant tumors. Supplementary Figure S14. Eliminated mutations for case CGHN2. Supplementary Figure S15. Comparison of five methods for estimation of tumor purity. Supplementary Figure S16. CD8+ T cell density in resistant tumors.

Supplementary Table S1. Summary of Patient and Sample Characteristics. Supplementary Table S2. Summary of Next-Generation Sequencing Analyses. Supplementary Table S3. Somatic Sequence Alterations*. Supplementary Table S4. Somatic Copy Number Alterations. Supplementary Table S5. Neoantigen Predictions. Supplementary Table S6. Characteristics of a Subset of Eliminated Candidate Neoantigens in the NSCLC patients*. Supplementary Table S7. Summary of Functionally Validated Eliminated, Gained, and Retained cMANAs. Supplementary Table S8. Eliminated MANA-specific T Cell Clonotypes in CGLU116, CGLU127 and CGLU161. Supplementary Table S9. Retained and Gained MANA-specific T Cell Clonotypes in CGLU116. Supplementary Table S10. Allelic Imbalance Analysis and Cellularity Estimates for CGLU116. Supplementary Table S11. Allelic Imbalance Analysis and Cellularity Estimates for CGLU117. Supplementary Table S12. Allelic Imbalance Analysis and Cellularity Estimates for CGLU127. Supplementary Table S13. Allelic Imbalance Analysis and Cellularity Estimates for CGLU161. Supplementary Table S14. Summary of Eliminated Neoantigens in NSCLC Cases. Supplementary Table S15. TCR-beta Sequencing Analysis. Supplementary Table S16. PD-L1 and CD8 Immunohistochemistry. Supplementary Table S17. Regions of Allelic Imbalance. Supplementary Table S18. Tumor Purity and Ploidy Estimates.

Supplementary Figure Legends

mp3 file (49.6MB). Patients with recurrent metastatic non-small cell lung cancer have a morbid prognosis, but a new epigenetic therapy may have potential for this population. Cancer Discovery Editors-in-Chief Jose Baselga, M.D., Ph.D. and Lewis C. Cantley, Ph.D. moderated a press conference about this research on Wednesday, Nov. 9, 2011.

Figure S4

Gene Methylation Detection, Sensitivity, Specificity Using Detectable vs. Non-detectable cutoff in blacks

Variable importance plot for random forest prediction

Purpose: CT screening can reduce death from lung cancer. We sought to improve the diagnostic accuracy of lung cancer screening using ultrasensitive methods and a lung cancer–specific gene panel to detect DNA methylation in sputum and plasma.Experimental Design: This is a case–control study of subjects with suspicious nodules on CT imaging. Plasma and sputum were obtained preoperatively. Cases (n = 150) had pathologic confirmation of node-negative (stages I and IIA) non–small cell lung cancer. Controls (n = 60) had non-cancer diagnoses. We detected promoter methylation using quantitative methylation-specific real-time PCR and methylation-on-beads for cancer-specific genes (SOX17, TAC1, HOXA7, CDO1, HOXA9, and ZFP42).Results: DNA methylation was detected in plasma and sputum more frequently in people with cancer compared with controls (P < 0.001) for five of six genes. The sensitivity and specificity for lung cancer diagnosis using the best individual genes was 63% to 86% and 75% to 92% in sputum, respectively, and 65% to 76% and 74% to 84% in plasma, respectively. A three-gene combination of the best individual genes has sensitivity and specificity of 98% and 71% using sputum and 93% and 62% using plasma. Area under the receiver operating curve for this panel was 0.89 [95% confidence interval (CI), 0.80–0.98] in sputum and 0.77 (95% CI, 0.68–0.86) in plasma. Independent blinded random forest prediction models combining gene methylation with clinical information correctly predicted lung cancer in 91% of subjects using sputum detection and 85% of subjects using plasma detection.Conclusions: High diagnostic accuracy for early-stage lung cancer can be obtained using methylated promoter detection in sputum or plasma. Clin Cancer Res; 23(8); 1998–2005. ©2016 AACR.

Supplemental Tables and figures legends

Purpose:Low-dose CT screening can reduce lung cancer–related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation–based detection of non–small cell lung cancer (NSCLC).Experimental Design:This nested case–control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation–specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42).Results:DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively.Conclusions:DNA methylation–based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

Gene promoter methylation amplification curves

Receiver operator classification curves for lung cancer detection for the genes obtained from Plasma

Receiver operator classification curves for lung cancer detection for the genes obtained from sputum

Cancer recurrence after surgery remains an unresolved clinical problem1–3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4–6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy. In mouse models of pulmonary metastasis, adjuvant epigenetic therapy targeting myeloid-derived suppressor cells disrupts the premetastatic microenvironment after resection of primary tumours and inhibits the dissemination of residual tumour cells.

Despite progress in immunotherapy, identifying patients that respond has remained a challenge. Through analysis of whole-exome and targeted sequence data from 5,449 tumors, we found a significant correlation between tumor mutation burden (TMB) and tumor purity, suggesting that low tumor purity tumors are likely to have inaccurate TMB estimates. We developed a new method to estimate a corrected TMB (cTMB) that was adjusted for tumor purity and more accurately predicted outcome to immune checkpoint blockade (ICB). To identify improved predictive markers together with cTMB, we performed whole-exome sequencing for 104 lung tumors treated with ICB. Through comprehensive analyses of sequence and structural alterations, we discovered a significant enrichment in activating mutations in receptor tyrosine kinase (RTK) genes in nonresponding tumors in three immunotherapy treated cohorts. An integrated multivariable model incorporating cTMB, RTK mutations, smoking-related mutational signature and human leukocyte antigen status provided an improved predictor of response to immunotherapy that was independently validated.

Objectives: Myeloid derived suppressor cells (MDSCs) are immature cells that aid in cancer progression and dissemination via immune system suppression. Previous work has shown that CCR 2 (C-C chemokine receptor) and CCR 5 expression on MDSCs is increased in non-small cell lung cancer (NSCLC). We hypothesized that patients with lung cancer will have detectable peripheral MDSCs with CCR2 and CCR5 expression preoperatively, that it would decrease immediately postoperatively, and then increase longitudinally if tumor recurs. Materials & Methods: As part of a prospective longitudinal study, whole blood samples were obtained from patients suspected to have primary lung cancer prior to surgery. Patients were excluded if they were minors, could not provider consent, had malignancy within the past 10 years or any immunosuppressive condition. Blood samples were obtained prior to surgery or at follow-up in clinic and processed within 1 hour of acquisition. We stained samples via 2 different methods: 1) whole blood and 2) peripheral blood mononuclear cells (PBMC) extracted from Ficoll density gradient and determined that whole blood staining had superior results. Samples were analyzed via flow cytometer and gated after defining MO (monocytic)-MDSCs as CD33+HLADRlow/-CD14+ and PMN-MDSCs as CD33+HLADR-CD15+. MDSCs were reported as a percentage of live leukocytes and means were reported with T-test performed for statistical analysis. Results: A total of 18 patients were recruited with a median age of 69 years (63.8-75) and 61% (11/18) females. Adenocarcinoma was present in 16, carcinoid tumor in 1 and both adenocarcinoma and carcinoid tumor in 1 patient. Stage I and Stage II were the most common (66.7% and 22.2%, respectively). Majority of the tumors were in the right upper lobe (55.6%). There were 7 healthy controls with a median age of 29 years (28-43) and 71% females. There was a significantly increased proportion of MO-MDSCs in NSCLC patients preoperatively compared to healthy controls (11.64% versus 5.02%, p = 0.02). CCR2+CCR5+ MO-MDSCs were 0.85% in patients versus 0.06% in controls (p=0.04). No differences were noted with PMN-MDSCs. Five patients had post-operative follow up (mean 152 days) with an average decrease of 63% in MO-MDSCs, 68% in CCR2+CCR5+ MO-MDSCs and no recurrence of tumor on CT scans. Conclusion: Early results of this on-going study demonstrate the detection of circulating CCR2+CCR5+ MO-MDSCs in the preoperative whole blood of NSCLC patients compared to healthy controls. Resection of the tumor is associated with a decrease of these MO-MDSCs after treatment. We are evaluating if any increase in CCR2+CCR5+ MO-MDSC in long term will allow us to use it as an adjuvant tool along with CT monitoring as a biomarker of residual or recurrent disease. Citation Format: Hamza Khan, Anas Awan, Maria Shishikura, Carley Blevins, Kristen Rodgers, Yuping Mei, Wasay Nizam, Shun Ishiyama, Yun Chen, Richard Battafarano, Errol Bush, Stephen Broderick, Stephen Yang, Hajime Orita, Peng Huang, Ada Tam, Jinny Ha, Franck Housseau, Malcolm Brock. Monitoring of CCR2 and CCR5 expression on circulating myeloid derived suppressor cells (MDSCs) in non-small cell lung cancer as a correlate of minimum residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 271.

Purpose: Low-dose CT screening can reduce lung cancer–related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation–based detection of non–small cell lung cancer (NSCLC). Experimental Design: This nested case–control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation–specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). Results: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. Conclusions: DNA methylation–based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

BACKGROUND AND OBJECTIVE Hyperhidrosis (HH) is associated with impairments in quality of life (QOL) and elevated anxiety. Microwave thermolysis is a newer treatment that reduces sweating, yet effects on QOL and emotional symptoms have not been examined. Two treatment sessions are recommended to achieve 80% amelioration of clinical HH. We hypothesized that microwave thermolysis would reduce sweat severity, improve QOL, and reduce anxiety in young adults suffering from axillary HH in a prospective clinical trial. STUDY DESIGN/MATERIALS AND METHODS We enrolled 24 young adults (mean age = 23.57 years, 54% female) with elevated scores on the Hyperhidrosis Disease Severity Scale. All participants received one session of microwave thermolysis, and 83% received two sessions. Participants completed measures of sweat severity, QOL, generalized anxiety, social anxiety, social avoidance, and anxious/depressive mood symptoms at baseline; post-first treatment; and following second treatment. RESULTS At baseline, all participants had severe sweating; 87.5% had impaired QOL, 75% had elevated social anxiety, 50% with generalized anxiety, 48% with social avoidance, and 38% with anxious/depressed mood. Paired samples t tests indicated significant improvements from baseline to first procedure, including decreased sweating (t(21) = 5.68, P

Simple Summary The role of exosomes in HIV (human immunodeficiency virus) as well as in cancer patients seems to be pivotal. The aim of our retrospective study was to assess the potential clinical value of CD9-positive plasma exosomes in lung cancer patients, patients with lung granulomas, healthy individuals, and HIV-positive patients with or without lung cancer. This study shows that CD9-positive plasma exosome concentrations differ between healthy controls, patients with immunocompetent pulmonary granulomas and patients with lung cancer. In addition, CD9-positive plasma exosomes are increased in HIV seropositive and HIV seronegative lung cancer patients compared to healthy controls, while chemotherapy-treated lung cancer patients have lower plasma exosome levels. This study also shows that in chemotherapy-naïve patients, plasma exosome levels are directly correlated with a prognosis with higher concentrations being associated with a longer, overall survival. These findings further support previous literature on the translational significance of total plasma exosomes in cancer patients, despite different immunological contexts. Abstract Recently, the role of exosomes in the progression of both cancer and HIV (human immunodeficiency virus) has been described. This study investigates the clinical significance of CD9-positive plasma exosomes in lung cancer patients, healthy individuals, and HIV-positive patients with or without lung cancer. Using a verified with transmission electron microscopy double-sandwich ELISA technique, plasma-derived exosomes were isolated and quantified from 210 lung cancer patients (including 44 metastatic patients with progressive disease after chemotherapy), 49 healthy controls, 20 patients with pulmonary granulomas, 19 HIV+ patients with lung cancer, 31 HIV+ patients without cancer, and 3 HIV+ patients with pulmonary granulomas. Plasma exosome concentrations differed between healthy controls, patients with immunocompetent pulmonary granulomas and patients with lung cancer even after chemotherapy (p < 0.001). Lung cancer patients after chemotherapy had lower exosome concentrations compared to patients with untreated lung cancer or granuloma (p < 0.001 for both). HIV+ patients without lung cancer had significantly higher exosome concentrations compared to HIV+ patients with lung cancer (p = 0.016). Although exosome concentrations differed between all different lung cancer histologies and healthy controls (p < 0.001 for all histologies), adjusted statistical significance was oµy retained for patients with granulomas and SCLC (Small-cell lung cancer, p < 0.001). HIV-induced immunodeficient patients with or without lung cancer had lower plasma exosomes compared to immunocompetent granuloma and lung cancer patients (p < 0.001). Finally, higher plasma exosomes were associated both on univariate (p = 0.044), and multivariate analysis (p = 0.040) with a better 3-year survival in stage II and III NSCLC (Non-small-cell lung carcinoma) patients. In conclusion, our study shows that CD9-positive plasma exosomes are associated with both lung cancer and HIV, prior chemotherapy, as well as with survival, suggesting a possible prognostic value.

Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non–small cell lung cancer after initial response to immune checkpoint blockade with anti–PD-1 or anti–PD-1/anti–CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens. Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264–76. ©2017 AACR. See related commentary by Yang, p. 250. This article is highlighted in the In This Issue feature, p. 235

Cancer recurrence after surgery remains an unresolved clinical problem

Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA – Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints – in particular the PD-1/PD-L1 pathway – may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade.

Background The purpose of this study was to assess the effect of human immunodeficiency virus (HIV) infection on postoperative survival among non-small cell lung cancer (NSCLC) patients. Methods A retrospective cohort study compared 22 HIV-infected lung cancer patients to 2,430 lung cancer patients with HIV-unspecified status who underwent resection at Johns Hopkins Hospital from 1985 to 2009. Subcohort comparative analyses were performed using individual matching methods. Results Thirty-day mortality rates did not differ between HIV-infected and HIV-unspecified patients. Survival rates for HIV-infected lung cancer patients were significantly shorter than for HIV-unspecified patients (median, 26 versus 48 months; p = 0.001). After adjustment, the relative hazard of mortality among HIV-infected NSCLC patients was more than threefold that of HIV-unspecified patients (adjusted hazard ratio, 3.08; 95% confidence interval: 1.85 to 5.13). When additional surgical characteristics were modeled in a matched subcohort, the association remained statistically significant (adjusted hazard ratio, 2.31; 95% confidence interval: 1.11 to 4.81). Moreover, HIV-infected lung cancer patients with CD4 counts less than 200 cells/mm 3 had shortened median survival compared with patients whose CD4 counts were 200 cells/mm 3 or greater (8 versus 40 months; p = 0.031). Postoperative pulmonary and infectious complications were also elevated in the HIV-infected group ( p = 0.001 and p p = 0.061). Conclusions The HIV-infected NSCLC patients have more postoperative complications, rapid progression to disease recurrence, and poorer postoperative survival. Optimizing immune status before surgery and careful patient selection based on diffusion capacity of lung for carbon monoxide may improve patient outcomes.

Purpose: The National Lung Cancer Screening Trial showed mortality reduction on patients with NSCLC by the use of Low-dose CT screening. The study exhibits an unreasonably high false positive rate of 96.6%, which can to lead to significant morbidity and mortality from unnecessary tests. This study aim to determine if methylated promoter regions of a panel of genes that are correlated with Non-Small Cell Lung Cancer can be detected in urine from patients with lung cancer versus those without. Materials and Methods: We conducted a prospective case-control study recruiting subjects from the Lung Cancer Spore Trial. Urine and pre-operative CT scans were obtained from all patients. We processed the urine using the Methylation on Beads assay to isolate and bisulfite treat circulating DNA and then employed Quantitative Methylation Specific Real-Time PCR to detect promoter methylation status of the genes: CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZFP42. Sensitivity, specificity, PPV and NPV values were calculated for each gene methylation status. Results: 34 patients were studied, including 23 patients with NSCLC and 11 patients with benign non-cancerous lesions. The sensitivity, specificity, PPV and NPV values for lung cancer detection in urine are shown in Table 1. Sensitivities ranged 43-96%, Specificities 64-91%, PPV 71-100% and NPV 40-67%. Promoter methylation of the gene panel CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZPF42 has a 78% sensitivity, 91% specificity, 95% PPV and 67% NPV for lung cancer detection. Conclusion: Our study suggests that urine can provide a highly sensitive and specific non-invasive route for lung cancer detection. These results are very promising and could potentially reduce unnecessary morbidity and mortality in people undergoing screening for NSCLC by use of a non-invasive method that could be more accessible on primary care centers. However, further studies and validation with larger sample sizes and different populations are necessary before its application to clinical practice. Patients with positive gene methylation from urine samples and its accuracy for NSCLC detection.Cancer (n=23)Cancer (n=23)Control (n=11)Control (n=11)nSensitivitynSpecificityPPVNPVCDO114611919353TAC11565010010058SOX1716704648050HOXA711483737940HOXA910431919143ZPF4222969187167Panel of genes18781919567 Citation Format: Lane Lerner, Lily Zheng, Anastasia Kottorou, Chen Chen, Tomoaki Ito, Kristen Rodgers, Beverly Lee, Robert Winn, Enrico Benedetti, Tza- Huei Wang, Malcolm V. Brock, James G. Herman, Alicia Hulbert. Urine epigenetic biomarkers for NSCLC diagnosis [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 3308.

Tumor cells contain nonsynonymous somatic mutations that alter the amino acid sequences of the proteins encoded by the affected genes. Those alterations are foreign to the immune system and may therefore represent tumor-specific neoantigens capable of inducing antitumor immune responses. Somatic mutational and neoantigen density has recently been shown to correlate with long-term benefit from immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma, suggesting that a high density of neoepitopes stemming from somatic mutations may enhance clinical benefit from blockade of immune checkpoints that unleash endogenous responses to these mutation-associated neoantigens (MANAs). Expression of the programmed cell death ligand 1 (PD-L1) in tumors or tumor-infiltrating immune cells has been associated with responses to PD-1 blockade; however, PD-L1 expression or other immune biomarkers have not been sufficient to fully explain therapeutic outcomes. Among the patients that initially respond to PD-1 blockade, some become resistant to the therapy. Up-regulation of alternate immune checkpoints, loss of HLA haplotypes, or somatic mutations in HLA or JAK1/JAK2 genes have been proposed as mechanisms of evasion to immune recognition in some patients, but the mechanisms underlying response and acquired resistance to immune checkpoint blockade have remained elusive. To examine mechanisms of resistance to immunotherapy, we performed genome-wide sequence analysis of protein coding genes and T-cell receptor (TCR) clonotype analysis, followed by functional assays of autologous T-cell activation of patients who demonstrated initial response to immune checkpoint blockade but ultimately developed progressive disease.Of a cohort of 42 NSCLC patients treated with single-agent PD-1 or combined PD-1 and CTLA4 blockade, we identified all consecutive cases that at the time of the analysis developed acquired resistance (two subjects treated with nivolumab and two with ipilimumab and nivolumab) and where paired tumor specimens were available both before and after therapy. To examine the landscape of genomic alterations and associated neoantigens, we performed whole exome sequencing of tumors from these patients. Pretreatment and postprogression specimens were obtained from the same anatomic location or from sites in close anatomic proximity. We examined multiple immune-related parameters of peptides stemming from somatic alterations using a computational multidimensional neoantigen prediction platform. This approach allowed for identification of peptides within mutated genes that were predicted to be processed and presented by MHC class I proteins and therefore had the potential to elicit an immune response. The algorithm evaluated the binding of mutant peptides (8-11mers) to patient-specific HLA class I alleles and ranked the neoantigens according to MHC binding affinity, antigen processing, and self-similarity. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. While algorithm-based predictions of antigenicity are valuable in narrowing down the large number of peptides capable of being generated by a mutation to a set potential antigenic peptides presented by self-MHC alleles, functional T cell recognition is critical to evaluate immune responsiveness. To this end, we developed a sensitive approach for assessing T-cell response to candidate MANAs (cMANAs) that utilized next-generation sequencing of TCR-Vb CDR3 regions as a measure of T-cell clonality. To evaluate T-cell recognition of eliminated neoantigens, purified peripheral blood T cells from the patients described above were stimulated with autologous peripheral blood mononuclear cells (PBMCs) loaded with cMANA peptides in a ten-day culture system. We subsequently used TCR next-generation sequencing to assess the differential abundance of neoantigen-specific T cell clonotypes in these expanded T cell populations. In order to further investigate the importance of eliminated MANAs, we generated peptides from retained and gained MANAs and assessed their potential to elicit a MANA-specific T-cell expansion. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. These findings indicate that patient-derived T cells recognized the eliminated neoantigens and suggest that these neoantigens were relevant targets for the achievement of initial therapeutic response to checkpoint blockade. Conceptually, neoantigen loss occurs through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations. To evaluate the contribution of these mechanisms to the loss of neoantigens, we analyzed the tumors both before and after therapy using the SCHISM pipeline and incorporating mutation frequency, tumor purity, and copy number variation to infer the fraction of cells containing a specific mutation (mutation cellularity). Consistent with our hypothesis, we observed both mechanisms of neoantigen elimination: loss of truncal changes through genetic events involving chromosomal deletions and loss of heterozygosity (LOH) and loss of subclonal neoantigens either by LOH or through elimination of tumor subclones. Both truncal and subclonal changes were among the eliminated neoantigens that were functionally validated. To evaluate the impact of changes in neoantigen landscape on cytotoxic T-cell receptor repertoire, we analyzed serially collected PBMCs, prior to immunotherapy initiation, at clinical response, and at resistance. We hypothesized that loss of neoantigens would lead to a decrease in clonality of cytotoxic TCR clonotypes, thus reflecting tumor immune evasion at the time of emergence of resistance. We observed peripheral T-cell expansion of a subset of the top 100 most frequent intratumoral clones, with the most frequent clones reaching up to a 44-fold increase in abundance in the blood at the time of response, followed by a decrease to pretreatment levels at the time of resistance. As a comparison, such decreases in TCR frequencies were not observed in a NSCLC patient with durable response to PD-1 blockade and no change in intratumoral TCR frequencies was seen in a NSCLC patient with primary resistance to PD-1 blockade. Taken together, these observations suggest that TCR expansion may be both a useful measure of response to checkpoint blockade and an indicator of acquired therapeutic resistance through neoantigen loss. As immune checkpoint therapy has become standard of care for many cancer types, the development of acquired resistance is being recognized more commonly. Through our comprehensive genomic analyses, we have identified changes in the genomic landscape of tumors during immune checkpoint blockade. These analyses show that emergence of acquired resistance is associated with loss of mutations encoding for putative tumor-specific neoantigens, through both elimination of tumor subclones and chromosomal loss of truncal alterations. Using a new approach to assess neoantigen reactivity by T cells, we found that some of these eliminated mutations indeed encoded peptides recognized by T cells in the peripheral circulation of the respective patients. Given that the antitumor efficacy of checkpoint blockade likely involves release of endogenous T-cell responses to tumor antigens generated by coding mutations, our findings are consistent with a mechanism of acquired resistance to immune checkpoint blockade that involves therapy-induced immune editing of MANAs. Acquisition of somatic resistance mutations is a common mechanism of therapeutic resistance to targeted therapies. However, elimination of genomic alterations and more specifically loss of somatic mutations through subsequent genetic events is uncommon in the context of natural tumor evolution or therapeutic resistance. Elimination of mutation associated antigens by a T-cell-dependent immunoselection process has been proposed as a mechanism of cancer immunoediting in melanoma after adoptive T cell transfer; however, the evolution of neoantigen loss as an escape mechanism under the selective pressure of immune checkpoint blockade in lung cancer has not been previously studied. In addition, we examined a variety of other mechanisms that have been proposed in the development of resistance to immunotherapies. We did not observe any differences in PD-L1 expression in tumor cells between responsive and resistant tumor samples. Likewise, we evaluated known potential genomic mechanisms of immunotherapy resistance; however, there were no new genomic alterations in the CD274 gene encoding for PD-L1, PDCD1 encoding for PD-1, CTLA4, JAK1 or JAK2 genes, in HLA genes, beta 2 microglobulin, or other antigen presentation-associated genes. This work demonstrates for the first time that acquired resistance to anti-PD-1 or anti-PD1/anti-CTLA4 therapy in lung cancer can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of tumor neoantigen reactivity may mitigate the development of acquired resistance. Citation Format: Valsamo Anagnostou, Kellie N. Smith, Patrick M. Forde, Noushin Niknafs, Rohit Bhattacharya, James White, Vilmos Adleff, Jillian Phallen, Neha Wali, Carolyn Hruban, Violeta B. Guthrie, Kristen Rodgers, Jarushka Naidoo, Hyunseok Kang, William Sharfman, Christos Georgiades, Franco Verde, Peter Illei, Qing Ka Li, Edward Gabrielson, Malcolm Brock, Cynthia Zahnow, Stephen B. Baylin, Rob Scharpf, Julie R. Brahmer, Rachel Karchin, Drew M. Pardoll, Victor E. Velculescu. Evolution of neoantigen landscape during immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr NG01. doi:10.1158/1538-7445.AM2017-NG01

Background: Epidemiological evidence suggests that HIV-infected individuals are at increased risk of lung cancer, but no data exist because large computed tomography (CT) screening trials routinely exclude HIV-infected participants. Methods: From 2006 to 2013, we conducted the world's first lung cancer screening trial of 224 HIV-infected current/former smokers to assess the CT detection rates of lung cancer. We also used 130 HIV-infected patients with known lung cancer to determine radiographic markers of lung cancer risk using multivariate analysis. Results: Median age was 48 years with 34 pack-years smoked. During 678 person-years, one lung cancer was found on incident screening. Besides this lung cancer case, 18 deaths (8%) occurred, but none were cancer related. There were no interim diagnoses of lung or extrapulmonary cancers. None of the pulmonary nodules detected in 48 participants at baseline were diagnosed as cancer by study end. The heterogeneity of emphysema across the entire lung as measured by CT densitometry was significantly higher in HIV-infected subjects with lung cancer compared with the heterogeneity of emphysema in those without HIV ( p ⩽ 0.01). On multivariate regression analysis, increased age, higher smoking pack-years, low CD4 nadir, and increased heterogeneity of emphysema on quantitative CT imaging were all significantly associated with lung cancer. Conclusions: Despite a high rate of active smoking among HIV-infected participants, only one lung cancer was detected in 678 patient-years. This was probably because of the young age of participants suggesting that CT screening of high-risk populations should strongly consider advanced age as a critical inclusion criterion. Future screening trials in urban American must also incorporate robust measures to ensure HIV patient compliance, adherence, and smoking cessation.

Purpose: Many patients die of recurrent NSCLC. Current clinical and molecular tests fail to predict patients at high risk for recurrence. We investigated the relationship between gene promoter methylation and recurrence of NSCLC and its prediction accuracy. Materials and Methods: We prospectively enrolled patients who underwent surgery for T1-2N0 NSCLC. We collected primary tumors, regional lymph nodes (RLN) and mediastinal lymph nodes (MLN). A total of 112 patients were included; cases (n = 39) and controls (n = 73) were defined based on cancer recurrence within 40 months of surgery. Promoter methylation of eight cancer-related genes (CDO1, TAC1, SOX17, p16, CDH13, RASSF1A, APC and MGMT) was obtained by using nanoparticle-based DNA extraction followed by qMSP. Logistic regression analysis adjusted by age, gender, race, tumor size and pack year was used to study the relationship between the gene promoter methylation and the risk of recurrence of NSCLC; machine learning assessed its prediction accuracy. Results: Cases had significantly higher p16 methylation levels than controls in all three tissue types by univariate and multivariate analysis after adjusting for confounders (age, gender, race, tumor size, smoking history, and other gene methylation levels). SOX17 was significantly methylated among cases in both RLN and MLN samples, and remained significant in MLN after adjusting for confounders. Table 1 shows the prediction accuracy of p16 and SOX17 using tumor tissue alone and combined with RLN and/or MLN. Prediction accuracies were increased after adding biomarkers from lymph nodes. Conclusion: Increased methylation status in the promoter region of p16 and SOX17 is associated with an increased risk of cancer recurrence among patients with T1-2N0 NSCLC treated with curative resection. Adding molecular analyses of lymph nodes to primary tumor samples significantly increased the diagnostic accuracy of predicting recurrence in these patients. Table 1.Prediction accuracy using tumor and combined with RLN and/or MLN.Tissue SamplesSensitivitySpecificityPPVNPVTumor0.410.840.520.76Tumor+RLN0.680.960.900.84Tumor+MLN0.560.880.710.79Tumor+RLN+MLN0.690.900.790.85 Citation Format: Chen Chen, Andrew Yang, Devlin Danielle, Kristen Rodgers, Peng Huang, Zhihao Lu, Candace Griffin, Beverly Lee, Richard Battafarano, Fenglei Yu, Tza-Huei Wang, Stephen Baylin, James Herman, Alicia Hulbert, Malcolm Brock. DNA methylation as biomarkers to predict early recurrence of T1-2N0 lung cancer. [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 4455.

e23016Background: Lung cancer remains the leading cause of cancer-related deaths worldwide. In the last few years, there has been a growing number of studies which implicate exosomes in lung cancer...

Epigenetic alterations are strongly associated with the development of cancer. We conducted a phase I/II trial of combined epigenetic therapy with azacitidine and entinostat, inhibitors of DNA methylation and histone deacetylation, respectively, in extensively pretreated patients with recurrent metastatic non–small cell lung cancer. This therapy is well tolerated, and objective responses were observed, including a complete response and a partial response in a patient who remains alive and without disease progression approximately 2 years after completing protocol therapy. Median survival in the entire cohort was 6.4 months (95% CI 3.8–9.2), comparing favorably with existing therapeutic options. Demethylation of a set of 4 epigenetically silenced genes known to be associated with lung cancer was detectable in serial blood samples in these patients and was associated with improved progression-free (P = 0.034) and overall survival (P = 0.035). Four of 19 patients had major objective responses to subsequent anticancer therapies given immediately after epigenetic therapy. Significance: This study demonstrates that combined epigenetic therapy with low-dose azacitidine and entinostat results in objective, durable responses in patients with solid tumors and defines a blood-based biomarker that correlates with clinical benefit. Cancer Discovery; 1(7); 598–607. ©2011 AACR. Read the Commentary on this article by Rodríguez-Paredes and Esteller, p. 557 This article is highlighted in the In This Issue feature, p. 539

Significant questions exist concerning the effectiveness of U.S. policies with nations and peoples across this region. An address of these questions begs an analysis of the economic and sociopolitical conditions in countries in the Middle East, for these conditions inform their relationships with the U.S. This report provides such an analysis for countries chosen for particular strategic interest to the U.S., namely Iraq, Iran, and Afghanistan. Current military conflicts in both Iraq and Afghanistan seek to provide a transition to stable, self-reliant, democratic states--a goal requiring economic development. In Iran, the government's lack of responsiveness to both U.S. and international pressures on key policy issues requires new thinking. Because the three countries are dissimilar cases, a separate evaluation of the conditions in each country was necessary. In order to ensure that analysis of core impediments to economic development and recommendations follow directly from these evaluations, what follows is presented as three standalone country studies.

Purpose: The National Lung Cancer Screening Trial provides hope that early detection can result in a reduction of mortality from lung cancer. This study was to determine if molecular alterations of sputum and plasma in patients with CT detected pulmonary nodules could predict patients with malignancy from those without. Materials and Methods: We conducted a prospective case-control study obtaining cases and controls from the Lung Cancer Spore Trial at Johns Hopkins Hospital. Plasma, sputum and pre-operative CT scans were obtained from all patients. We processed the plasma, sputum and tumor by using promoter Methylation on beads followed by quantitative methylation specific real-time PCR to investigate promoter methylation status of six cancer-related genes, CDO1, TAC1, HOXA7, HOXA9, SOX17 and ZFP42. Results: 223 patients were analyzed, including 162 patients with NSCLC and 61 patients with benign lesions. The sensitivity and specificity values for lung cancer detection in plasma, sputum or in both are shown. Plasma methylation seems to be more specific than sputum methylation. Both concomitant methylation in both plasma and sputum yielded the highest specificity for all gene combinations. Promoter methylation of CDO1, TAC1, HOXA9 and SOX17 has a high specificity but low sensitivity to detect lung cancer. Sensitivity and specificity for lung cancer detection using methylation changes in plasma and sputumPlasmaPlasmaSputumSputumPlasma & SputumPlasma & SputumGeneSpecificitySensitivitySpecificitySensitivitySpecificitySensitivityCDO180%45%67%63%93%26%TAC167%52%40%65%80%31%HOXA7100%11%80%27%100%6%HOXA960%66%7%90%67%60%SOX1767%56%73%52%93%27%ZFP4247%69%33%74%60%52%4 or more genes67%50%67%63%97%26%CDO1, TAC1, HOXA9, SOX1787%39%80%40%93%15% Conclusion: Our gene panel of promoter methylation has a high enough specificity to be clinical useful but its sensitivity is low. The integration of this panel with other biomarkers would be needed to create a platform of high enough specificity and sensitivity to be clinically applicable. Citation Format: Chen Chen, Alicia Hulbert, Alejandro Stark, Kristen Rodgers, Beverly Lee, Malcolm Brock, James Herman. Ultrasensitive DNA methylation detection using sputum and plasma for early detection of NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2015-3820

76 Background: Preoperative chemoradiotherapy using a platinum plus either a taxane or 5FU results in a pathological complete response rate (pCR) of approximately 25% to 30% in patients with operable esophageal cancer. A predictive biomarker to personalize chemotherapy in an effort to increase pCR would be a significant advance. Checkpoint with forkhead and ring finger domains (CHFR) is an E3 ligase which plays a role in response to mitotic stress. In our retrospective data, hypermethylation and silencing of CHFR, correlates with improved response and prolonged survival following therapy with taxanes. The primary endpoint of this study is to determine if there is a higher pCR rate when CHFR methylation status (MS) is used to decide if patients receive a taxane or 5FU. Methods: All patients presenting to Johns Hopkins with operable T2-T4/N0-N3 esophageal cancer are eligibile. A methylation-specific PCR which demonstrates CHFR MS has been developed by our lab. Methylated (M) patients receive weekly cisplatin (30mg/m2)/paclitaxel (50mg/m2) and unmethylated (U) patients receive previously described regimens of folfox or cisplatin/5FU. All patients receive concurrent radiation. Results: From July 2011 to September 2013, 35 patients have been assessed, 18 U (51%), 14 M (40%) and 3 unknown (9%). Twenty-four patients (22 males and 2 females) have been treated on study (age 48 – 74 years old, median 63.8) with 14 tumors U (58%) and 10 tumors M (42%) respectively. To date, 2/13 U patients (15%) have had a pCR and 3/8 M patients (38%) have had a pCR (p=0.32). Toxicities of each regimen are consistent with previous reports. Conclusions: Trimodality therapy is standard of care for operable esophageal cancer in the United States. Different chemotherapies have been combined with radiation prior to surgery to improve pCR rates but no biomarkers to personalize chemotherapy exist at present. Epigenetic biomarkers such as CHFR MS potentially may be used to decide which patients are more sensitive to taxane based regimens. Accrual to this study is ongoing. Clinical trial information: NCT01372202.

Epigenetic alterations driving carcinogenesis and cancer progression can be specifically targeted by the demethylating agent azacitidine (Aza) and the histone deacetylase inhibitor entinostat. While this treatment combination has been effective in a limited number of patients (pts) with treatment-refractory non-small cell lung cancer (NSCLC), we observed clinical benefit in 5 of 5 patients who received immunotherapy with PD-1/PD-L1 pathway blockade immediately following epigenetic therapy. Three of 5 pts developed partial tumor regressions (RECIST criteria, duration 10+ to 20+ mo.) and 2 pts had stable disease ≥6 mo. This compares to the objective response + SD rates of NSCLC to monotherapy with anti-PD-1 (18% + 5%) or anti-PD-L1 (10% + 12%). To understand how epigenetic therapy may synergize with blockade of the immunosuppressive PD-1 pathway, we used genome wide methylation and expression profiling on 8 NSCLC cell lines treated with low dose Aza. We discovered complex immunomodulatory effects of Aza with up-regulation of diverse immune related pathways including Jun/Jnk, NFKB, viral defense, type I interferon signaling, the inflammasome, antigen processing and presentation and immune evasion including up-regulation of PD-L1 expression. Multiple cancer-testes antigens were also up-regulated, thereby conferring de novo antigenicity. Supporting the idea that Aza acts specifically through inhibition and degradation of DNA methyltransferase proteins, colon cancer cells genetically haplo-insufficient for DNMT1 and devoid of DNMT3b mirror the immunomodulatory effects of Aza. Upstream events potentially controlling these pathways were defined, and prominent among them was up-regulation of the transcription factor, interferon regulatory factor 7 (IRF7), a DNA hypermethylated gene. These data were used to query hundreds of primary NSCLC samples from the Cancer Genome Atlas project (TCGA). A low basal expression signature of interferon pathway related genes was significantly associated with low IRF7 expression and promoter methylation in squamous tumors. Another hypermethylated transcription factor, PITX1, which inhibits a subset of type I interferon signaling genes, tracked with non-squamous cancers. Together, these findings support a model in which epigenetic modulation activates innate and adaptive immune responses within the tumor microenvironment together with induction of counter-regulatory immune checkpoint ligands which can be therapeutically blocked with antibodies. Based on these findings, a clinical trial testing the efficacy of DNMT and HDAC inhibition combined with PD-1 pathway blockade is under development. This work will form the basis for an immune-classification of NSCLC, as well as biomarker discovery for a novel therapeutic paradigm combining epigenetic and immunotherapy with potentially synergistic activity against the world's most deadly malignancy. Supported by Stand Up to Cancer. Citation Format: John Wrangle, Wei Wang, Alexander Koch, Hariharan Easwaran, Helai Mohammad, Princy Parsana, Frank Vendetti, Kristen Rodgers, Xiaoyu Pan, Kirsten Harbom, Cynthia Zahnow, Janis Taube, Julie Brahmer, Peter Jones, Suzanne Topalian, Charles Rudin, Malcolm Brock, Drew Pardoll, Stephen Baylin. Epigenetic therapy and sensitization of lung cancer to immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2013-4619

e18147 Background: A completed clinical trial has demonstrated activity of a combination of epigenetic therapies: entinostat, a histone deacetylase inhibitor, and azacytidine (AZA), a hypomethylating agent. A plasma-based biomarker assaying methylation status of key gene promoters from circulating DNA was developed. In order to test the relationship of activating mutations to sensitivity to epigenetic therapy, we treated non-small cell lung cancer (NSCLC) lines with AZA and assessed tumor growth in xenografts. Tumor from patients on trial was analyzed for mutational status. Methods: Nine NSCLC lines were treated in vitro for 72 hours with 500 nM AZA and injected to form flank tumors in NOG mice. Four lines contain no activating mutations. Of the five mutant lines, three were KRAS and two were EGFR mutations. Lines with no activating mutations are termed “mutation-negative” for known oncogenic drivers. Growth of mock versus treated tumors was compared over time to determine treatment phenotypes. Lines were grouped into four categories: augmented growth compared to mock, no treatment effect, mild inhibition of tumor growth, and marked inhibition. Seventeen tumors from the clinical trial were analyzed by SNaPshot multiplex PCR. Results: Among mutation negative lines three showed marked tumor growth inhibition and one showed mild inhibition. Among cell lines with activating mutations, one showed mild inhibition, three showed no effect, and one showed augmented tumor growth. Among 17 tumor samples from patients on study, four driver mutations were identified: 3 KRAS and one PTEN mutation. All four patients experienced progressive disease on study. Among thirteen patients with mutation negative tumors, three had stable disease and one had a complete response. Conclusions: The association between driver mutation negative status and treatment outcome in NSCLC cell lines in patients receiving AZA and entinostat merits further exploration. Future trials of epigenetic therapy in NSCLC may benefit from a biomarker enrichment strategy and the data presented here represents a potential way forward.