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Purpose: Patients with small-cell lung cancer (SCLC) have an exceptionally poor prognosis, calling for improved real-time noninvasive biomarkers of therapeutic response. Experimental Design: We performed targeted error-correction sequencing on 171 serial plasmas and matched white blood cell (WBC) DNA from 33 patients with metastatic SCLC who received treatment with chemotherapy (n = 16) or immunotherapy-containing (n = 17) regimens. Tumor-derived sequence alterations and plasma aneuploidy were evaluated serially and combined to assess changes in total cell-free tumor load (cfTL). Longitudinal dynamic changes in cfTL were monitored to determine circulating cell-free tumor DNA (ctDNA) molecular response during therapy. Results: Combined tiered analyses of tumor-derived sequence alterations and plasma aneuploidy allowed for the assessment of ctDNA molecular response in all patients. Patients classified as molecular responders (n = 9) displayed sustained elimination of cfTL to undetectable levels. For 14 patients, we observed initial molecular responses, followed by ctDNA recrudescence. A subset of patients (n = 10) displayed a clear pattern of molecular progression, with persistence of cfTL across all time points. Molecular responses captured the therapeutic effect and long-term clinical outcomes in a more accurate and rapid manner compared with radiographic imaging. Patients with sustained molecular responses had longer overall (log-rank P = 0.0006) and progression-free (log-rank P < 0.0001) survival, with molecular responses detected on average 4 weeks earlier than imaging. Conclusions: ctDNA analyses provide a precise approach for the assessment of early on-therapy molecular responses and have important implications for the management of patients with SCLC, including the development of improved strategies for real-time tumor burden monitoring.

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non–small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

Tumor mutation burden is an imperfect proxy of tumor foreignness and has therefore failed to consistently demonstrate clinical utility in predicting responses in the context of immunotherapy. We evaluated mutations in regions of the genome that are unlikely to undergo loss in a pan-cancer analysis across 31 tumor types (n = 9,242) and eight immunotherapy-treated cohorts of patients with non-small-cell lung cancer, melanoma, mesothelioma, and head and neck cancer (n = 524). We discovered that mutations in single-copy regions and those present in multiple copies per cell constitute a persistent tumor mutation burden (pTMB) which is linked with therapeutic response to immune checkpoint blockade. Persistent mutations were retained in the context of tumor evolution under selective pressure of immunotherapy and tumors with a high pTMB content were characterized by a more inflamed tumor microenvironment. pTMB imposes an evolutionary bottleneck that cancer cells cannot overcome and may thus drive sustained immunologic tumor control in the context of immunotherapy.

Introduction Neuroendocrine neoplasms (NEN) are heterogeneous malignancies that can arise at almost any anatomical site and are classified as biologically distinct well-differentiated neuroendocrine tumors (NET) and poorly differentiated neuroendocrine carcinomas (NEC). Current systemic therapies for advanced disease, including targeted therapies, chemotherapy, and immunotherapy, are associated with limited duration of response. New therapeutic targets are needed. One promising target is delta-like ligand 3 (DLL3), an inhibitory ligand of the Notch receptor whose overexpression on the surface of NEN is associated with tumorigenesis. Methods This article is a narrative review that highlights the role of DLL3 in NEN progression and prognosis, the potential for therapeutic targeting of DLL3, and ongoing studies of DLL3-targeting therapies. Classification, incidence, pathogenesis, and current management of NEN are reviewed to provide biological context and illustrate the unmet clinical needs. Discussion DLL3 is overexpressed in many NENs, implicated in tumor progression, and is typically associated with poor clinical outcomes, particularly in patients with NEC. Targeted therapies using DLL3 as a homing beacon for cytotoxic activity mediated via several different mechanisms (eg, antibody-drug conjugates, T-cell engager molecules, CAR-Ts) have shown promising clinical activity in small-cell lung cancer (SCLC). DLL3 may be a clinically actionable target across NEN. Conclusions Current treatment options for NEN do not provide sustained responses. DLL3 is expressed on the cell surface of many NEN types and is associated with poor clinical outcomes. Initial clinical studies targeting DLL3 therapeutically in SCLC have been promising, and additional studies are expanding this approach to the broader group of NEN.

Introduction Anti-PD-(L)1 immune checkpoint inhibitors (ICI) improve survival in patients with advanced non-small cell lung cancer (aNSCLC). The clinical features, survival, and burden of toxicities of patients with aNSCLC alive >1 year from ICI initiation are poorly understood. Materials and Methods We defined ICI survivors as patients alive >1 year after ICI start and retrospectively reviewed demographics, treatment, and immune-related adverse events (irAEs). Long-term irAEs were defined as ongoing irAEs lasting >1 year; burden of toxicity measures were based on percentage of days a patient experienced toxicity. Using linear and logistic regression, we evaluated association between demographics and disease characteristics with burden of toxicity. Results We identified 114 ICI survivors from 317 patients with aNSCLC. Half (52%) experienced an irAE of any grade, and 23.7% developed long-term irAEs. More ICI survivors with irAES in the first year had never smoked (P = .018) or received ICIs as frontline therapy (P = .015). The burden of toxicity in the first year significantly correlated with the burden of toxicity afterward (ρ = 0.72; P < .001). No patients with progressive disease had a high burden of toxicity, and they experienced 30.6% fewer days with toxicity than those with stable disease. Increased duration of therapy was associated with higher odds of experiencing toxicity. Half of ICI survivors with irAEs were still receiving treatment for unresolved irAEs at time of death or last follow-up. Conclusion Significant proportions of ICI survivors have unresolved long-term toxicities. These data support a growing need to understand long-term toxicity to optimize management of those treated with ICIs.

NUT carcinoma is a rare, aggressive cancer defined by rearrangements of the NUTM1 gene. No routinely effective treatments of NUT carcinoma exist, despite harboring a targetable oncoprotein, most commonly BRD4-NUT. The vast majority of cases are fatal. Poor awareness of the disease is a major obstacle to progress in the treatment of NUT carcinoma. While the incidence likely exceeds that of Ewing sarcoma, and BRD4-NUT heralded the bromodomain and extra-terminal domain (BET) inhibitor class of selective epigenetic modulators, NUT carcinoma is incorrectly perceived as “impossibly rare,” and therefore receives comparatively little private or governmental funding or prioritization by pharma. To raise awareness, propagate scientific knowledge, and initiate a consensus on standard and targeted treatment of NUT carcinoma, we held the First International Symposium on NUT Carcinoma on March 3, 2021. This virtual event had more than eighty attendees from the Americas, Europe, Asia, and Australia. Patients with NUT carcinoma and family members were represented and shared perspectives. Broadly, the four areas discussed by experts in the field included (1) the biology of NUT carcinoma; (2) standard approaches to the treatment of NUT carcinoma; (3) results of clinical trials using BET inhibitors; and (4) future directions, including novel BET bromodomain inhibitors, combinatorial approaches, and immunotherapy. It was concluded that standard chemotherapeutic approaches and first-generation BET bromodomain inhibitors, the latter complicated by a narrow therapeutic window, are only modestly effective in a minority of cases. Nonetheless, emerging second-generation targeted inhibitors, novel rational synergistic combinations, and the incorporation of immuno-oncology approaches hold promise to improve the prognosis of this disease.

(A) Comparison between ctDNA molecular responses and clinical benefit determined through radiographic imaging for patients treated with immunotherapy-containing regimens revealed concordance between each assessment (p = 0.019, Fisher’s exact test). For these comparisons, durable clinical benefit (DCB) was defined as a lack of progression or death within 6 months of treatment initiation; non-durable benefit (NDB) was defined as progression or death within 6 months of treatment initiation. Comparison between ctDNA molecular responses and radiographic assessments (PR/CR vs SD/PD/MR) for the determination of (B) overall survival (OS) at 64 months (maximum OS observed in patients with date of death recorded) and progression-free survival (PFS) at (C) 6 months, (D) 9 months and (E) 14 months (maximum PFS observed in patients with clinically documented progression following systemic treatment), is shown for all 33 patients included in the study cohort.

Supplementary Tables S1-S9

Purpose:Patients with small-cell lung cancer (SCLC) have an exceptionally poor prognosis, calling for improved real-time noninvasive biomarkers of therapeutic response.Experimental Design:We performed targeted error-correction sequencing on 171 serial plasmas and matched white blood cell (WBC) DNA from 33 patients with metastatic SCLC who received treatment with chemotherapy (n = 16) or immunotherapy-containing (n = 17) regimens. Tumor-derived sequence alterations and plasma aneuploidy were evaluated serially and combined to assess changes in total cell-free tumor load (cfTL). Longitudinal dynamic changes in cfTL were monitored to determine circulating cell-free tumor DNA (ctDNA) molecular response during therapy.Results:Combined tiered analyses of tumor-derived sequence alterations and plasma aneuploidy allowed for the assessment of ctDNA molecular response in all patients. Patients classified as molecular responders (n = 9) displayed sustained elimination of cfTL to undetectable levels. For 14 patients, we observed initial molecular responses, followed by ctDNA recrudescence. A subset of patients (n = 10) displayed a clear pattern of molecular progression, with persistence of cfTL across all time points. Molecular responses captured the therapeutic effect and long-term clinical outcomes in a more accurate and rapid manner compared with radiographic imaging. Patients with sustained molecular responses had longer overall (log-rank P = 0.0006) and progression-free (log-rank P < 0.0001) survival, with molecular responses detected on average 4 weeks earlier than imaging.Conclusions:ctDNA analyses provide a precise approach for the assessment of early on-therapy molecular responses and have important implications for the management of patients with SCLC, including the development of improved strategies for real-time tumor burden monitoring.

Figure S1 shows basal centrosome amplification in cell lines. Figure S2 shows the effect of GF on cell viability. Figure S3 shows HSET expression in cell lines. Figure S4 shows the effect of GF on clonogenic potential after RT. Figure S5 shows the induction of micronuclei after GF and/or RT. Figure S6 shows the expression of IFN-β after GF and/or RT.

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non–small cell lung cancer (NSCLC). Griseofulvin (GF; FDA-approved treatment) inhibits CC, and combined with radiation treatment (RT), resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and radiation treatment resulted in a significant tumor growth delay. Both GF and radiation treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased radiation treatment efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, radiation treatment, and immunotherapy could be a promising strategy to treat NSCLC.

The Notch signaling pathway is a critical embryonic developmental regulatory pathway that has been implicated in oncogenesis. In non–small cell lung cancer (NSCLC), recent evidence suggests that Notch signaling may contribute to maintenance of a cancer stem or progenitor cell compartment required for tumorigenesis. We explored whether intact Notch signaling is required for NSCLC clonogenic and tumorigenic potential in vitro and in vivo using a series of genetically modified model systems. In keeping with previous observations, we find that Notch3 in particular is upregulated in human lung cancer lines and that downregulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003) is associated with decreased proliferation and clonogenic capacity in vitro. We show that this phenotype is rescued with the expression of NICD3, a constitutively active cleaved form of Notch3 not affected by γ-secretase inhibition. Using an inducible LSL-KRASG12D model of lung cancer in vivo, we show a transient upregulation of Notch pathway activity in early tumor precursor lesions. However, a more rigorous test of the requirement for Notch signaling in lung oncogenesis, crossing the LSL-KRASG12D mouse model with a transgenic with a similarly inducible global dominant-negative suppressor of Notch activity, LSL-DNMAML (dominant-negative mastermind-like), reveals no evidence of Notch pathway requirement for lung tumor initiation or growth in vivo. Distinct Notch family members may have different and potentially opposing activities in oncogenesis, and targeted inhibition of individual Notch family members may be a more effective anticancer strategy than global pathway suppression. Mol Cancer Res; 9(12); 1746–54. ©2011 AACR.

Small cell lung cancer (SCLC) is an aggressive disease with one of the highest case-fatality rates among cancer. The recommended therapy for SCLCs has not changed significantly over the past 30 years; new therapeutic approaches are a critical need. TP53 is mutated in the majority of SCLC cases and its loss is required in transgenic mouse models of the disease. We synthesized an array of biodegradable poly(β-amino ester) (PBAE) polymers that self-assemble with DNA and assayed for transfection efficiency in the p53-mutant H446 SCLC cell line using high-throughput methodologies. Two of the top candidates were selected for further characterization and TP53 delivery in vitro and in vivo. Nanoparticle delivery of TP53 resulted in expression of exogenous p53, induction of p21, induction of apoptosis, and accumulation of cells in sub-G1 consistent with functional p53 activity. Intratumoral injection of subcutaneous H446 xenografts with polymers carrying TP53 caused marked tumor growth inhibition. This is the first demonstration of TP53 gene therapy in SCLC using nonviral polymeric nanoparticles. This technology may have general applicability as a novel anticancer strategy based on restoration of tumor suppressor gene function. Mol Cancer Ther; 12(4); 405–15. ©2013 AACR.

PDF file - 86 KB, High-throughput screen of a library of PBAE polymers

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PDF file - 927K, Supplemental Figure 1 (associated with Figure 2). Expression of IGF-1R, IR, ERK, and AKT does not correlate with sensitivity to OSI-906 in SCLC cell lines. Supplemental Figure 2 (associated with Figure 4). Treatment with OSI-906 results in inhibition of phosphorylated IGF-1R and Akt, but not phosphorylated ERK, in both sensitive and resistant cell lines. Supplemental Figure 3. Inhibition of MEK or overexpression of an active MEK mutant does not alter SCLC sensitivity to OSI-906. Supplemental Figure 4. OSI-906 treatment induces cell cycle arrest in sensitive cell lines.

PDF - 295K, Table S1. In vitro effects of CLO prodrug in NCI-60 cell screen. Table S2. In vitro effects of the CLO prodrug on SK-Mel-5 and UACC-62 melanoma cells. Figure S1. Bisphosphonamidate prodrugs are cell permeable and undergo intracellular activation in UACC-62 cells. Figure S2. CLO prodrug does not affect the viability of human keratinocytes and peripheral blood mononuclear cells. Figure S3. Intratumoral delivery of clodronate has no antitumor activity.

PDF file - 167 KB, The transfection efficiency of 456 polymers is limited by events downstream of binding and uptake.

Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for the treatment of extraskeletal disease is limited because of low cell permeability. We recently described a bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in non–small cell lung cancer cells. To evaluate anticancer activity of this prodrug class across many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an antitumor effect of CLO prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and potential to selectively target melanoma cells. Mol Cancer Ther; 13(2); 297–306. ©2013 AACR.

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Supplementary Materials & Methods

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PDF file - 25 KB, The transfection efficiency of 456 polymers in two non-transformed human lung fibroblast cell lines.

New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). Insulin-like growth factor 1 receptor (IGF-1R) inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways, including phosphatidylinositol-3-kinase-Akt and mitogen-activated protein kinase. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R, and the closely related insulin receptor. Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 μmol/L. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (P = 0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell-cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared with mock-treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach. Mol Cancer Ther; 12(6); 1131–9. ©2013 AACR.

Purpose: The hedgehog (Hh) signaling pathway, a key regulator of cell growth and differentiation during development is implicated in pathogenesis of certain cancers. Vismodegib (GDC-0449) is a small-molecule inhibitor of smoothened, a key component of Hh signaling. This phase I trial assessed GDC-0449 treatment in patients with solid tumors refractory to current therapies or for which no standard therapy existed.Experimental Design: Sixty-eight patients received GDC-0449 at 150 mg/d (n = 41), 270 mg/d (n = 23), or 540 mg/d (n = 4). Adverse events, tumor responses, pharmacokinetics, and pharmacodynamic down-modulation of GLI1 expression in noninvolved skin were assessed.Results: Thirty-three of 68 patients had advanced basal cell carcinoma (BCC), 8 had pancreatic cancer, 1 had medulloblastoma; 17 other types of cancer were also represented. GDC-0449 was generally well-tolerated. Six patients (8.8%) experienced 7 grade 4 events (hyponatremia, fatigue, pyelonephritis, presyncope, resectable pancreatic adenocarcinoma, and paranoia with hyperglycemia), and 27.9% of patients experienced a grade 3 event [most commonly hyponatremia (10.3%), abdominal pain (7.4%), and fatigue (5.9%)]. No maximum tolerated dose was reached. The recommended phase II dose was 150 mg/d, based on achievement of maximal plasma concentration and pharmacodynamic response at this dose. Tumor responses were observed in 20 patients (19 with BCC and 1 unconfirmed response in medulloblastoma), 14 patients had stable disease as best response, and 28 had progressive disease. Evidence of GLI1 down-modulation was observed in noninvolved skin.Conclusions: GDC-0449 has an acceptable safety profile and encouraging anti-tumor activity in advanced BCC and medulloblastoma. Further study in these and other cancer types is warranted. Clin Cancer Res; 17(8); 2502–11. ©2011 AACR.

Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57–18.35; P = 0.007 and HR 6.91; 95% CI, 1.37–34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non–small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies.Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer.See related commentary by Zou and Meyerson, p. 1038

Related CCR Translation from Phase I Trial of Hedgehog Pathway Inhibitor Vismodegib (GDC-0449) in Patients with Refractory, Locally Advanced or Metastatic Solid Tumors

Supplementary Table S1: Efficacy by investigator assessment. Supplementary Table S2: Objective response rate by Central Radiographic Assessment in key subgroups of interest. Supplementary Table S3: Efficacy in 3L patients by Central Radiographic Assessment. Supplementary Table S4: Deaths attributable to drug-related, treatment-emergent adverse events (TEAEs). Supplementary Table S5. Overview of pleural effusions. Supplementary Table S6. Overview of pericardial effusions. Supplementary Table S7. Overview of edema. Supplementary Table S8: Overview of cutaneous reactions.

Purpose: Seneca Valley Virus (SVV-001) is a novel naturally occurring replication-competent picornavirus with potent and selective tropism for neuroendocrine cancer cell types, including small cell lung cancer. We conducted a first-in-human, first-in-class phase I clinical trial of this agent in patients with cancers with neuroendocrine features, including small cell lung cancer.Experimental Design: Clinical evaluation of single intravenous doses in patients with cancers with neuroendocrine features was performed across five log-increments from 107 to 1011 vp/kg. Toxicity, viral titers and clearance, neutralizing antibody development, and tumor response were assessed.Results: A total of 30 patients were treated with SVV-001, including six with small cell carcinoma at the lowest dose of 107 vp/kg. SVV-001 was well tolerated, with no dose-limiting toxicities observed in any dose cohort. Viral clearance was documented in all subjects and correlated temporally with development of antiviral antibodies. Evidence of in vivo intratumoral viral replication was observed among patients with small cell carcinoma, with peak viral titers estimated to be >103-fold higher than the administered dose. One patient with previously progressive chemorefractory small cell lung cancer remained progression-free for 10 months after SVV-001 administration, and is alive over 3 years after treatment.Conclusions: Intravenous SVV-001 administration in patients is well tolerated at doses up to 1011 vp/kg, with predictable viral clearance kinetics, intratumoral viral replication, and evidence of antitumor activity in patients with small cell lung cancer. Phase II clinical evaluation in small cell lung cancer is warranted, and has been initiated. Clin Cancer Res; 17(4); 888–95. ©2011 AACR.

Supplementary Table S1. Summary of patient characteristics Supplementary Table S2. Summary of plasma samples analyzed Supplementary Table S3. Summary of peripheral blood lymphocytes and tumor infiltrating lymphocytes samples analyzed Supplementary Table S4. Genes analyzed by TEC-Seq Supplementary Table S5. Summary of TEC-Seq Characteristics Supplementary Table S6. Somatic sequence alterations detected in cfDNA Supplementary Table S7. Mutation Cellularity Analysis for tumor-specific cfDNA variants Supplementary Table S8. TCR-beta sequencing analysis Supplementary Table S9. Differentially abundant clones for CGLU111 Supplementary Table S10. Differentially abundant clones for CGLU117 Supplementary Table S11. Differentially abundant clones for CGLU127 Supplementary Table S12. Differentially abundant clones for CGLU212 Supplementary Table S13. Differentially abundant clones for CGLU135 Supplementary Table S14. Differentially abundant clones for CGLU161 Supplementary Table S15. Differentially abundant clones for CGLU159 Supplementary Table S16. Differentially abundant clones for CGLU162 Supplementary Table S17. Differentially abundant clones for CGLU203 Supplementary Table S18. Differentially abundant clones for CGLU243

Supplementary Figure S1.

Supplementary Figure S1. ctDNA clonal dynamics during anti-PD1 for patients with a clinical response. Supplementary Figure S2. ctDNA clonal dynamics during anti-PD1 for patients with molecular primary resistance. Supplementary Figure S3. ctDNA molecular responses precede radiologic responses. Supplementary Figure S4. Molecular responses correlate with a favorable prognosis. Supplementary Figure S5. Duration of molecular response correlates with progression-free and overall survival. Supplementary Figure S6. Disease prognostication by radiologic imaging at first assessment. Supplementary Figure S7. Differential clinical outcome by molecular responses in patients with radiologically stable disease. Supplementary Figure S8. Disease prognostication by tumor mutation burden. Supplementary Figure S9. ctDNA molecular response more accurately distinguish clinical responders from non-responders compared to clonal tumor mutation burden. Supplementary Figure S10. Molecular responses mirror pathologic responses to anti-PD1 therapy in early stage operable NSCLC. Supplementary Figure S11. TCR clonal dynamics during response and acquired resistance to anti-PD1. Supplementary Figure S12. TCR clonal dynamics for patients with clinical primary resistance. Supplementary Figure S13. Differential VJ gene usage for a patient with clinical response compared to a patient with clinical primary resistance. Supplementary Figure S14. Dynamic changes in pro-inflammatory and immunosuppressive cytokines in early stage NSCLC patients with differential responses to anti-PD1 therapy.

Purpose:Although extensive-stage small-cell lung cancer (SCLC) is highly responsive to first-line therapy, virtually all patients develop resistance with short survival. Rovalpituzumab tesirine (Rova-T) is an antibody–drug conjugate targeting delta-like 3 protein (DLL3). This open-label, single-arm, phase II study (TRINITY) assessed safety and efficacy of Rova-T in patients with DLL3-expressing SCLC in the third-line and beyond (3L+) setting.Patients and Methods:Patients with DLL3-expressing SCLC (determined by mouse antibody immunohistochemistry [IHC] assay), and ≥2 prior regimens, received 0.3 mg/kg Rova-T once every 6 weeks for two cycles. During study, a rabbit antibody IHC assay was developed and used for the final analysis, with DLL3-positive and DLL3-high defined as ≥25% and ≥75% of tumor cells positive for DLL3, respectively. The primary endpoints were objective response rate (ORR) and overall survival (OS).Results:Among 339 patients enrolled, 261 (77%) had two prior lines of therapy and 78 (23%) had ≥3. DLL3-high and DLL3-positive tumors by rabbit IHC were seen in 238 (70%) and 287 (85%) patients, respectively. The remaining 52 (15%) were DLL3-negative only by rabbit IHC or had missing results. ORR was 12.4%, 14.3%, and 13.2% in all, DLL3-high, and DLL3-positive patients, respectively. Median OS was 5.6 months in all patients and 5.7 months in DLL3-high patients. The most common adverse events (AE) were fatigue, photosensitivity reaction, and pleural effusion. Grade 3–5 AEs were seen in 213 (63%) patients.Conclusions:Rova-T is the first targeted agent in SCLC to use DLL3, a novel biomarker. However, results demonstrate modest clinical activity in 3L+ SCLC, with associated toxicities.

Supplementary Figure 1 from Therapeutic Efficacy of ABT-737, a Selective Inhibitor of BCL-2, in Small Cell Lung Cancer

XLSX file - 138KB, Supplemental Table 1. Tumor sample information, ABT-737 versus vehicle tumors submitted for microarray analysis. Supplemental Table 2. ABT-737 Treatment Response - Differentially Expressed Genes. Supplemental Table 3. ABT-737 Treatment Response - Gene Sets. Supplemental Table 4. Connectivity Map output ranked list.

PDF file - 399KB, Supplemental Figure 3. SCLC PDXs and cell lines express mTOR pathway components and the combination of ABT-737 and rapamycin is well tolerated in PDX-bearing mice.

PDF file - 527KB, Supplemental Figure 2. Rapamycin responses in a panel of SCLC cell lines and synergy matrix plots of ABT-737 plus inhibitors of PI3K and mTOR in SCLC cell lines.

PDF file - 187KB, Supplemental Figure 4. Dose-dependent effects of rapamycin and ABT-737 on LX33 PDXs.

PDF file - 126KB, Supplemental Figure 1. The combination of ABT-737 and etoposide is poorly tolerated in mice bearing SCLC PDXs.

Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of small cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. We compared primary SCLC xenografts prepared from de novo human tumors with standard cell line–based xenografts in the evaluation of a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent dichotomy may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significant decreases in tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, we isolated resistant derivatives of an initially sensitive cell line–based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling reveals 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. Taken together, these data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the testing of novel anticancer strategies in relevant preclinical models. [Cancer Res 2008;68(7):2321–8]

Supplementary Table 1 from Therapeutic Efficacy of ABT-737, a Selective Inhibitor of BCL-2, in Small Cell Lung Cancer