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Different subtypes of non-small cell lung cancer (NSCLC) are associated with different patterns of metastatic spread. Anatomic location of lesions in the chest may influence patterns of cancer growth and the shrinkage to therapy. Consequently, lesion location could affect apparent response rates per RECIST. We sought to explore this and develop, as needed, treatment response assessments less affected by the location.Cases of advanced oncogene-addicted NSCLC (EGFR, ALK, and ROS1) with pre- and on-therapy imaging during initial targeted therapy were identified. Lesions located in the lung parenchyma, pleural space or intra-thoracic lymph nodes were identified and analyzed separately from each other by RECIST 1.1 (unidimensional measurements) and by a novel MAX methodology (bidimensional measurements) which takes the axis with the greatest absolute percentage change on therapy in each location as the representative measurement.Three hundred three patients with 446 unidimensional measured lesions were included for RECIST analysis. Two hundred forty nine patients with 386 bidimensional measured lesions were included for MAX analysis, as well as the analysis comparing RECIST and MAX. Intrathoracic location significantly impacted percentage shrinkage and the response rate per RECIST. The response rates for pleural, intra-parenchymal and nodal lesions were 34.1%, 49.6%, and 68.3%, respectively (P = .0002). The MAX methodology both increased the apparent treatment effect and made it consistent between intrathoracic locations. For pleural, parenchymal and nodal lesions, the MAX calculated response rate were 83.7%, 72.2%, and 75.4%, respectively (P-value = .24).Intrathoracic lesion location affects RECIST-based treatment effectiveness estimations. The MAX methodology neutralizes location effect when examining impact of treatment and should be explored further.

Purpose: A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma. Patients and Methods: Anti–PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS). Results: Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs. Conclusions: With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation. See related commentary by Olson and Luke, p. 1167

Defining clinically relevant MET amplification levels in non-small cell lung cancer (NSCLC) remains challenging. We hypothesize that oncogene overlap and MET amplicon size decline with increase in MET plasma copy number (pCN), thus enriching for MET-dependent states.We interrogated cell-free DNA NGS results of 16,782 patients with newly diagnosed advanced NSCLC to identify those with MET amplification as reported using Guardant360. Co-occurring genomic mutations and copy number alterations within each sample were evaluated. An exploratory method of adjusting for tumor fraction was also performed and amplicon size for MET was analyzed when available.MET amplification was detected in 207 (1.2%) of samples. pCN ranged from 2.1 to 52.9. Of these, 43 (20.8%) had an overlapping oncogenic driver, including 23 (11.1%) METex14 skipping or other MET mutations. The degree of (non-MET) oncogene overlap decreased with increases in pCN. Patients with MET pCN ≥ 2.7 had lower rates of overlapping drivers compared to those with MET pCN2.7 (6.1% vs. 16.3%, P = .033). None of the 7 patients with pCN6.7 had an overlapping driver. After adjusting for tumor fraction, adjusted pCN (ApCN) was also lower for those with overlapping drivers than those without (median ApCN 4.9 vs. 7.3, P =.024). There was an inverse relationship between amplicon size and pCN.We propose that a high MET pCN and/or ApCN, together with the absence of overlapping oncogenic drivers and small MET amplicon size, will enrich for patients most likely to derive benefit from MET targeted therapy.

BACKGROUND: Women diagnosed with breast cancer prior to age 45 years (

Background Ependymoma (EPN) posterior fossa group A (PFA) has the highest rate of recurrence and the worst prognosis of all EPN molecular groups. At relapse, it is typically incurable even with re-resection and re-irradiation. The biology of recurrent PFA remains largely unknown, however, the increasing use of surgery at first recurrence has now provided access to clinical samples to facilitate a better understanding of this. Methods In this large longitudinal international multicenter study, we examined matched samples of primary and recurrent disease from PFA patients to investigate the biology of recurrence. Results DNA methylome derived copy number variants (CNVs) revealed large scale chromosome gains and losses at recurrence. CNV changes were dominated by chromosome 1q gain and/or 6q loss, both previously identified as high-risk factors in PFA, which were present in 23% at presentation but increased to 61% at 1 st recurrence. Multivariate survival analyses of this cohort showed that cases with 1q gain or 6q loss at 1 st recurrence were significantly more likely to recur again. Predisposition to 1q+/6q- CNV changes at recurrence correlated with hypomethylation of heterochromatin-associated DNA at presentation. Cellular and molecular analyses revealed that 1q+/6q- PFA had significantly higher proportions of proliferative neuroepithelial undifferentiated progenitors and decreased differentiated neoplastic subpopulations. Conclusions This study provides clinically and preclinically-actionable insights into the biology of PFA recurrence. The hypomethylation predisposition signature in PFA is a potential risk-classifier for trial stratification. We show that the cellular heterogeneity of PFAs evolves largely because of genetic evolution of neoplastic cells.

Introduction Historical reservations regarding radiosurgery (SRS) for small-cell-lung-cancer (SCLC) brain metastases (BrM) include concerns for short-interval/diffuse CNS-progression, poor prognoses, and increased neurological mortality specific to SCLC histology. We compared SRS outcomes for SCLC and non-small-cell-lung-cancer (NSCLC) where SRS is well established. Methods Multicenter first-line SRS outcomes for SCLC and NSCLC from 2000-2022 were retrospectively collected (N=892-SCLC/N=4,785-NSCLC). Data from the prospective JLGK0901 SRS trial were analyzed as a comparison cohort (N=98-SCLC/N=794-NSCLC). OS and CNS-progression were analyzed using Cox-Proportional-Hazard and Fine-Gray models, respectively, with multivariable (MV) adjustment (including age/sex/performance-status/year/extracranial disease/BrM-number/BrM-volume). Mutation-stratified analyses were performed in propensity score-matched (PSM) retrospective cohorts of EGFR/ALK-positive-NSCLC, mutation-negative-NSCLC, and SCLC. Results OS was superior with NSCLC over SCLC in the retrospective dataset (median-OS, 10.5 vs 8.6 months, MV-p Conclusion After SRS, SCLC was associated with shorter OS compared to NSCLC. CNS progression occurred earlier in SCLC overall but was similar in patients matched on baseline characteristics. Neurological mortality, lesions at CNS-progression, and leptomeningeal-progression were comparable. These findings may better inform clinical decision-making for SCLC patients.

PURPOSE: Radiation pneumonitis remains a major limitation in the radiation therapy treatment of patients with lung cancer. Functional avoidance radiation therapy uses functional imaging to reduce pulmonary toxic effects by designing radiation therapy plans that reduce doses to functional regions of the lung. Lung functional imaging has been developed that uses 4-dimensional computed tomography (4DCT) imaging to calculate 4DCT-based lung ventilation (4DCT-ventilation). A phase 2 multicenter study was initiated to evaluate 4DCT-ventilation functional avoidance radiation therapy. The study hypothesis was that functional avoidance radiation therapy could reduce the rate of grade ≥2 radiation pneumonitis to 12% compared with a 25% historical rate, with the trial being positive if ≤16.4% of patients experienced grade ≥2 pneumonitis. METHODS AND MATERIALS: Lung cancer patients receiving curative-intent radiation therapy (prescription doses of 45–75 Gy) and chemotherapy were accrued. Patient 4DCT scans were used to generate 4DCT-ventilation images. The 4DCT-ventilation images were used to generate functional avoidance plans that reduced doses to functional portions of the lung while delivering the prescribed tumor dose. Pneumonitis was evaluated by a clinician at 3, 6, and 12 months after radiation therapy. RESULTS: Sixty-seven evaluable patients were accrued between April 2015 and December 2019. The median prescription dose was 60 Gy (range, 45–66 Gy) delivered in 30 fractions (range, 15–33 fractions). The average reduction in the functional volume of lung receiving ≥20 Gy with functional avoidance was 3.5% (range, 0%–12.8%). The median follow-up was 312 days. The rate of grade ≥2 radiation pneumonitis was 10 of 67 patients (14.9%; 95% upper CI, 24.0%), meeting the phase 2 criteria. CONCLUSIONS: 4DCT-ventilation offers an imaging modality that is convenient and provides functional imaging without an extra procedure necessary. This first report of a multicenter study of 4DCT-ventilation functional avoidance radiation therapy provided data showing that the trial met phase 2 criteria and that evaluation in a phase 3 study is warranted.

Background: The clinical implications of the androgen receptor (AR), particularly in the context of aromatase inhibitor (AI) refractory metastatic breast cancer (MBC), are unclear. While AR is associated with more indolent primary tumors, in the absence of low estradiol/blocked ER, AR can exert a pro-survival signal. Thus, in a phase II trial of Fulvestrant (Fulv) plus Enzalutamide (Enza) in ER+/Her2- MBC (NCT02953860) we analyzed serial biopsies pre- and post-treatment. Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- MBC. Fulv at 500 mg IM days 1, 15, 29 and every 4 weeks thereafter and Enzaat 160 mg po daily on a continual basis were administered. Biopsies were required at study entry and at ~4 weeks on therapy. The clinical benefit rate at 24 weeks (CBR24) was the primary endpoint for efficacy. We performed mutational analysis using a modified Archer VariantPlex Solid Tumor Assay to detect mutations in ESR1 exon 8 and 67 other gene hotspots. We examined estrogen, progesterone, androgen and glucocorticoid receptor protein by IHC and multiplex for immune cells and PD-L1. Frozen cores were utilized to perform reverse phase protein array (RPPA) based protein pathway activation analysis of over 150 proteins from LCM-enriched tumor in baseline and post-treatment metastatic biopsies. Comparisons of Responders (progression free survival (PFS) equal to or longer than 24 weeks) and Non-Responders were performed using moderated t-tests on log2 transformed data. Results: 32 patients were eligible, median age was 61 years (46-87), and 90.6% had visceral disease with an average of 4 prior non-hormonal therapies and 3 prior hormonal agents (including 37.5% with prior Fulv). PFS >24 weeks was observed in ~22% of patients treated with Fulv plus Enza, including 42% of those who had prior Fulv. When stratified by both AR and ER protein levels, median time to progression was 59 days (95% CI: 55 to Inf) when both targets were high (greater than or equal to 10%), but only 14 days (95% CI: 13 to Inf) when both were less than 10%. Metastases with ESR1 mutations in the ligand binding domain had significantly higher levels of ER and PR protein than those with wild type ESR1 (p24 weeks was observed in 22% of patients treated with Fulv plus Enza, including 42% who had prior Fulv treatment, suggesting contribution of the anti-androgen. Response was significantly better when metastases were >10% for ER and AR. Poor response to Fulv plus Enza was significantly associated with mTOR pathway activation and patients with PIK3CA and or PTEN mutated metastases had a significantly shorter PFS. Mutation status also affected hormone receptor expression and immune infiltrates. The increase in PD-L1 protein following treatment with Fulv plus Enza warrants further pre-clinical investigation into whether the addition of anti-androgen can enhance efficacy of checkpoint inhibitor therapy in ER+ metastatic disease resistant to standard endocrine therapy approaches. Citation Format: Jennifer Richer, Nicole Spoelstra, Alyse Winchester, Julia Wulfkuhlue, Rosa Gallagher, Sharon Sams, Gregory Vidal, Peter Kabos, Jennifer Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Tessa McSpadded, Gloria Crawford, Virginia Borges, Dexiang Gao, Emanuel Petricoin, Anthony Elias. Response of persistent metastatic ER+/Her2- breast cancer treated with fulvestrant plus enzalutamide [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-01.

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)‐approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin‐based bivalent human epidermal growth factor fusion toxin (bi‐EGF‐IT) to treat EGFR‐expressing HNSCC. Bi‐EGF‐IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR‐expressing HNSCC cell lines and three human EGFR‐negative cancer cell lines. Bi‐EGF‐IT had increased binding affinity for EGFR‐expressing HNSCC compared with the monovalent version (mono‐EGF‐IT), and both versions specifically depleted EGFR‐positive HNSCC, but not EGFR‐negative cell lines, in vitro. Bi‐EGF‐IT exhibited a comparable potency to that of the FDA‐approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi‐EGF‐IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono‐EGF‐IT. In addition, in vivo off‐target toxicities were significantly reduced in the bi‐EGF‐IT treatment group compared with the mono‐EGF‐IT group. These results demonstrate that bi‐EGF‐IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono‐EGF‐IT and erlotinib. Thus, the novel bi‐EGF‐IT is a promising drug candidate for further development., In this study, we have developed a diphtheria toxin‐based bivalent human EGF fusion toxin for the treatment of HNSCC with significantly improved efficacy and markedly less in vivo off‐target toxicity compared with the monovalent human EGF fusion toxin. The bivalent human EGF fusion toxin is a promising novel drug candidate for the treatment of EGFR‐positive HNSCC.

Background: Almost all ER+ breast cancers express androgen receptor (AR), but its function is uncertain. AR expression is associated with more indolent tumors, however high AR expression relative to ER is associated with endocrine resistance, and in the absence of estradiol or if ER function is blocked, preclinical data would suggest that AR can take over to signal cell survival and proliferation. This non-blinded randomized phase II trial of neoadjuvant fulvestrant with or without enzalutamide was performed in women with T2 or greater ER+/Her2- primary breast cancer. Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- primary breast cancer cT2 or greater. A total of 4 months of therapy was given (fulvestrant 500 mg IM weeks 1, 3, 5, 9, and 13). Patients were randomized to receive enzalutamide 160 mg po daily on a continual basis for 16 weeks. Stratification factors were clinical node status (N0 vs N1/2) and T-stage (T2 vs T3/4). Surgery was planned for week 17. Fresh tumor biopsies were required at study entry and at ~4 weeks on therapy. Tissue, both fresh frozen and FFPE, was also obtained at time of surgery. The PEPI score at time of surgery was the primary endpoint for efficacy. The statistical design were parallel phase II trials with the experimental arm designed as a Simon 2-stage. The expectation for the control arm was a PEPI score of 0 in 16% and 32% for the combination arm. Additional enrollment of 12 patients would ensue if 4 or more achieved a PEPI score of 0 within the first 22 patients enrolled onto the combination arm. 27 patients would be enrolled into the fulvestrant alone arm. Results: The Simon 2-stage criteria were met and the trial continues to accrue up to 11 more evaluable patients. Thus far, 58 patients were consented of whom 50 were treated. Median age was 61.5 years (45-83); PS 0 (0-2). Among the 46 patients with information on AEs, there are 25 patients with Grade 2 AEs, including 5 with fatigue, 3 with headache, and 2 with hot flashes. There are 5 patients with grade 3 AEs including abdominal pain, gallbladder obstruction, ALT elevation, hyperglycemia, and hypertension. We also have 2 patients with grade 4 AEs , chest pain-cardiac and cardiac disorder-other. There is no grade 5 event. Paired samples at baseline and at 4 weeks are collected so far from 49 patients. 42 patients have completed surgery, while two patients have not undergone surgery. Conclusions: The combination of fulvestrant plus enzalutamide had manageable side effects. The trial is now in its extended stage since PEPI score = 0 was achieved in at least 4 of the first 22 patients on the combination arm. Extensive molecular studies of paired fresh biopsies from pretreatment and at 4 weeks are underway. These analyses and correlations with clinical outcome will be described. Citation Format: Anthony D Elias, Monica Fournier, Gregory A Vidal, Sharon Sams, Nicole Spoelsta, Peter Kabos, Jennifer R Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Julia Wulfkuhle, Emanuel Petricoin, Lisa Carter, Kathryn Zolman, Stephanie Armstead, Alyse Winchester, Tessa McSpadden, Gloria Crawford, Virginia Borges, Dexiang Gao, Jennifer Richer. Phase II randomized trial of fulvestrant with or without enzalutamide in ER+/Her2- primary breast cancer (BC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-06.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood–derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg−/− (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non–HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)–related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. Implications: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Patients with metastatic breast cancer (MBC) refractory to standard of care therapies have a poor prognosis. The purpose of this study was to assess patient characteristics and clinical outcomes for patients with MBC treated on phase I clinical trials. We performed a retrospective review of all patients with MBC who were enrolled in phase I clinical trials at the University of Colorado Cancer Center from January 2012 to June 2018. A total of 208 patients were identified. Patients had a mean age of 57 years and received on average 2.1 (range 0‐10) prior lines of chemotherapy. The majority of patients had hormone receptor‐positive/HER2‐negative breast cancer (58.6%) and 30.3% had triple‐negative breast cancer. The median progression free survival (PFS) was 2.8 months (95% CI, 2.3‐3.9) and median overall survival (OS) was 11.5 months (95% CI, 9.6‐13.2). Independent factors associated with longer PFS in multivariable analysis were treatment in a breast cancer‐selective trial or cohort (p = 0.016), age >50 years (p = 0.002), and ≤2 prior lines of chemotherapy in the metastatic setting (p = 0.025). Phase I clinical trials remain a valuable option for select patients with MBC and enrollment should be encouraged when available., We performed a retrospective review of all patients with metastatic breast cancer (MBC) who were enrolled in phase I clinical trials at the University of Colorado Cancer Center. Independent factors associated with longer PFS in multivariable analysis were treatment in a breast cancer‐selective trial or cohort, age >50 years, and ≤2 prior lines of chemotherapy in the metastatic setting. Phase I clinical trials remain a valuable option for selecting patients with MBC and enrollment should be encouraged when available.

Background: Most estrogen receptor alpha positive (ER+) breast cancers (BCs) express androgen receptor (AR) protein, but its function is unclear. High AR relative to ER is associated with endocrine resistance. This randomized phase II trial of neoadjuvant fulvestrant (F) with or without the anti-androgen enzalutamide (E) was designed to determine if the addition of E to F in women with ≥T2 ER+/Her2- primary BC would increase the % patients (pts) with limited residual disease at time of surgery as measured by modified preoperative endocrine predictive index (PEPI). Methods: 4 months of therapy was given prior to surgery (F 500 mg IM weeks 1, 3, 5, 9, and 13) and pts were randomized to receive E 160 mg po daily for 16 weeks, stratified by node status and T-stage. Tumor biopsies were required at study entry and after 4 weeks on therapy (Wk5). PEPI score at time of surgery was the primary endpoint for efficacy. The minimax two-stage design had 80% power with type I error rate of 0.08. Reverse phase phosphoprotein array (RPPA), IHC for ER/PR/AR/GR/Ki67, and Polaris multiplex immunofluorescence (IF) panel for myeloid lineage immune cells were performed. Average signal levels were compared across arms, PEPI score (0, >0), PEPI by arm, and Ki67 ( Results: 69 pts consented; 59 evaluable. 95% completed surgery. PEPI=0 observed in 10 (17%). Of 33 pts on FE arm, 21 had T3/T4 tumors, 91% ER+/PR+, median AR expression 80%, Ki67 15%. Of 26 patients on F arm, 19% had T3/T4 tumors, 96% ER+/PR+, median AR expression 85%, Ki67 10%. PEPI=0 was achieved more frequently on the FE arm (8, 24%) than the F arm (2, 8%) (p=0.16). Toxicity was as expected with endocrine therapy. IHC of baseline vs Wk5 biopsies showed decreased ER, PR, and Ki67 levels by Wk5 that remained decreased at time of surgery. AR and GR were significantly decreased only in the FE arm at the time of surgery. RPPA revealed that baseline EGFR (Y1604 and Y992) was higher in tumors with PEPI=0 tumors, whereas the average baseline mTOR activation was higher among pts with PEPI>0. Significant changes detected by RPPA upon treatment included a significant decrease in AR. Myeloid-derived suppressor cells (MDSC) were significantly reduced by treatment only in the FE arm. Conclusions: The combination FE had manageable side effects. PEPI=0 was achieved more frequently on the FE arm (8/33) than the F arm (2/26). Activated EGFR was higher pretreatment in tumors achieving PEPI=0. mTOR pathway was elevated pretreatment in PEPI>0 tumors (also observed with resistance to FE in ER+/HER2- metastatic disease (SABCS 2021). When comparing pretreatment tumor to Wk5, total AR by RPPA was the most differentially decreased protein in PEPI=0 tumors. AR signaling is known to support immunosuppressive cells and we observed a marked decrease in MDSCs with treatment only on the FE arm. Citation Format: Anthony D. Elias, Alyse Staley, Monica Fornier, Gregory A. Vidal, Sharon Sams, Nicole Spoelstra, Peter Kabos, Jennifer R. Diamond, Elena Shagisultanova, Rosa I. Gallagher, Julia Wulfkuhle, Emanuel Petricoin, Kathryn Zolman, Stephanie Biller, Vida Alami, Tessa McSpadden, Virginia Borges, Lyndsey S. Crump, Dexiang Gao, Jennifer K. Richer. Randomized phase II trial of preoperative fulvestrant with or without enzalutamide for ER+/Her2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT144.

In cancer-free tissues, somatic mutations accumulate in all of us over time. In most instances, these mutations are inconsequential to cellular fitness or to overall health. However, some mutations can result in a cell having a fitness advantage over its surroundings, enabling proliferation. In the blood, this proliferation - known as clonal hematopoiesis (CH) - is associated with an increased risk of hematologic malignancies, most notably leukemia, and is commonly observed in the elderly. People with Down syndrome (DS) also exhibit CH and have a markedly higher risk of leukemia (up to 400x); therefore, we investigated CH in people with DS to understand how mutations in the blood can lead to disrupted hematopoiesis and lead to varying susceptibility, onset, and severity of hematologic malignancies. To detect CH, I used a rare-mutation detection technique called DuplexSeq to analyze leukocytic genomic DNA from blood draws. This targeted-sequencing tool spans 37 genes implicated in leukemia - including leukemias seen in those with DS - and implements a double-stranded tag to incorporate mutational data from both DNA strands, enabling higher sensitivity than most sequencing methods. We characterized detected mutations using bioinformatic pipelines, various databases, and statistical methods. By comparing CH between people with and without DS, we observe an overrepresentation of protein-altering mutations in people with DS in genes important for hematopoiesis. Notably, people with DS exclusively harbor CH with JAK2 mutations, many of which have been associated with myeloid proliferative neoplasms. Mutations in NPM1 that have been previously seen in cancer are also exclusively observed in people with DS with potential to lead to differentiation block, a hallmark of leukemia. Finally, mutations with the highest variant allele frequency in TET2 are largely observed in people with DS, indicating preferential expansion in the DS context. These fledgling expansions in TET2 may be indicative of modified hematopoietic differentiation. Ongoing work is centered around understanding the consequences of these mutations and how they distinguish phenotypes among people with DS. Using a database for DS research, we are performing multi-omics analyses to understand which biological pathways may be disrupted in the same individuals with CH of interest. Additionally, we will use mouse models of DS to investigate the selective advantage of the observed mutations in the DS context, how they impact differentiation fate, and how cell intrinsic and extrinsic factors can influence clonal expansions. Overall, these studies offer a means by which risk of hematologic malignancies can be monitored by CH with elucidation of potential mechanisms. By characterizing CH, early markers of disrupted hematopoiesis can be established to determine susceptibility of leukemia and be used to monitor those who are most at risk. Citation Format: Edward J. Evans, Neetha Paul-Eduthan, Junxiao Hu, Dexiang Gao, Matthew Galbraith, Joaquin Espinosa, James DeGregori. Clonal hematopoiesis in Down syndrome and its potential impact on hematopoietic lineages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1510.

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and are commonly associated with poor outcomes in melanoma patients treated with immune checkpoint inhibitors. Inducing the differentiation of MDSCs using all-trans retinoic acid (ATRA) alters their activity and reduces MDSC frequency. This trial seeks to assess the safety and efficacy of combining ATRA and pembrolizumab in metastatic melanoma patients. In 24 stage IV melanoma patients, treatment with pembrolizumab Q3W plus the supplemental treatment of ATRA orally for three days surrounding each of the first four pembrolizumab infusions effectively lowered the frequency of circulating PMN-MDSCs and enhanced melanoma-specific T cell activity. The combination was well tolerated. Median progression free survival was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response. Targeting MDSCs remains a promising mechanism to enhance the efficacy of anti-PD-1 therapies and this combination merits further investigation.

Background While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. Method To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). Results We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. Conclusions We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.

Up to 91% of ER+ breast cancers express androgen receptor (AR), but its function is uncertain. Although AR expression is associated with more indolent tumors, high AR expression relative to ER is associated with endocrine resistance, and in the absence of estradiol or if ER function is blocked, preclinical studies suggest that AR can take over to signal cell survival and proliferation. Following extensive preclinical studies and a brief phase I to demonstrate a lack of significant PK interaction, this phase II trial of fulvestrant plus enzalutamide in ER+/Her2- metastatic breast cancer was conducted. Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- measurable or evaluable metastatic breast cancer without CNS disease. Prior fulvestrant was allowed, if clinically indicated as per treating physician. Fulvestrant was administered in standard dosing at 500 mg IM days 1, 15, 29 and every 4 weeks thereafter. Enzalutamide was given at 160 mg po daily on a continual basis. Fresh tumor biopsies were required at study entry and at about 4 weeks on therapy. The primary efficacy endpoint of the trial was clinical benefit rate at 24 weeks (CBR24). Assuming the undesirable rate of 10% and desirable rate of 30%, a sample size of 24 provided 89% power to detect this 25% rate difference using an exact binomial test with a one-sided alpha of 0.085. Due to the exploratory nature of biomarker analysis, the type I error rate was not adjusted for exploring multiple biomarkers. Results: A total of 38 patients were consented, of whom 32 were eligible. Median age was 61 years (46-87); PS 1 (0-1); a median of 2 prior chemotherapy and 2 prior hormonal therapies for metastatic disease. Twelve patients had prior fulvestrant, and 90% had visceral disease. TEAEs >20% included fatigue, nausea/vomiting, constipation, headache, anorexia, although most were low grade. There were no G4 or G5 toxicities. Median PFS was 2.0 months (0.5-12). CBR24 was 25% (7/28 evaluable).Conclusions: In a heavily pretreated population of women with metastatic ER+/Her2- BC, the combination of fulvestrant plus enzalutamide had manageable side effects, and modest activity. About 25% reached the primary endpoint of clinical benefit of more than 6 months on therapy. Extensive molecular studies of paired fresh biopsies from pretreatment and at 4 weeks are underway. These analyses and correlations with clinical outcome will be described. Citation Format: Anthony D Elias, Nicole Spoelsta, Gregory A Vidal, Sharon Sams, Peter Kabos, Jennifer R Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Tessa McSpadden, Gloria Crawford, Lisa Carter, Kathryn Zolman, Stephanie Armstead, Alyse Winchester, Virginia Borges, Julia Wulfkuhle, Emanuel Petricoin, Dexiang Gao, Jennifer Richer. Phase II trial of fulvestrant plus enzalutamide in ER+/Her2- advanced breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-14.

Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-β, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.

OBJECTIVE: To evaluate the role of curative intent concurrent chemoradiation (CCRT) vs radiation (RT) alone for T1-T3N0 HPV-positive and HPV-negative oropharyngeal squamous cell cancer (OPSCC). METHODS: The NCDB was queried for patients diagnosed between 2010 and 2017 with CT1-3N0M0 OPSCC treated with definitive RT or CCRT. Univariable analysis (UVA) and multivariable analysis (MVA) Cox regression analysis was performed with OS as the endpoint. Propensity score matching (PSM) 1:1 was performed. Interaction test to assess heterogeneity of treatment effect. RESULTS: A total of 2830 patients were queried. On MVA, CCRT was associated with improved OS for T3N0 tumors (HR 0.49; 95% CI 0.39–0.63) but not for T1N0 (HR 1.43; 95% CI 0.99–2.07) and T2N0 (HR 0.92; 95% CI 0.75–1.13). For T3 patients, CCRT improved OS for HPV-negative (HR 0.43; 95% CI 0.31–0.59) and HPV-positive tumors (HR 0.39; 95% CI 0.25–0.61). After PSM, CCRT was not statistically different to RT for patients with T1-2N0 HPV-negative tumors (HR 1.10; 95% CI 0.85–1.43; p = 0.48) and T1-2N0 HPV-positive tumors (HR 1.15; 95% CI 0.79–1.68; p = 0.45). After PSM, CCRT improved OS compared to RT alone for patients with T3N0 HPV-negative (HR 0.43; 95% CI 0.31–0.59; p < 0.01) and HPV-positive tumors (HR 0.39; 95 %CI 0.25–0.61; p < 0.01). CONCLUSIONS: CCRT is associated with improved OS in HPV-positive and HPV-negative T3N0 OPSCC. RT alone vs. CCRT demonstrated similar OS for T1-T2N0 OPSCC for both HPV negative and HPV positive tumors.

BackgroundImmuno-oncology (IO) agents have demonstrated efficacy across many tumor types and have led to change in standard of care. In breast cancer, atezolizumab and pembrolizumab were recently FDA-approved in combination with chemotherapy specifically for patients with PD-L1-positive metastatic triple-negative breast cancer (TNBC). However, the single agent PD-1/PD-L1 inhibitors demonstrate only modest single agent efficacy in breast cancer. The purpose of this study was to investigate the efficacy of novel IO agents in patients with metastatic breast cancer (MBC), beyond TNBC, treated in phase I clinical trials at the University of Colorado.MethodsWe performed a retrospective analysis using a database of patients with MBC who received treatment with IO agents in phase I/Ib clinical trials at the University of Colorado Hospital from January 1, 2012 to July 1, 2018. Patient demographics, treatments and clinical outcomes were obtained.ResultsWe identified 43 patients treated with an IO agent either as a single agent or in combination. The average age was 53 years; 55.8% had hormone receptor-positive/HER2-negative breast cancer, 39.5% TNBC and 4.7% HER2-positive. Patients received an average of 2 prior lines of chemotherapy (range 0-7) in the metastatic setting. Most patients (72.1%) received IO alone and 27.9% received IO plus chemotherapy. Median progression-free survival (PFS) was 2.3 months and median overall survival (OS) was 12.1 months. Patients remaining on study ≥ 6 months (20.9%) were more likely to be treated with chemotherapy plus IO compared to patients with a PFS < 6 months (77.8% v. 14.7%). No differences in number of metastatic sites, prior lines of chemotherapy, breast cancer subtype, absolute lymphocyte count, or LDH were identified between patients with a PFS ≥ 6 months vs. < 6 months.ConclusionsOur phase I experience demonstrates benefit from IO therapy that was not limited to patients with TNBC and confirms improved efficacy from IO agents in combination with chemotherapy. A subset of patients with MBC treated in phase I clinical trials with an IO agent derived prolonged clinical benefit. Predictors of response to immunotherapy in breast cancer remain uncharacterized and further research is needed to identify these factors.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

IMPORTANCE: Although stereotactic radiosurgery (SRS) is preferred for limited brain metastases from most histologies, whole-brain radiotherapy (WBRT) has remained the standard of care for patients with small cell lung cancer. Data on SRS are limited. OBJECTIVE: To characterize and compare first-line SRS outcomes (without prior WBRT or prophylactic cranial irradiation) with those of first-line WBRT. DESIGN, SETTING, AND PARTICIPANTS: FIRE-SCLC (First-line Radiosurgery for Small-Cell Lung Cancer) was a multicenter cohort study that analyzed SRS outcomes from 28 centers and a single-arm trial and compared these data with outcomes from a first-line WBRT cohort. Data were collected from October 26, 2017, to August 15, 2019, and analyzed from August 16, 2019, to November 6, 2019. INTERVENTIONS: SRS and WBRT for small cell lung cancer brain metastases. MAIN OUTCOMES AND MEASURES: Overall survival, time to central nervous system progression (TTCP), and central nervous system (CNS) progression-free survival (PFS) after SRS were evaluated and compared with WBRT outcomes, with adjustment for performance status, number of brain metastases, synchronicity, age, sex, and treatment year in multivariable and propensity score–matched analyses. RESULTS: In total, 710 patients (median [interquartile range] age, 68.5 [62-74] years; 531 men [74.8%]) who received SRS between 1994 and 2018 were analyzed. The median overall survival was 8.5 months, the median TTCP was 8.1 months, and the median CNS PFS was 5.0 months. When stratified by the number of brain metastases treated, the median overall survival was 11.0 months (95% CI, 8.9-13.4) for 1 lesion, 8.7 months (95% CI, 7.7-10.4) for 2 to 4 lesions, 8.0 months (95% CI, 6.4-9.6) for 5 to 10 lesions, and 5.5 months (95% CI, 4.3-7.6) for 11 or more lesions. Competing risk estimates were 7.0% (95% CI, 4.9%-9.2%) for local failures at 12 months and 41.6% (95% CI, 37.6%-45.7%) for distant CNS failures at 12 months. Leptomeningeal progression (46 of 425 patients [10.8%] with available data) and neurological mortality (80 of 647 patients [12.4%] with available data) were uncommon. On propensity score–matched analyses comparing SRS with WBRT, WBRT was associated with improved TTCP (hazard ratio, 0.38; 95% CI, 0.26-0.55; P

Introduction Preclinically, high epidermal growth factor receptor 1 (FGFR1) messenger RNA (FGFR1-MRNA) and FGFR1 amplification (FGFR1-AMP) predicted sensitivity to fibroblast growth factor receptor inhibitors in non–small-cell lung cancer and small-cell lung cancer cell lines. KRAS mutations did not preclude sensitivity. Patients and Methods Metastatic EGFR- and ALK-negative lung cancers were screened for FGFR1-MRNA by in-situ hybridization (ISH) and FGFR1-AMP by silver in-situ hybridization (SISH). Patients with positive findings were offered ponatinib, a multi–kinase inhibitor of FGFR1-4. Differences in overall survival (OS) between cohorts were assessed by the log-rank test. Association of FGFR1 positivity with clinicopathologic features were assessed by Fisher exact test and Kruskal-Wallis rank sum test. Results A total of 171 cases were prescreened: 9 (7.3%) of 123 SISH+; 53 (42.1%) of 126 ISH+; and 6 cases concordantly positive for SISH and ISH. SISH+ cases had fewer coincident KRAS mutations (P = .03) than SISH− cases, and ISH+ cases had worse OS (P = .020) than ISH− cases. Data distributions suggested a distinct higher positivity cut point for FGFR1 ISH (≥ 20%), occurring in 29 (23%) of 126 cases, was associated with small-cell lung cancer histology (P = .022), soft tissue metastases (P = .050) and shorter OS (P = .031). Four patients received ponatinib on study: All ISH+ by the initial cut point, 2 of 4 by higher cut point, 1 of 4 SISH+. Tolerability was poor. The best response for the 2 higher ISH cases was stable disease and progressive disease for the 2 lower ISH cases. Conclusion Elevated FGFR1-MRNA is more common than FGFR1-AMP and associated with worse OS. Higher FGFR1 mRNA expression may be associated with a specific phenotype and is worthy of further exploration. Ponatinib's poor tolerance suggests further fibroblast growth factor receptor exploration in ISH+ cases should utilize more selective FGFR1 inhibitors.

Background: Metastatic triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking hormone receptor expression and HER2 over-expression. Metastatic TNBC has been difficult to treat due to a general lack of targeted therapies, however, immunotherapy has shown substantial activity in a subset of patients. In metastatic TNBC patients with PD-L1-positive (combined positive score (CPS) ≥ 10) disease, KEYNOTE-355 demonstrated that addition of pembrolizumab (PD-1 inhibitor) to chemotherapy (nab-paclitaxel, paclitaxel, or carboplatin/gemcitabine) prolonged median progression free survival (PFS) compared to chemotherapy alone (9.7 months vs 5.6 months). Median 1-year PFS was also increased from 12.0% to 39.1% in these patients treated with pembrolizumab. These data led to the FDA approval of pembrolizumab with chemotherapy in PD-L1-positive metastatic TNBC patients. Despite these encouraging results, the majority of patients do not have long-term disease control. Radiotherapy (RT) in combination with immunotherapy and chemotherapy represents a promising avenue to prolong long-term response. RT can stimulate cellular damage and cause the release of tumor antigens, promoting a local T cell response. In addition, localized RT can result in the shrinkage of distant sites of metastasis via the abscopal effect when used with immunotherapy. The purpose of this study is to investigate the benefit of combining RT with pembrolizumab and chemotherapy in patients with metastatic PD-L1-positive TNBC. Methods: This two-stage, single-arm phase II study will assess the efficacy of RT in combination with nab-paclitaxel/paclitaxel plus pembrolizumab in PD-L1-positive unresectable or metastatic TNBC patients aged ≥18 years. To be included, patients must have only received < 1 prior line of systemic therapy in the metastatic setting or adjuvant/neoadjuvant setting if metastatic recurrence was within 12 months of treatment. The primary endpoint of the study is the 1-year PFS rate and a total of 29 subjects will be enrolled. Patients will be treated first with RT followed by the initiation of systemic therapy within seven days. Ablative RT will be directed at 1-4 sites of metastatic disease in 3 fractions of 8 Gy each. Nab-paclitaxel or paclitaxel will be given weekly day 1, day 8 every 3 weeks and pembrolizumab will be given every 3 weeks. Imaging will be repeated every 9 weeks to assess response based on RECIST 1.1. Systemic treatment will be continued until disease progression or intolerable toxicity. Treatment beyond progression will be allowed in certain patients who had initial response followed by progression. In these patients, repeat RT to new sites of disease can be administered with continuation of pembrolizumab to investigate the potential of re-sensitizing patients to immunotherapy. Blood will be collected before and after treatment for immune profiling. The sample size was determined using a null hypothesis for the 1-year PFS rate of 39% and an alternate hypothesis of 60%. The sample size of 29 yields a power of 80% to detect this difference with an alpha of 0.1 (1-sided). An interim analysis will be performed after enrollment of seventeen subjects in stage one. Citation Format: Anna R. Schreiber, Jodi Kagihara, Andrew Nicklawsky, Dexiang Gao, Anosheh Afghahi, Anthony Elias, Peter Kabos, Elena Shagisultanova, Todd Pitts, Julie Lang, Sana Karam, Virginia Borges, Christine Fisher, Jennifer R. Diamond. Phase II study of radiotherapy in combination with chemotherapy and immunotherapy in patients with PD-L1-positive metastatic triple-negative breast cancer [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 CT120.

Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (B- and T-lymphocyte attenuator, BTLA) or activation of immune activity (LIGHT). The aim of this study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in NSCLC.A TMA of 527 resected NSCLC samples and 56 NSCLC cell lines were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was optimized on the Dako Link48 autostainer using a polyclonal antibody from RD Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 28-8 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used.HVEM expression in the NSCLC resected samples and cell lines revealed a positive H-score more than 1 was 18.6% (77/415) and 48.2% (27/56) respectively. HVEM expression was significantly higher in patients with lymph node N2 metastasis (25.5% vs 7.9% vs 17.5%, p = 0.046) when comparing with N1 or no lymph node metastasis, and was marginally significantly higher in patients with stage III/IV disease (24.5% vs 16.4%, p = 0.059). Subgroup analysis showed that HVEM (median 45 vs 36 months, p = 0.706) and PD-L1 expression (median 45 vs 48 months, p = 0.178) status was not predictive of overall survival. HVEM was found to have a significant negative correlation with PD-L1 expression (r=-0.274, p 0.001) in patients with NSCLC and also a weak negative correlation in NSCLC cell lines (r=-0.162, p = 0.352).HVEM was found to be overexpressed in NSCLC patients of N2 lymph node metastasis or later stage and has a negative co-relationship with PD-L1 expression. HVEM was not prognostic for NSCLC patients.

Objectives Small cell lung cancer (SCLC) patients of all stages are treated with etoposide and cisplatin or carboplatin with or without surgery or chest radiotherapy. Initial response rates are ≥70% however the majority of patients relapse and are resistant to additional therapies due to pan-resistance to these salvage therapies. Therefore, new treatments are urgently needed. The non-taxane microtubule inhibitor eribulin has produced responses in heavily pretreated breast cancer patients. We evaluated the efficacy of eribulin alone and in combination with radiation in a panel of SCLC cell lines established from patients prior to or after receiving chemotherapy and or radiation. Material and methods Growth inhibition by eribulin alone, radiation alone and the combination was assessed by MTS assay and clonogenic survival. Eribulin induced cell cycle arrest was evaluated by FACS. Apoptosis was evaluated by using the Caspase-GLO 3/7 luminescent plate assay and by the Vybrant apoptosis assay with analysis by FACS. Results Eribulin mesylate inhibited the growth of all 17-SCLC lines at concentrations of ≤10 nM which is a clinically achievable dose. Growth inhibition was not significantly different between cell lines established prior to or after chemotherapy (p = .5). Concurrent eribulin + radiation induced a greater G2-M arrest, an increase in apoptotic cells and increased growth inhibition over radiation alone. Conclusions Eribulin was highly active alone and in combination with radiation in treatment naive SCLC lines and lines established from previously treated patients. In vivo pre-clinical studies of eribulin alone and in combination with radiation should be considered in SCLC cell lines.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora and angiogenic kinases with pro-apoptotic and antiproliferative activity in preclinical models of TNBC. Methods: This two institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC refractory to 1-3 prior lines of chemotherapy in the advanced setting. Patients had ECOG PS ≤ 1, measureable disease by RECIST 1.1 and no evidence of brain metastasis. Patients were treated with ENMD-2076 250 mg PO daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary end point was 6-month clinical benefit rate (6-CBR) and secondary endpoints included time to progression (TTP), PK profile, safety and biologic correlatives in archival and fresh serial tumor biopsies in a subset of patients. Results: Between July 2012 and October 2016, 41 patients were enrolled (median age 54; range 30-73; female 40; male 1). Patients received a mean 1.7 prior lines of chemotherapy for locally advanced unresectable or metastatic disease and 80.5% received prior neoadjuvant or adjuvant chemotherapy (N=33). Thirty-six patients were evaluable per protocol for the primary efficacy analysis. Five patients (12.2%) were not included in the efficacy analysis due to: adverse events (AE) leading to discontinuation prior to objective efficacy assessment (N=3), not meeting eligibility criteria on day 1 (N=1) and withdraw of consent in cycle 1 (N=1). The study proceeded to the second stage of enrollment based on observing three 6-CBR events in Stage 1 (N=18 patients). The 6-CBR in the overall trial was 16.7% (95% exact CI: 6%-32.8%; 2 patients with PR and 4 patients with SD > 6 mos). The median duration of response or clinical benefit in these patients was 32 weeks (8 cycles). 4-CBR was 27.8% (95% exact CI: 14%-45.2%). Dose reduction occurred in 8 patients (20%) for fatigue, hypertension and proteinuria. The most common grade 3 treatment-related adverse events were hypertension (37.5%) and fatigue (10%). One patient experienced grade 4 hypertension. Analysis of serial tumor biopsies prior to and following 2 weeks of ENMD-2076 (N=8 patients), demonstrated a treatment-induced decrease in cellular proliferation (Ki-67) and microvessel density (CD34) as assessed by IHC. Immunofluorescence performed on a subset of samples demonstrated an increase in p53-family member expression following treatment, consistent with changes observed in preclinical TNBC patient-derived tumor xenograft models. Conclusions: ENMD-2076 has durable clinical activity in a subset of patients with pretreated, advanced or metastatic triple-negative breast cancer. Predictive biomarker development using archival and fresh tumor tissue is underway. Exploration of lower doses of ENMD-2076 in future clinical trials may improve tolerability. Citation Format: Diamond JR, Eckhardt SG, Pitts TM, van Bokhoven A, Aisner D, Gustafson DL, Capasso A, Elias AD, Storniolo AM, Schneider BP, Gao D, Tentler JJ, Borges VF, Miller KD. Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-16.

We sought to identify tumor-secreted factors that altered the frequency of MDSCs and correlated with clinical outcomes in advanced melanoma patients. We focused our study on several of the many factors involved in the expansion and mobilization of MDSCs. These were identified by measuring circulating concentrations of 13 cytokines and growth factors in stage IV melanoma patients (n = 55) and healthy controls (n = 22). Based on these results, we hypothesized that IL-6 and IL-8 produced by melanoma tumor cells participate in the expansion and recruitment of MDSCs and together would be predictive of overall survival in melanoma patients. We then compared the expression of IL-6 and IL-8 in melanoma tumors to the corresponding plasma concentrations and the frequency of circulating MDSCs. These measures were correlated with clinical outcomes. Patients with high plasma concentrations of either IL-6 (40%) or IL-8 (63%), or both (35%) had worse median overall survival compared to patients with low concentrations. Patients with low peripheral concentrations and low tumoral expression of IL-6 and IL-8 showed decreased frequencies of circulating MDSCs, and patients with low frequencies of MDSCs had better overall survival. We have previously shown that IL-6 is capable of expanding MDSCs, and here we show that MDSCs are chemoattracted to IL-8. Multivariate analysis demonstrated an increased risk of death for subjects with both high IL-6 and IL-8 (HR 3.059) and high MDSCs (HR 4.265). Together these results indicate an important role for IL-6 and IL-8 in melanoma patients in which IL-6 potentially expands peripheral MDSCs and IL-8 recruits these highly immunosuppressive cells to the tumor microenvironment. This study provides further support for identifying potential therapeutics targeting IL-6, IL-8, and MDSCs to improve melanoma treatments.

Background: The clinical implications of the androgen receptor (AR), particularly in the context of aromatase inhibitor (AI) refractory metastatic breast cancer (MBC) are unclear. While AR is associated with more indolent primary tumors, high AR relative to ER in primary breast cancer is associated with endocrine resistance, and in the absence of estradiol or low or blocked ER, AR can exert a pro-survival signal. In a phase II trial of fulvestrant plus enzalutamide in ER+/Her2- MBC we analyzed serial biopsies pre- and post-treatment Methods: Eligible patients were women with ECOG 0-2, ER+/Her2- MBC. Fulvestrant 500 mg IM days 1, 15, 29 and every 4 weeks thereafter and Enzalutamide at 160 mg po daily on a continual basis were administered. Biopsies were required at study entry and at ~4 weeks on therapy. The clinical benefit rate at 24 weeks (CBR24) was the primary endpoint for efficacy. We performed mutational analysis to detect mutations including ESR1 exon 8 mutations.We examined estrogen, progesterone, androgen and glucocorticoid receptors, multiplex analysis of immune cells and PD-L1, and performed reverse phase protein array (RPPA) based protein pathway activation analysis of over 150 proteins/phosphoprotein drug targets from LCM-enriched tumor epithelium in baseline and post-treatment metastatic biopsies. Comparisons of PFS Responders (PFS longer than or equal to 24 weeks) and PFS Non-Responders (PFS shorter than or equal to 5 weeks) were performed using moderated t-tests on log2 transformed data. Results: 32 were eligible and median age was 61 years (46-87); PS 1 (0-1); a median of 2 prior chemotherapy and 2 prior hormonal therapies for metastatic disease (including 7 with prior Fulvestrant) and 90% had visceral disease. ESR1 mutant metastases had higher levels of ER and PR than those with wild type ESR1 (p Conclusions: Clinical benefit lasting 6-12 months was observed in 23% of patients. Our studies show important differences in hormone receptor expression and immune infiltrates in ESR1 mutated disease. RPPA based pathway activation mapping showed that mTOR pathway activation was associated with shorter PFS (p Citation Format: Jennifer K. Richer, Nicole S. Spoelstra, Alyse Winchester, Julia Wulfkuhle, Sharon B. Sams, Gregory Vidal, Peter Kabos, Jennifer Diamond, Elena Shagisultanova, Anosheh Afghahi, Jose Mayordomo, Tessa McSpadden, Gloria Crawford, Virginia Borges, Dexiang Gao, Emanuel Petricoin, Anthony D. Elias. Laboratory analyses of metastatic ER+/Her2- breast cancer treated with fulvestrant plus enzalutamide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2867.

9536 Background: Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and are commonly associated with poor outcomes in melanoma patients treated with immune checkpoint inhibitors. Inducing the differentiation of MDSCs using all-trans retinoic acid (ATRA) reduces MDSC frequency. This analysis seeks to assess the safety and efficacy of combining ATRA and pembrolizumab in advanced melanoma patients. Methods: This single arm, single institution, phase I/II study (NCT03200847) enrolled 24 patients diagnosed with stage IV melanoma. Eligible patients were over the age of 18 and had not been previously treated anti-PD-1 therapy. Treatment consisted of 200mg Q3W pembrolizumab plus the supplemental treatment of 150 mg/m2 ATRA orally for 3 days surrounding each of the first four infusions of pembrolizumab, with patients continuing pembrolizumab for up to two years until confirmed disease progression or unacceptable toxicity. The primary endpoints were safety and reduction in circulating MDSCs. Secondary endpoints were overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) according to RECIST v1.1. Results: At data cut off (Feb, 2021) 22 patients were evaluable for tumor response. Median follow-up was 1.0 years (0.3-2 years). In general, the combination of pembrolizumab and ATRA was well tolerated. The most common treatment-related adverse events (AEs) were grade 1 or 2, including headache (22 pts, 92%), fatigue (18 pts, 75%), rash (16 pts, 66%), and nausea (8 pts, 33%), most of which corresponded with the 3-day course of ATRA treatment. Ten patients had grade 3 or higher AEs with most being common ICI-related AEs. The ORR was 60% and DCR was 83%. Six-month PFS rate was 62%. Excluding patients diagnosed with uveal melanoma (n = 2) the ORR was 72%, DCR was 86%, and the six-month PFS rate was 68%. Paired analysis showed sustained decreases in absolute numbers ( p = 0.002) and percentage ( p = 0.007) of circulating MDSCs (CD3-CD19-CD56-CD11b+CD33+HLA-DR-/low) 4-6 weeks after stopping ATRA. The study is ongoing and further data will be presented in the future. Conclusions: This study demonstrates that the combination of ATRA and pembrolizumab is well tolerated and suggests that reducing MDSCs with ATRA may enhance the efficacy of pembrolizumab. This strategy of targeting MDSCs in combination with pembrolizumab warrants further development. Research Funding: Merck. Clinical trial information: NCT03200847.

10531 Background: Genomic assays, such as RS, are used to determine chemotherapy benefit in early-stage, estrogen receptor (ER)- and/or progesterone receptor (PR)-positive, HER2 negative BC patients (pts). Currently, guidelines to use pts’ germline genetic testing results to guide adjuvant therapy are lacking. Several reports have indicated worse outcomes for BC pts with g CHEK2 pathogenic variants (PV). We investigated whether PV in CHEK2 were associated with increased RS. Methods: Patient-level clinical data and RS were derived from electronic medical records of seven medical centers between years 2013-17. Confirmation of RS using the Genomic Health provider portal was performed. 38 pts with germline PV in CHEK2 (15 pts/39.5% with c.1100delC mutation) and RS score (cases) were matched with BC pts whose genetic testing did not identify PV (controls) using a 1:2 matching schema. Pts were matched based on age at diagnosis and lymph node (LN) status. LN negative pts were further matched based on T-stage. A multivariate random intercept linear mixed model of CHEK2 mutation status on RS was performed, adjusting for PR. A secondary ordinal univariate analysis was conducted that categorized RS into low, intermediate and high risk ( < 18, 18-30, and > 30, respectively). P-values were reported based on a null hypothesis of no effect against a two-sided alternative. Results: The median RS for cases was 19.5 (interquartile range [IQR]: 15 to 25) and the median RS for controls was 18 (IQR: 12 to 22). A greater proportion of cases were categorized as high risk (10.5%) compared to controls (5.6%), and a smaller proportion of cases were categorized as low risk (36.8%) compared to controls (49.3%). Cases had higher grade and increased proportion of PR-negative BC as compared with controls (grade 1: 12.1% of cases versus 32.4% of controls; PR-negative: 7.9% of cases versus 5.6% of controls). The variables used to match cases and controls (age, lymph node status, and T-stage) had similar summary statistics. The RS was 1.97-point higher in pts with g CHEK2 PV compared to controls, after adjusting for PR (95% confidence interval [CI]: 1.02-point lower to 4.96-point higher; p = 0.194). The secondary analysis of CHEK2 mutation status on an ordinal RS risk group yielded comparable results; on average, the odds of being high risk compared to the combined intermediate/low risk groups was 1.72 times higher in cases compared to controls (95% CI: 0.77 to 3.80; p = 0.181), but these differences were not significant. Conclusions: Our case-control study did not show a statistically higher RS for BC that develops in pts with g CHEK2 PV. Further studies are warranted to evaluate the association between type of CHEK2 PV (frameshift versus missense) and other modifying genetic variables and RS.

Therapeutic antibodies to immune checkpoints show promising results. Programmed death-ligand 1 (PD-L1), an immune checkpoint ligand, blocks the cancer immunity cycle by binding the PD-L1 receptor (programmed death 1). We investigated PD-L1 protein expression and messenger RNA (mRNA) levels in SCLC.PD-L1 protein expression and mRNA levels were determined by immunohistochemistry (IHC) with SP142 and Dako 28-8 PD-L1 antibodies and in situ hybridization in primary tumor tissue microarrays in both tumor cells and tumor-infiltrating immune cells (TIICs) obtained from a limited-disease SCLC cohort of 98 patients. An additional cohort of 96 tumor specimens from patients with extensive-disease SCLC was assessed for PD-L1 protein expression in tumor cells with Dako 28-8 antibody only.The overall prevalence of PD-L1 protein expression in tumor cells was 16.5%. In the limited-disease cohort, the prevalences of PD-L1 protein expression in tumor cells with SP142 and Dako 28-8 were 14.7% and 19.4% (tumor proportion score cutoff ≥1%) and PD-L1 mRNA ISH expression was positive in 15.5% of tumor samples. Increased PD-L1 protein/mRNA expression was associated with the presence of more TIICs (p0.05). The extensive-disease cohort demonstrated a 14.9% positivity of PD-L1 protein expression in tumor cells with Dako 28-8 antibody.A subset of SCLCs is characterized by positive PD-L1 and/or mRNA expression in tumor cells. Higher PD-L1 and mRNA expression correlate with more infiltration of TIICs. The prevalence of PD-L1 in SCLC is lower than that published for NSCLC. The predictive role of PD-L1 expression in SCLC treatment remains to be established.

Small-cell lung cancer (SCLC) cells have rapid proliferation, universal Rb inactivation, and high rates of MYC family amplification, making aurora kinase inhibition a natural target. Preclinical studies have demonstrated activity for Aurora A and pan-Aurora inhibitors with some relationship to MYC family expression. A clinical trial showed activity for an Aurora kinase A inhibitor, but no biomarkers were evaluated. We screened a panel of 23 SCLC lines with and without MYC family gene amplification or high MYC family gene expression for growth inhibition by the highly potent, selective aurora kinase B inhibitor barasertib. Nine of the SCLC lines were very sensitive to growth inhibition by barasertib, with IC50 values of 75% growth inhibition at 100 nmol/L. Growth inhibition correlated with cMYC amplification (P = 0.018) and cMYC gene expression (P = 0.026). Sensitive cell lines were also enriched in a published MYC gene signature (P = 0.042). In vivo, barasertib inhibited the growth of xenografts established from an SCLC line that had high cMYC gene expression, no cMYC amplification, and was positive for the core MYC gene signature. Our studies suggest that SCLC tumors with cMYC amplification/high gene expression will frequently respond to Aurora B inhibitors and that clinical studies coupled with predictive biomarkers are indicated. Mol Cancer Ther; 15(10); 2314–22. ©2016 AACR.

BackgroundAntitumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in immune infiltration and therapeutic responses between patients, demanding a mouse model with appropriate heterogeneity to investigate mechanistic differences.MethodsWe developed a unique HNSCC mouse model to investigate underlying mechanisms of heterogeneous antitumor immunity. This model system may provide a better control for tumor-intrinsic and host-genetic variables, thereby uncovering the contribution of the adaptive immunity to tumor eradication. We employed single-cell T-cell receptor (TCR) sequencing coupled with single-cell RNA sequencing to identify the difference in TCR repertoire of CD8 tumor-infiltrating lymphocytes (TILs) and the unique activation states linked with different TCR clonotypes.ResultsWe discovered that genetically identical wild-type recipient mice responded heterogeneously to the same squamous cell carcinoma tumors orthotopically transplanted into the buccal mucosa. While tumors initially grew in 100% of recipients and most developed aggressive tumors, ~25% of recipients reproducibly eradicated tumors without intervention. Heterogeneous antitumor responses were dependent on CD8 T cells. Consistently, CD8 TILs in regressing tumors were significantly increased and more activated. Single-cell TCR-sequencing revealed that CD8 TILs from both growing and regressing tumors displayed evidence of clonal expansion compared with splenic controls. However, top TCR clonotypes and TCR specificity groups appear to be mutually exclusive between regressing and growing TILs. Furthermore, many TCRα/TCRβ sequences only occur in one recipient. By coupling single-cell transcriptomic analysis with unique TCR clonotypes, we found that top TCR clonotypes clustered in distinct activation states in regressing versus growing TILs. Intriguingly, the few TCR clonotypes shared between regressors and progressors differed greatly in their activation states, suggesting a more dominant influence from tumor microenvironment than TCR itself on T cell activation status.ConclusionsWe reveal that intrinsic differences in the TCR repertoire of TILs and their different transcriptional trajectories may underlie the heterogeneous antitumor immune responses in different hosts. We suggest that antitumor immune responses are highly individualized and different hosts employ different TCR specificities against the same tumors, which may have important implications for developing personalized cancer immunotherapy.

Prostate cancer patients (pts) with non-metastatic prostate specific antigen (PSA) progression after local therapy and a long PSA doubling time (PSADT) usually have a period of PSA observation prior to starting on androgen deprivation therapy (ADT). We report results of an investigator-initiated, IRB-approved Phase II clinical trial (CT.gov-NCT03087903) investigating grape seed extract (GSE) in pts with PSA-only recurrence after maximum local therapy for prostate cancer. Pts with baseline PSA ≥ 0.2 ng/mL and rising PSA on 2 separate occasions and no evidence of metastases were eligible. Pts with baseline PSADT < 4 weeks (if absolute PSA > 2 ng/mL) were excluded. Pts were given 150mg of GSE orally twice daily for 12 months. GSE was administered as a formulation Leucoselect Phytosome-Indena S.p.A. (~100% proanthocyanidins, enriched in lower procyanidin oligomers complexed with soy phospholipids, about 1:2.6 w/w), with increased bioavailability. Study endpoints included PSA response (defined as an increase in PSADT by 30%) and change in PSA velocity. PSA was checked at baseline, 6 weeks and 3, 6, 9, and 12 months after starting GSE. Pts were removed from the trial with clinical/radiographic progression or if PSADT was less than 3 months. Pts completed dietary surveys and provided biologic specimens for tissue banking. The association of time and PSA level was assessed using a mixed effect modeling approach with the intention of calculating the doubling time. The natural log of PSA was taken to satisfy the linearity assumption of this method. Notably, pts on observation with a rising PSA after treatment for prostate cancer could be accrued rapidly to clinical trials; 27 pts were screened for this clinical trial at two sites (between January 2018 - August 2018) with 20 pts registered and started on treatment. Median (range) age and baseline PSA of pts enrolled were 71 (60 - 83) and 2.65 (0.44 - 17.44), respectively. Mean pretreatment PSADT was 5.4 months. GSE was well tolerated without serious adverse effects. 8 patients were withdrawn early due to PSADT progression of Citation Format: Paul Maroni, Elizabeth R. Kessler, Rodrigo Rodrigues-Pessoa, Andrew Nicklawsky, Thomas Flaig, Elaine Lam, Brandon Bernard, James Moore, Kyra Anderson, Dexiang Gao, Komal Raina, Rajesh Agarwal. A phase II trial using grape seed extract for prostate cancer patients with non-metastatic PSA progression after local therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT207.

Head and neck squamous cell carcinoma (HNSCC) is a biologically aggressive cancer in which high rates of recurrence (local, locoregional, or distant) contribute significantly to poor patient survival. One of the primary goals for current clinical practice of HNSCC surveillance is to detect recurrence at an early stage. However, current methods are either subjective, invasive, hard to access, not able to detect recurrence in a timely manner, or expensive. Saliva, given its directly adjacence to the anatomic location of HNSCC, may represent an opportunity to monitor HNSCC progression in a completely noninvasive, easy-to-access, and cost-effective method for HNSCC surveillance in a timely manner. We have developed a panel of seven biomarkers through screening of methylated microRNA loci (i.e., 9-1, 124-1, 124-2, 124-3, 129-2, 137, and 148) for HNSCC, and hereafter referred to as HNKlear. HNKlear demonstrated 92.1% sensitivity and 97.8% specificity in 189 HNSCC and 92 control tissue samples and 84.9% sensitivity and 95.4% in 86 pretreatment HNSCC and 108 control saliva samples. HNKlear is able to detect 93.5% T1 tumor and 91.2% stage I tumor in tissue, and 85.0% or 80.0% of HNSCC saliva when tumor is at T1 or stage I, respectively. In our prospective study, the value of HNKlear in patients’ saliva correlates with the tumor burden in 42 patients’ saliva before and after surgery within 3 months. HNKlear value is associated with progression-free survival in 57 primary tumor tissues and 38 treatment-naïve saliva. More importantly, HNKlear in saliva was shown to detect recurrence ranging from 90-337 days ahead of clinical detection of recurrence in the longitudinal follow-up of 43 HNSCC patients. Although these findings need to be validated in a large clinical trial, they suggest that analysis of certain methylated mciroRNA in saliva could have clinical significance for surveillance of HNSCC patients. Citation Format: Ling Lv, Steve Quattlebaum, Dexiang Gao, Yu Cao, Ben Milam, Francis Hall, Derek Smith, Elyse Handley, Sophia Bornstein, Neil Gross, John Song, Shi-Long Lu. Potential clinical value of methylated microRNAs in saliva-liquid biopsy for surveillance of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B07.

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non–small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. Implications: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

To test whether a microRNA (miRNA) panel may serve as an alternative biomarker of fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor sensitivity in lung cancer.Histologically diverse lung cancer cell lines were submitted to assays for ponatinib and AZD4547 sensitivity. miRNAs, FGFR1 messenger RNA, gene copy number, and protein expression were detected by real-time quantitative PCR, fluorescence in-situ hybridization, and immunoblotting in 34 lung cancer cell lines.Among 34 cell lines, 14 exhibited ponatinib sensitivity and 20 exhibited AZD4547 sensitivity (drug concentration causing 50% inhibition values 100 nmol/L). A total of 39 of the 377-miRNA set were initially identified from the 4 paired ponatinib-sensitive or -insensitive cell lines to have at least an 8-fold differential expression and then were detected in all the 34 cell lines. A predictive panel of 3 miRNAs (let-7c, miRNA155, and miRNA218) was developed that had an area under the curve (AUC) of 0.886 with a sensitivity of 71.4% and specificity of 77.3% to predict response to ponatinib. The miRNA panel performed similar to FGFR1 protein expression (AUC = 0.864) and messenger RNA expression (AUC = 0.939), and better than FGFR1 amplification (AUC = 0.696). Furthermore, we validated this panel using data for sensitivity to AZD4547 in the cell line cohort with an AUC of 0.931 and a sensitivity of 73.3% and specificity of 76.2%, respectively.The developed miRNA panel (let-7c, miRNA155, and miRNA218) may be useful in predicting response to FGFR tyrosine kinase inhibitors, either ponatinib or AZD4547 in lung cancer cell lines, and warrants further validation in the clinical setting.

Background To identify aberrant promoter methylation of genomic loci encoding microRNA (mgmiR) in head and neck squamous cell carcinoma (HNSCC) and to evaluate a biomarker panel of mgmiRs to improve the diagnostic accuracy of HNSCC in tissues and saliva. Methods Methylation of promoter regions of mgmiR candidates was initially screened using HNSCC and control cell lines and further selected using HNSCC and control tissues by quantitative methylation-specific PCR (qMS-PCR). We then examined a panel of seven mgmiRs for validation in an expanded cohort including 189 HNSCC and 92 non-HNSCC controls. Saliva from 86 pre-treatment HNSCC patients and 108 non-HNSCC controls was also examined using this panel of seven mgmiRs to assess the potentials of clinical utilization. Results Among the 315 screened mgmiRs, 12 mgmiRs were significantly increased in HNSCC cell lines compared to control cell lines. Seven out of the 12 mgmiRs, i.e., mgmiR9-1, mgmiR124-1, mgmiR124-2, mgmiR124-3, mgmiR129-2, mgmiR137, and mgmiR148a, were further found to significantly increase in HNSCC tumor tissues compared to control tissues. Using multivariable logistic regression with dichotomized variables, a combination of the seven mgmiRs had sensitivity and specificity of 92.6 and 92.4% in tissues and 76.7 and 86.1% in saliva, respectively. Area under the receiver operating curve for this panel was 0.97 in tissue and 0.93 in saliva. This model was validated by independent bootstrap validation and random forest analysis. Conclusions mgmiR biomarkers represent a novel and promising screening tool, and the seven-mgmiR panel is able to robustly detect HNSCC in both patient tissue and saliva. Electronic supplementary material The online version of this article (10.1186/s13148-018-0470-7) contains supplementary material, which is available to authorized users.

Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.

PURPOSE: Functional imaging has been proposed that uses 4DCT images to calculate 4DCT-based lung ventilation (4DCT-ventilation). We have started a 2-institution, phase 2 prospective trial evaluating the feasibility, safety, and preliminary efficacy of 4DCT-ventilation functional avoidance. The trial hypothesis is that the rate of grade ≥2 radiation pneumonitis could be reduced to 12% with functional avoidance, compared with a 25% rate of pneumonitis with a historical control. The trial employed a Simon 2-stage design with a planned futility analysis after 17 evaluable patients. The purpose of this work is to present the trial design and implementation, dosimetric data, and clinical results for the planned futility analysis. METHODS AND MATERIALS: Eligible patients were patients with lung cancer who were prescribed doses of 45 to 75 Gy. For each patient, the 4DCT data were used to generate a 4DCT-ventilation image using the Hounsfield unit technique along with a compressible flow-based image registration algorithm. Two intensity modulated radiation therapy treatment plans were generated: (1) a standard lung plan and (2) a functional avoidance treatment plan that aimed to reduce dose to functional lung while meeting target and normal tissue constraints. Patients were treated with the functional avoidance plan and evaluated for thoracic toxicity (presented as rate and 95% confidence intervals [CI]) with a 1-year follow-up. RESULTS: The V20 to functional lung was 21.6% ± 9.5% (mean ± standard deviation) with functional avoidance, representing a decrease of 3.2% (P < .01) relative to standard, nonfunctional treatment plans. The rates of grade ≥2 and grade ≥3 radiation pneumonitis were 17.6% (95% CI, 3.8%−43.4%) and 5.9% (95% CI, 0.1%−28.7%), respectively. CONCLUSIONS: Dosimetrically, functional avoidance achieved reduction in doses to functional lung while meeting target and organ at risk constraints. On the basis of Simon’s 2-stage design and the 17.6% grade ≥2 pneumonitis rate, the trial met its futility criteria and has continued accrual.

Introduction The role of serum tumor markers in the modern management of advanced NSCLC remains poorly described. Methods A single-center retrospective analysis of available carcinoembryonic antigen, CA125, CA19.9, and CA27.29 levels at baseline and during treatment of stage IV lung adenocarcinoma by oncogenic driver was conducted. Results A total of 142 patients were analyzed (60 with anaplastic lymphoma kinase gene [ ALK ] rearrangement, 50 with EGFR mutation, four with ROS1 rearrangement, and 29 with KRAS mutation). Of these, 82% had at least one marker (95% if all four markers were measured), with CA27.29 being the most commonly increased and CA19.9 the rarest. Only CA27.29 differed significantly by oncogene (it was less common in KRAS ) ( p = 0.016). The median times to nadir during tyrosine kinase inhibitor (TKI) therapy in EGFR and ALK cases were 16.4 and 20 weeks, respectively. Of the 41 patients with EGFR mutation or ALK or ROS1 rearrangement, 24 (59%) demonstrated an initial increase within the first 4 weeks of TKI therapy, 58% of whom then had their levels fall below baseline. An increase in marker level of 10% or more from nadir occurred in 53% of systemic and 22% of central nervous system–only progression. Conclusions Serum tumor markers are frequently increased in lung adenocarcinoma regardless of driver oncogene. Changes within the first 4 weeks of therapy may be misleading. Progression is associated with marker increases, especially in sites other than the central nervous system.

Background: Immuno-oncology (IO) agents have demonstrated exceptional clinical benefit in patients with many tumor types, including melanoma and lung cancer. The PD-L1 inhibitor, atezolizumab, was recently FDA approved in combination with nab-paclitaxel for patients with PD-L1 positive triple-negative breast cancer (TNBC); however, single-agent PD-1/PD-L1 inhibitors demonstrate modest efficacy in breast cancer. The purpose of this study was to investigate the efficacy of IO agents in patients with metastatic breast cancer treated in phase I clinical trials. Methods: We performed a retrospective analysis using a database of patients with metastatic breast cancer who received treatment with IO agents in phase I/Ib clinical trials at the University of Colorado Anschutz Medical Campus from January 1, 2012 to July 1, 2018. Abstracted data included patient demographics, baseline characteristics, and clinical outcomes. Results: 208 patients with metastatic breast cancer were treated in phase I/Ib clinical trials; 43 were treated with an IO agent. The average age was 53 years old and 55.8% had hormone receptor-positive/HER2-negative breast cancer, 39.5% TNBC, and 4.6% HER2-positive disease. On average, patients received two prior lines of chemotherapy (range 0-7) in the metastatic setting. 31/43 patients (72.1%) received single agent or combination IO, and 12/43 (27.9%) received IO + chemotherapy. Median progression-free survival (PFS) was 2.3 months and median overall survival (OS) was 12.1 months in all patients. 9/43 (21%) of patients remained on study > 6 months and had a median PFS of 8.6 months. Patients remaining on study > 6 months were more likely to be treated with IO + chemotherapy compared to patients with a PFS < 6 months (77.8% v. 14.7%, p=0.0007). There was no difference in sites of metastasis, prior lines of chemotherapy, breast cancer subtype, absolute lymphocyte count, or LDH between patients with a PFS > 6 months compared to < 6 months. Conclusions: A subset of patients with metastatic breast cancer treated in phase I clinical trials at our center with an IO agent had derived prolonged clinical benefit. The benefit was not limited to patients with TNBC and was associated with receipt of chemotherapy in combination with IO. Predictors of response to immunotherapy in breast cancer beyond PD-L1 expression remain uncharacterized, and further research is needed to identify these factors. Citation Format: Jodi A. Kagihara, Jennifer A. Weiss, Andrew Nicklawsky, Dexiang Gao, Virginia F. Borges, Peter Kabos, Antonio Jimeno, Jennifer R. Diamond. Efficacy of immunotherapy agents in patients with metastatic breast cancer treated in phase I clinical trials [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A76.

Although tyrosine kinase inhibitors (TKI) are the most common first-line therapy for metastatic renal cell carcinoma, high-dose interleukin-2 (HD-IL2) remains the only agent that provides durable complete responses. The optimal sequence of these agents remains uncertain. This retrospective multi-institutional study examined the safety and efficacy of HD-IL2 following TKI therapy. After IRB approval at 7 HD-IL2 centers, data relating to patient, disease, and treatment characteristics among 40 consecutive patients with metastatic renal cell carcinoma who were treated with HD-IL2 after at least 1 prior TKI therapy were retrospectively collected. The most common cardiac adverse events were grade 3 hypotension and vascular leak syndrome. Six patients (15%) experienced other grade ≥3 cardiac adverse events. There were 2 treatment-related deaths due to congestive heart failure, occurring in 1 patient with short TKI to HD-IL2 interval and another patient with an abnormal baseline cardiac stress test. Best responses included 2 CRs (5%, duration 40+ and 62+ mo), 3 PRs (8%, duration 6, 11, and 24 mo), 13 SD (32%, median duration 12 mo), 20 PD (50%), and 2 not evaluable patients. Median overall survival was 22 months. Administration of HD-IL2 could be safe and effective after TKI therapy; however, careful selection of patients is critical. We recommend baseline cardiac risk factor assessment, screening with both cardiac stress test and echocardiogram, and allowing a TKI to HD-IL2 interval of at least 2 months.

Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. Clin Cancer Res; 20(12); 3299–309. ©2014 AACR.