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Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. Significance: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. See related video: https://vimeo.com/720726054 This article is highlighted in the In This Issue feature, p. 1397

Supplementary Data from Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors.Significance:JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155.See related video: https://vimeo.com/720726054This article is highlighted in the In This Issue feature, p. 1397

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m2 daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8–5.7]; and median overall survival was 8.5 months (95% CI, 5.1–11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond.Significance:We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC.See related commentary by Pacheco and Byers, p. 1340.This article is highlighted in the In This Issue feature, p. 1325

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC.Significance:Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

DOC- 25K, Supplementary Tables 1-6

Supplementary Tables S3-S8

Supplementary Figure Legends

Supplementary Figure 1. "Phase 2-Eligible" Population. Supplementary Figure 2. ALKA-372-001 and STARTRK-1 Dosing Regimens.

Clinical trial protocol

Supplementary Figures 1-11

Text for supplement: Supplementary methods, supplementary figure legends

Supplementary Figure 1-5, Supplementary Tables 1-3

Supplementary Tables and Figures

Supplementary Tables 4-5

Supplemental Figure Legends

Immunohistochemical Analysis of CD8+ Tumor Infiltrating Lymphocytes. Representative images of CD8 scoring: 0 (A), 1+ (B), 2+ (C), and 3+ (D).

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:Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here, we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs.Experimental Design:We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations.Results:By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs. 63%), but plasma specimens were more likely to harbor ≥2 ALK mutations (24% vs. 2%, P = 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with ≥2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, P = 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired ≥1 new ALK mutations on lorlatinib.Conclusions:ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

Purpose: PD-1 inhibitors are established agents in the management of non–small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, anaplastic lymphoma kinase (ALK)-positive, and EGFR wild-type/ALK-negative patients.Experimental Design: We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORR) were assessed using RECIST v1.1. PD-L1 expression and CD8+ tumor-infiltrating lymphocytes (TIL) were evaluated by IHC.Results: Objective responses were observed in 1 of 28 (3.6%) EGFR-mutant or ALK-positive patients versus 7 of 30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced EGFR-mutant (N = 68) and ALK-positive (N = 27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre–tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5%, and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI–resistant biopsies (N = 57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8+ TILs (grade ≥2) were observed in only 1 pretreatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens and was not observed in any ALK-positive, pre- or post-TKI specimens.Conclusions: NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8+ TILs within the tumor microenvironment may underlie these clinical observations. Clin Cancer Res; 22(18); 4585–93. ©2016 AACR.See related commentary by Gettinger and Politi, p. 4539

Clinicopathologic Features of EGFR-Mutant and ALK-Positive Patients Included in PD-L1 Expression Analysis

Purpose:Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion–associated colorectal cancer.Experimental Design:We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results.Results:We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair–deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E–mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy.Conclusions:RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Supplementary Figures

Supplementary Tables

Supplementary Figure 1. Pan-TRK IHC in four cases identified as having NTRK fusions by OncoPanel analysis shows strong cytoplasmic staining confirming the presence of functional TRK fusion protein (40Ã-). Top left image demonstrates the specificity of TRK staining, which is present only in neoplastic cells, whereas adjacent normal crypts are negative. Supplementary Figure 2A. Two cases of ALK fusion in our cohort shared clinical and molecular features but differed in their microsatellite status. Both tumors were APC wild-type (wt) and contained biallelic RNF43 frameshift (fs) mutations. B. Copy number variation by chromosome is color-coded for two ALK fusion-associated colon cancers in our cohort. ALK+MMR-D cancer (top) displays a diploid genome, whereas ALK+MSS cancer (bottom) displays frequent copy number gains and losses. ALK fusion breakpoints are shown in both profiles (arrow). The vertical axis is the ratio of the number of reads for each specimen and a panel of normals in log base 2 scale. A value of 0 denotes no difference from normal (diploid). Supplementary Figure 3A. MMR-D RTK fusion CRC cases display a diploid genome similar to those in other MMR-D cases: BRAF V600E+mutated and Lynch syndrome. Copy number profile plots of RTK fusions or Wnt fusions with MSS CRC show extensive gains and losses and are shown for comparison. 3B. All MMR-D subgroups: BRAF V600E+, Lynch syndrome and RTK fusion show a significantly higher homopolymer indel rate (top) and TMB (bottom) than do MSS tumors with either RTK fusions or Wnt fusions. Supplementary Figure. 4. Histologic analysis with hematoxylin and eosin stained slides for two patients with NTRK1-LMNA fusions. Morphological analysis shows: A. mucinous and B. medullary histology.

Supplementary Figure Legends

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.

The outcomes of patients with SCLC have not yet been substantially impacted by the revolution in precision oncology, primarily owing to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. Although, these results are encouraging, many patients do not respond to, or rapidly recur after, current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion, is a snapshot of the current biomarker and clinical trial landscapes for SCLC. Finally, we identify key knowledge gaps that should be addressed to advance the field in pursuit of reduced SCLC mortality. This review largely summarizes work presented at the Third Biennial International Association for the Study of Lung Cancer SCLC Meeting.

BackgroundThe selective TRK inhibitor larotrectinib was approved for paediatric and adult patients with advanced TRK fusion-positive solid tumours based on a primary analysis set of 55 patients. The aim of our analysis was to explore the efficacy and long-term safety of larotrectinib in a larger population of patients with TRK fusion-positive solid tumours.MethodsPatients were enrolled and treated in a phase 1 adult, a phase 1/2 paediatric, or a phase 2 adolescent and adult trial. Some eligibility criteria differed between these studies. For this pooled analysis, eligible patients were aged 1 month or older, with a locally advanced or metastatic non-CNS primary, TRK fusion-positive solid tumour, who had received standard therapy previously if available. This analysis set includes the 55 patients on which approval of larotrectinib was based. Larotrectinib was administered orally (capsule or liquid formulation), on a continuous 28-day schedule, to adults mostly at a dose of 100 mg twice daily, and to paediatric patients mostly at a dose of 100 mg/m2 (maximum of 100 mg) twice daily. The primary endpoint was objective response as assessed by local investigators in an intention-to-treat analysis. Contributing trials are registered with ClinicalTrials.gov, NCT02122913 (active not recruiting), NCT02637687 (recruiting), and NCT02576431 (recruiting).FindingsBetween May 1, 2014, and Feb 19, 2019, 159 patients with TRK fusion-positive cancer were enrolled and treated with larotrectinib. Ages ranged from less than 1 month to 84 years. The proportion of patients with an objective response according to investigator assessment was 121 (79%, 95% CI 72-85) of 153 evaluable patients, with 24 (16%) having complete responses. In a safety population of 260 patients treated regardless of TRK fusion status, the most common grade 3 or 4 larotrectinib-related adverse events were increased alanine aminotransferase (eight [3%] of 260 patients), anaemia (six, 2%), and decreased neutrophil count (five [2%]). The most common larotrectinib-related serious adverse events were increased alanine aminotransferase (two [

In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.

Lurbinectedin is a synthetic marine-derived anticancer agent that acts as a selective inhibitor of oncogenic transcription. Lurbinectedin monotherapy (3·2 mg/mIn this phase 3, open-label, randomised study, adult patients aged 18 years or older with SCLC who relapsed after platinum-based chemotherapy were recruited from 135 hospitals across North America, South America, Europe, and the Middle East. Patients were randomly assigned (1:1) centrally by dynamic allocation to intravenous lurbinectedin 2·0 mg/mBetween Aug 30, 2016, and Aug 20, 2018, 613 patients were randomly assigned to lurbinectedin plus doxorubicin (n=307) or control (topotecan, n=127; CAV, n=179) and comprised the intention-to-treat population; safety endpoints were assessed in patients who had received any partial or complete study treatment infusions (lurbinectedin plus doxorubicin, n=303; control, n=289). After a median follow-up of 24·1 months (95% CI 21·7-26·3), 303 patients in the lurbinectedin plus doxorubicin group and 289 patients in the control group had discontinued study treatment; progressive disease was the most common reason for discontinuation (213 [70%] patients in the lurbinectedin plus doxorubicin group vs 152 [53%] in the control group). Median overall survival was 8·6 months (95% CI 7·1-9·4) in the lurbinectedin plus doxorubicin group versus 7·6 months (6·6-8·2) in the control group (stratified log-rank p=0·90; hazard ratio 0·97 [95% CI 0·82-1·15], p=0·70). 12 patients died because of treatment-related adverse events: two (1%) of 303 in the lurbinectedin plus doxorubicin group and ten (3%) of 289 in the control group. 296 (98%) of 303 patients in the lurbinectedin plus doxorubicin group had treatment-emergent adverse events compared with 284 (98%) of 289 patients in the control group; treatment-related adverse events occurred in 268 (88%) patients in the lurbinectedin plus doxorubicin group and 266 (92%) patients in the control group. Grade 3 or worse haematological adverse events were less frequent in the lurbinectedin plus doxorubicin group than the control group (anaemia, 57 [19%] of 302 patients in the lurbinectedin plus doxorubicin group vs 110 [38%] of 288 in the control group; neutropenia, 112 [37%] vs 200 [69%]; thrombocytopenia, 42 [14%] vs 90 [31%]). The frequency of treatment-related adverse events leading to treatment discontinuation was lower in the lurbinectedin plus doxorubicin group than in the control group (26 [9%] of 303 patients in the lurbinectedin plus doxorubicin group vs 47 [16%] of 289 in the control group).Combination therapy with lurbinectedin plus doxorubicin did not improve overall survival versus control in patients with relapsed SCLC. However, lurbinectedin plus doxorubicin showed a favourable haematological safety profile compared with control.PharmaMar.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy., Neuroendocrine carcinomas (NECs) arise from different anatomic sites, but have similar histological and clinical features. Here, the authors show that the epigenetic landscape of a range of NECs converges towards a common epigenetic state, while distinct subtypes occur within neuroendocrine prostate cancer contributing to intratumor heterogeneity in clinical samples.

Purpose: Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary ALK mutations. Here, we investigated utility of plasma genotyping for identifying ALK resistance mutations at relapse on next-generation ALK TKIs. Experimental Design: We analyzed 106 plasma specimens from 84 patients with advanced ALK-positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify ALK mutations. Results: By genotyping plasma, we detected an ALK mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of ALK mutations (67% vs. 63%), but plasma specimens were more likely to harbor ≥2 ALK mutations (24% vs. 2%, P = 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an ALK mutation in 22 (76%), including 14 (48%) with ≥2 ALK mutations. The most frequent combinations of ALK mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 ALK mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, P = 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired ≥1 new ALK mutations on lorlatinib. Conclusions: ALK resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of ALK resistance mutations leading to treatment-refractory compound ALK mutations.

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m2 daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8–5.7]; and median overall survival was 8.5 months (95% CI, 5.1–11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond. Significance: We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC. See related commentary by Pacheco and Byers, p. 1340. This article is highlighted in the In This Issue feature, p. 1325

Lung cancer patients with tumors harboring actionable alterations can achieve very durable responses to first-line targeted therapy. However, identifying targetable alterations using next-generation sequencing (NGS) is a complex and time-intensive process. As actionable genetic alterations are enriched in lung cancers arising in patients with limited smoking history, we designed a workflow to expedite NGS testing for this group.We developed a protocol to allow for next-day extraction of nucleic acids from frozen tissue. Specimens were designated as high priority during sequencing. We determined the interval between biopsy and NGS results to evaluate whether the workflow reduced the pre-analytical period and in-laboratory turnaround time and allowed for rapid initiation of genotype-matched therapy.Between January 2017 and May 2018, 21 patients participated in the expedited sequencing program. The median interval between biopsy and NGS results was 10.7 days. Six patients received results within 1 week of biopsy. Performing molecular analysis on frozen tissue and prioritizing sequencing and analysis of these specimens reduced the pre-analytical period from 3.5 to 1.3 days (p0.0001) and shortened in-laboratory turnaround time by 3 days (11.8 versus 8.4 business days, p0.0001). Ninety-three percent of patients with an actionable molecular alteration received first-line targeted therapy. The median time-to-initiation of treatment was 19.7 days from biopsy.Sequencing and analyzing nucleic acids from frozen tissue is a practical strategy for shortening the time to matched therapy. The significant advantage of upfront treatment with targeted therapies in subsets of lung cancer patients provides rationale for developing workflows that accelerate comprehensive molecular analysis.

3552 Background: Delta-like 3 (DLL3) is highly and specifically expressed in solid tumors, such as neuroendocrine carcinomas (NECs), malignant melanoma (MM), and medullary thyroid carcinoma (MTC). Rovalpituzumab tesirine (Rova-T) is a DLL3-targeting antibody-drug conjugate. Methods: This Phase 1/2 study (NCT02709889) enrolled patients with relapsed/refractory DLL3+ (>1% by IHC) advanced solid tumors and ECOG performance status of 0-1. Rova-T was given IV at 0.2, 0.3, or 0.4 mg/kg on d 1 of each 6-wk cycle (q6wk) for dose escalation (3+3 design) in disease-specific cohorts in Phase I. The recommended Phase 2 dose (RP2D) was tested in Phase II. Safety and dose-limiting toxicities (DLTs) were primary endpoints; efficacy outcomes were secondary endpoints. Results: The study enrolled 200 patients; 101 had NECs (large cell NEC [n=13], neuroendocrine prostate cancer [n=21], high-grade gastroenteropancreatic NEC [n=36], other [n=31]) and 99 had other solid tumors (MM [n=20], MTC [n=13], glioblastoma [GBM; n=23], other [n=43]). The median age was 61 y (range, 28-84); 63% were male. The RP2D was 0.3 mg/kg q6wk for 2 cycles in all cohorts. There were 7 DLTs in 5 patients: 2 with 0.2 mg/kg (Grade [Gr] 3 photosensitivity reaction, Gr 3 dyspnea), 2 with 0.3 mg/kg (1 with Gr 2 effusion, Gr 3 tumor lysis syndrome, and Gr 3 rhabdomyolysis; 1 with Gr 4 kidney injury), and 1 with 0.4 mg/kg (Gr 4 thrombocytopenia). Despite only 1 DLT identified with 0.4 mg/kg, the totality of the safety data suggested that this dose is not well tolerated. Common adverse events (AEs) in patients given 0.3 mg/kg (n=145) are shown (Table). Serious AEs occurred in 77/145 patients (53%), most commonly (≥3%) malignant neoplasm progression (n=18; 12%), pleural effusion (n=7; 5%), pericardial effusion (n=6; 4%), and dyspnea (n=5; 3%). The objective response rate (ORR) was 11% (21/200): 14 had NEC, 2 had MM, 2 had MTC, 2 had small cell carcinoma (SCC) not of lung origin (all partial responses), and 1 had GBM (complete response). In patients with NECs given 0.3 mg/kg, ORR, clinical benefit rate, and progression-free survival trended in favor of those with high DLL3-expressing tumors (≥50% by IHC) which represented 51% of NECs. Conclusions: Rova-T was tolerable in patients with advanced solid tumors at 0.3 mg/kg q6wk for 2 cycles. Antitumor activity was observed in patients with NEC, MM, MTC, SCC, and GBM. Clinical trial information: NCT02709889 . [Table: see text]

PURPOSE Genetic rearrangements of the tyrosine receptor kinase ROS proto-oncogene 1 ( ROS1) are oncogenic drivers in non-small-cell lung cancer (NSCLC). We report the results of an updated integrated analysis of three phase I or II clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2) of the ROS1 tyrosine kinase inhibitor, entrectinib, in ROS1 fusion–positive NSCLC. METHODS The efficacy-evaluable population included adults with locally advanced or metastatic ROS1 fusion–positive NSCLC with or without CNS metastases who received entrectinib ≥ 600 mg orally once per day. Co-primary end points were objective response rate (ORR) assessed by blinded independent central review and duration of response (DoR). Secondary end points included progression-free survival (PFS), overall survival (OS), intracranial ORR, intracranial DoR, intracranial PFS, and safety. RESULTS In total, 161 patients with a follow-up of ≥ 6 months were evaluable. The median treatment duration was 10.7 months (IQR, 6.4-17.7). The ORR was 67.1% (n = 108, 95% CI, 59.3 to 74.3), and responses were durable (12-month DoR rate, 63%, median DoR 15.7 months). The 12-month PFS rate was 55% (median PFS 15.7 months), and the 12-month OS rate was 81% (median OS not estimable). In 24 patients with measurable baseline CNS metastases by blinded independent central review, the intracranial ORR was 79.2% (n = 19; 95% CI, 57.9 to 92.9), the median intracranial PFS was 12.0 months (95% CI, 6.2 to 19.3), and the median intracranial DoR was 12.9 months (12-month rate, 55%). The safety profile in this updated analysis was similar to that reported in the primary analysis, and no new safety signals were found. CONCLUSION Entrectinib continued to demonstrate a high level of clinical benefit for patients with ROS1 fusion–positive NSCLC, including patients with CNS metastases.

Genetic rearrangements of the tyrosine receptor kinase ROS proto-oncogene 1 (The efficacy-evaluable population included adults with locally advanced or metastaticIn total, 161 patients with a follow-up of ≥ 6 months were evaluable. The median treatment duration was 10.7 months (IQR, 6.4-17.7). The ORR was 67.1% (n = 108, 95% CI, 59.3 to 74.3), and responses were durable (12-month DoR rate, 63%, median DoR 15.7 months). The 12-month PFS rate was 55% (median PFS 15.7 months), and the 12-month OS rate was 81% (median OS not estimable). In 24 patients with measurable baseline CNS metastases by blinded independent central review, the intracranial ORR was 79.2% (n = 19; 95% CI, 57.9 to 92.9), the median intracranial PFS was 12.0 months (95% CI, 6.2 to 19.3), and the median intracranial DoR was 12.9 months (12-month rate, 55%). The safety profile in this updated analysis was similar to that reported in the primary analysis, and no new safety signals were found.Entrectinib continued to demonstrate a high level of clinical benefit for patients with

8565 Background: Temozolomide has activity in small-cell lung cancer (SCLC), including patients (pts) with brain metastases (mets; Pietanza M, Clin Cancer Res 2012). Inhibition of Poly (ADP-ribose) polymerase (PARP) is another therapeutic strategy in SCLC. We hypothesized that olaparib plus temozolomide may be safe and effective for pts with relapsed SCLC and clinically active against CNS disease (Farago A, Cancer Discovery 2019). Here, we present an updated analysis of this combination in pts with relapsed SCLC, including a second cohort testing an alternative dosing strategy and an exploratory analysis of CNS-specific outcomes. Methods: In this phase I/II trial of olaparib plus temozolomide in pts with recurrent SCLC, pts were sequentially enrolled into two cohorts defined by dosing schedule. In cohort 1, olaparib was dosed on D1-7 of each 21d cycle. In cohort 2, olaparib was dosed on D1-21. Temozolomide was dosed on D1-7 in both cohorts. Each cohort had a phase I portion (conventional 3+3 dose-escalation) for determination of MTD and RP2D and a phase II portion with primary endpoint of ORR. Per protocol, eligible pts could have untreated asymptomatic brain mets < 1cm and, after mandatory baseline imaging, CNS imaging was performed at investigator’s discretion. A post-hoc exploratory analysis of CNS-specific outcomes was performed using modified RECIST criteria (Long GV, Lancet Oncol 2012) in which brain mets ≥5mm were considered measurable, and intracranial response was independently assessed by an attending radiologist. Results: 66 pts with median of 2 prior lines of therapy (range, 1-7) were enrolled, 50 pts in cohort 1 and 16 pts in cohort 2. 33/66 (50%) pts had history of brain mets, 15/66 (23%) pts had untreated brain mets at enrollment. The confirmed ORR of cohort 2 was 7% (1/14 evaluable pts, 95% CI: 0.2-33.9%), and the updated confirmed ORR of the entire study population was 34% (21/62 evaluable pts, 95% CI: 22.3-47.0%). The most common adverse events were hematologic toxicities (thrombocytopenia, anemia, and neutropenia). 22/50 (44%) of cohort 1 pts and 4/16 (25%) of cohort 2 pts required dose reduction. Of 15 pts with untreated brain mets, best overall intracranial response (including both confirmed and unconfirmed responses) was CR in 6 pts, PR in 4 pts, SD in 3 pts and PD in 1 for a CNS disease control rate of 87% (95% CI: 59.5-98.3%). Of 10 pts with CR/PR as their best intracranial response, 4 responses were confirmed. With non-CNS progression as a competing risk, the probability of CNS progression among the entire study population was 17% (95% CI: 8.8-26.7%) at 6 months and 21% (95% CI: 12.1-32.0%) at 12 months. Conclusions: Olaparib and temozolomide may be an effective therapy for relapsed SCLC, especially for pts with CNS disease where we observed a high rate of intracranial disease control. Ongoing analyses regarding optimal dosing schedule will inform potential for future use of this combination. Clinical trial information: NCT02446704.

BACKGROUND NTRK gene fusions occur in a range of tumor types, including those with CNS metastases. Larotrectinib, a highly selective FDA- and EMA- approved TRK inhibitor, demonstrated an objective response rate (ORR) of 79% across various cancers (Hong et al. Lancet Oncol. 2020). We report data on patients with TRK fusion cancer with CNS metastases treated with larotrectinib. METHODS Patients with solid tumors with CNS metastases at baseline harboring an NTRK gene fusion enrolled in two clinical trials (NCT02122913 and NCT02576431) were identified. Response was assessed by the investigator per RECIST v1.1. RESULTS As of July 15, 2019, 14 patients with CNS-metastases were enrolled: lung cancer (n=7), thyroid cancer (n=4), melanoma (n=2), and non-secretory breast cancer (n=1). Median age was 53 years (range 25–76). Including all sites of disease, the ORR was 71% (95% CI 42–92%): ten patients had a partial response (PR), two had stable disease (SD), and two had progressive disease (lung and melanoma). Of the three patients with measurable intracranial disease at baseline, the best intracranial response was 1 complete response, 1 PR, and 1 SD. Median duration of response for all patients was 14.8 months (range 1.9+ to 17.4+), median progression-free survival was 9.9 months (range 1.4 to 19.3+), and median overall survival was 27.8 months (range 1.4+ to 27.8). Treatment duration ranged from 1.4 to 25.0 months; six patients continued treatment beyond progression (lasting between 0.1+ and 8.5 months). Treatment-emergent adverse events (TEAEs) were mainly Grade 1 and 2; Grade 3 TEAEs occurred in seven patients (50%), with one (myalgia) attributed to larotrectinib (7%). There were no Grade 4 TEAEs. CONCLUSIONS Larotrectinib was highly active and well tolerated in TRK fusion cancer patients with CNS metastases. These results support testing for NTRK gene fusions across all cancers, including in patients with CNS metastases.

Introduction: The development of immune-related adverse events (irAEs) has been associated with improved efficacy of immune checkpoint inhibitors in patients with urothelial cancer, melanoma, and NSCLC. Whether this association exists in patients with SCLC is currently unknown. Methods: We conducted a multicenter retrospective study to evaluate the relationship between irAEs and immunotherapy efficacy in SCLC. To account for the lead-time bias resulting from the time-dependent nature of irAEs, the development of irAEs was considered as a time-varying covariate in univariate and multivariate Cox proportional hazard models. Results: Of the 183 patients treated with immunotherapy, 73 (39.9%) experienced at least one irAE. A total of 42 patients (22.9%) had grade 1 to 2 irAEs, whereas 31 patients (16.9%) had grade 3 to 4 irAEs. The median time of onset to the first irAE was 24 days (interquartile range: 14–55). The baseline clinicopathologic features were well-balanced between patients with and without irAEs. At a median follow-up of 24 months (95% confidence interval [CI]: 17.0–31.6), the median progression-free survival was significantly longer in the irAE group than the non-irAE group (3.8 versus 1.3 mo, p < 0.0001). The median overall survival was also significantly longer among patients with irAEs than patients without irAEs (13.8 versus 2.9 mo, p < 0.0001). When analyzed as a time-varying covariate, the development of irAEs was associated with a significant improvement in progression-free survival (hazard ratio: 0.44 [95% CI: 0.29–0.66], p < 0.001) and overall survival (hazard ratio: 0.47 [95% CI: 0.32–0.71], p < 0.001) in multivariate models. Conclusions: The development of irAEs is associated with improved clinical outcomes for immunotherapy in patients with advanced SCLC.

Purpose: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion–associated colorectal cancer. Experimental Design: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. Results: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair–deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E–mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. Conclusions: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyzed cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single cell analysis of human clinical samples exhibit a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

Introduction Lung cancer is associated with severe coronavirus disease 2019 (COVID-19) infections. Symptom overlap between COVID-19 and lung cancer may complicate diagnostic evaluation. We aimed to investigate the incidence, symptoms, differential diagnosis, and outcomes of COVID-19 in patients with lung cancer. Methods To determine an at-risk population for COVID-19, we retrospectively identified patients with lung cancer receiving longitudinal care within a single institution in the 12 months (April 1, 2019 to March 31, 2020) immediately preceding the COVID-19 pandemic, including an “active therapy population” treated within the last 60 days of this period. Among patients subsequently referred for COVID-19 testing, we compared symptoms, laboratory values, radiographic findings, and outcomes of positive versus negative patients. Results Between April 1, 2019 and March 31, 2020, a total of 696 patients received longitudinal care, including 406 (58%) in the active therapy population. Among 55 patients referred for COVID-19 testing, 24 (44%) were positive for COVID-19, representing a cumulative incidence of 3.4% (longitudinal population) and 1.5% (active therapy population). Compared with patients who were COVID-19 negative, those who were COVID-19 positive were more likely to have a supplemental oxygen requirement (11% versus 54%, p = 0.005) and to have typical COVID-19 pneumonia imaging findings (5 versus 56%, p = 0.001). Otherwise, there were no marked differences in presenting symptoms. Among patients who were COVID-19 negative, alternative etiologies included treatment-related toxicity (26%), atypical pneumonia (22%), and disease progression (22%). A total of 16 patients positive for COVID-19 (67%) required hospitalization, and seven (29%) died from COVID-related complications. Conclusions COVID-19 was infrequent in this lung cancer population, but these patients experienced high rates of morbidity and mortality. Oncologists should maintain a low threshold for COVID-19 testing in patients with lung cancer presenting with acute symptoms.