Search

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

Representativeness of study participants

Figure legend Supplemental Figure 1

Bronchial dysplasia (BD), a presumed precursor of pulmonary squamous cell carcinoma (SCC), rarely progresses to invasive cancer. A high-risk cohort at the University of Colorado provided an opportunity to directly sample airway epithelium at mapped sites on successive bronchoscopies. We have hypothesized that persistent dysplastic lesions showing a similar or higher level of dysplasia on follow-up biopsy, are associated with increased risk for the development of SCC. Endoscopic biopsies from 188 high-risk subjects were histologically classified according to the current WHO classification for BD using a numeric histology score ranging from 1 to 8 representing normal bronchial mucosa through invasive lung cancer. Differences in follow-up histology scores were compared between sites classified by clinical, histologic, and immunohistochemical variables. Subjects with a higher frequency of sites that persist or progress to high-grade dysplasia (≥37.5% persist/progress, N = 35 versus N = 114) show a significant association with development of incident invasive SCC (adjusted HR, 7.84; 95% confidence interval, 1.56–39.39), and those with incident lung SCC have adjusted mean follow-up histology scores 1.55 U higher than in subjects without lung cancer. Current smoking, elevated Ki67 growth fraction, histologic features of angiogenic squamous dysplasia (ASD) and higher histology score in baseline biopsies are significantly associated with increased follow-up histology scores. These results show that persistent BD is associated with the development of invasive SCC. Furthermore, increased expression of Ki67, the presence of angiogenic change and degree of baseline atypia are associated with persistence of BD. Cancer Prev Res; 9(1); 96–104. ©2015 AACR.

Supplementary figure 1. (A) Changes in other circulating cytokines. Colored box denotes the time when the patients were being treated with ATRA. (B) example gating strategy for CD8+ T cell activation.

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.

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

Characteristics of patients experiencing clinical benefit

Table S1. Expanded HSPC engraftment into mouse cohorts Table S2. Human immune cell presence in HM tissues Table S3a. CUHM003 mutations present at diagnosis b. Additional CUHM003 mutations present at progression c. CUHM005 mutations present at diagnosis d. Additional CUHM005 mutations present at progression Table S4. Enrichment of immunity-related genes in patient and HM tumors Table S5a. Genes expressed only in the CUHM003 patient and mHM model b. Genes expressed only in the CUHM003 patient and aHM model c. Genes expressed only in the CUHM005 patient and mHM model d. Genes expressed only in the CUHM005 patient and aHM model Table S6. Changes in cancer pathway GSEA expression in patients, their NSG, mHM, and aHM models Table S7. Changes in GSEA hallmark expression between mouse models Table 8a. Genes differentially expressed in CUHM003 after PD1 treatment b. Genes differentially expressed in CUHM005 after PD1 treatment

Supplemental figure S2. Univariable analysis comparing frequency of SCC in cases grouped by highest baseline (BL) histology score (HS) or mean baseline histology score using a cutoff of HS > 5 (moderate dysplasia).

Supplementary figure 2. Changes in other clinical hematologic measures. Colored box denotes the time when the patients were being treated with ATRA. * Denotes p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

SUPPLEMENTAL TABLE S1. BIOPSY AND CLINCAL CHARACTERISTICS. SUPPLEMENTAL TABLE S2. BASELINE HISTOLOGY AND OUTCOME.

Supplemental figure S1. Univariable analysis comparing frequency of SCC using four different definitions of persistent bronchial dysplasia (PBD; RBD = regressive BD) for sites with any baseline diagnosis (HS 1-7).

Aurora A (A) and Aurora B (B) kinase gene expression in SCLC cell lines.

Supplemental figure S3. Sites that show HGD in follow-up biopsies are more frequent in subjects that have prevalent (Prev) or develop incident (Inc) squamous cell carcinoma (SCC) as compared to those without cancer (No CA). *p < 0.05.

All adverse events experienced by patients, including those unrelated to treatment

Trial inclusion/exclusion criteria

Establishing recommended phase II dose (RP2D) and dose limiting toxicities (DLTs)

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.

XLSX file - 66KB, Table S1. Compiled and tabulated ponatinib IC50 values, FGF and FGFR expression, FGFR1 gene copy number and characteristics of the 58 lung cancer cell lines.

PDF file - 786KB, Figure S1. FGFR1 immunoblots of extracts from lung cancer cell lines representative of the 58 cell lines employed in the studies. Figure S2. Images and quantification of clonogenic growth assays in selected lung cancer cell lines following shRNA-mediated FGFR1 silencing. Figure S3. Dose response curve for inhibition of Colo699 cell growth by the FGF ligand trap, FP-1039. Figure S4. Venn diagram of the overlap of FGFR1, FGF2 and FGF9 expression in ponatinib-sensitive lung cancer cell lines and splicing status of FGFR2 in H1581 and H1048 cells. Figure S5. Graph of ROC analysis of FGFR1 GCN and mRNA expression in a TMA comprised of 21 lung cancer cell lines. Figure S6. Kaplan-Meier analysis of overall survival of lung cancer patients with increased FGFR1 GCN or high mRNA. Figure S7. Venn diagram showing overlap of FGF2 and FGF9 expression with FGFR1 mRNA positivity in primary lung squamous cell carcinomas and adenocarcinomas analyzed by the Cancer Genome Atlas.

XLSX file - 106KB, Table S5. Tabulated FGFR1, FGF2 and FGF9 mRNA expression values, FGFR1 CNV values and mutation status of TCGA lung adenocarcinomas and squamous cell carcinomas.

PDF file - 90KB, Table S2. Tabulated expression values for selected FGFs in lung cancer cell lines from the Cancer Cell Line Encyclopedia. Table S3. Tabulated FGFR1 gene copy number values determined by SISH and associated patient data for the surgical lung cancer cohort. Table S4. Tabulated FGFR1 mRNA expression values determined by ISH and associated patient data for the surgical lung cancer cohort.

PDF file - 191K, Western blotting for phosphorylated (Y1068) EGFR, total EGFR, pERK and total ERK in NSCLC cells treated with DMSO, gefitinib and/or Wnt pathway inhibitors, qPCR analyses of Axin-2 mRNA levels in cells in cells treated with canonical Wnt pathway inhibitors, western blotting for activated beta-catenin in stable cell lines, and determination of the ability of activated beta-catenin expression to mitigate the impact of gefitinib and/or XAV939 treatment in HCC4006 NSCLC cells

PDF file - 222K, Demonstration that beta-catenin or TCF-4 knockdown results in enhanced sensitivity of NSCLC cells to gefitinib treatment, that beta-catenin or TCF-4 knockdowns reduce the expression of the corresponding protein, that there is minimal correlation between the expression of canonical Wnt target genes and gefitinib sensitivity for a panel of NSCLC cell lines, that there is no significant correlation of SLuGs with gefitinib induced genes, and that SLuGs are not enriched in lung cancer specific genes

PDF file - 105K, Graphical representation of shRNA counts obtained by deep sequencing in the screen for synthetic lethality with gefitinib in H322C and HCC4006 NSCLC cells

XLS file - 683K, The table contains all screening results from the shRNA screen, including results for each of the two cell lines (H322C and HCC4006) and overlapping hits (SLuG2s). The shRNA sequences, the frequency that these sequences were detected in the different replicates, and the associated significance are provided

PDF file - 235K, Validation of shRNA-mediated knockdown of tankyrase-1 expression, demonstration of enhanced NSCLC cell killing by combined inhibition of tankyrase and EGFR in multiple NSCLC cell lines, and specificity of this synergistic cell killing for NSCLC cells with EGFR activation

PDF fiel - 67K

PDF file - 113K

XLS file - 34K, Lists of primers and probes for real-time RT-PCR, shRNA sequences and antibodies are provided

PDF file - 240K, Demonstration that CK1alpha or CK2alpha knockdown results in enhanced sensitivity of NSCLC cells to gefitinib treatment, that these knockdowns reduce the expression of the corresponding proteins, that treatment with the CK1� activator pyrvinium pamoate acts synergistically with gefitinib to eliminate NSCLC cells, and specificity of this synergistic cell killing for NSCLC cells following EGFR inhibition

Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC. Cancer Res; 72(16); 4154–64. ©2012 AACR.

PDF file - 76K, Second xenograft experiment utilizing a different shRNA for TNKS1

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Background: Recruitment and expansion of immunosuppressive myeloid cells present a significant barrier to the successful treatment of melanoma. We aimed to identify tumor-secreted factors that altered the frequency of MDSCs and correlated with clinical outcomes in advanced melanoma patients. We hypothesized that production of IL-6 and IL-8 by melanoma tumors would lead to expansion and accumulation of MDSCs and correlate with long-term clinical outcomes. Methods: Expression of IL-6 and IL-8 in melanoma tumors as well as the plasma concentration of IL-6 and IL-8 were measured and compared with the frequency of circulating MDSCs in a total of 52 stage IV melanoma patients. These measures were correlated with tumor burden, BRAF status, lactic acid dehydrogenase (LDH) levels and with clinical outcomes. Samples were collected beginning in January 2011 and clinical follow-up was collected through January 2018. Results: The plasma concentration of both IL-6 and IL-8 correlated with tumor burden (p < 0.0001 and p= 0.0122, respectively). Patients with high plasma concentrations of either IL-6 (40%) or IL-8 (63%) had worse median overall survival (MOS) (7.5 months, p < 0.0001 and 9.2 months, p = 0.0015, respectively) compared to patients with low concentrations, where MOS was not reached. Patients with high plasma concentrations of both IL-6 and IL-8 (35%) had even worse MOS (6.7 months) than patients with low levels of both cytokines (MOS not reached, 31%) p < 0.0001. We observed that patients with lower circulating concentrations and lower tumoral expression of IL-6 and IL-8 showed decreased frequencies of circulating MDSCs. Furthermore, patients with low frequencies of MDSCs (MOS not reached) had better overall survival than patients with high frequencies of these cells (MOS 11.9 months) p = 0.0224. Multivariable analysis showed that when adjusting for variables BRAF status and LDH in the model, the hazard for the event of death for subjects with both high IL-6 and IL-8 is 4.46 times (95% CI, 1.4, 14.2) that of the hazard for the subjects with low IL-6 and IL-8 (p = 0.01). Additionally, the hazard of death for subjects with higher total MDSCs is 3.3 times (95% CI, 1.2, 9.0) that for subjects with low total MDSCs. Conclusions: The durability of the nearly 7-year follow-up time for these advanced melanoma patients further strengthens the implied importance of IL-6 and IL-8 in the accumulation of MDSC’s in melanoma patients. These data provide clues for potential therapeutic targets in advanced melanoma patients. Citation Format: Richard P. Tobin, Kimberly R. Jordan, Dana Davis, Victoria M. Vorwald, Kasey Couts, Dexiang Gao, Derek E Smith, William A Robinson, Virginia Borges, Martin D McCarter. Tumor-produced IL-6 and IL-8 are associated with MDSC accumulation and correlate with long-term clinical outcomes in melanoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A117.

Introduction: Women diagnosed with breast cancer within 5 years postpartum have poor outcomes. In rodents, postpartum mammary gland involution promotes tumor progression and metastasis, implicating breast involution in the poor prognosis of breast cancers diagnosed in postpartum women. Rodent mammary gland involution is characterized by wound healing programs that include epithelial cell death, immune cell infiltrate, and fibrillar collagen deposition; all attributes associated with breast cancer progression. Thus, the gland remodeling of postpartum involution may provide a plausible explanation for how postpartum breast involution promotes breast cancer. Here, we characterize human breast tissue across pregnancy, lactation, and the postpartum time-period to determine if remodeling of the secretory competent breast to a quiescent state involves loss of secretory lobules, and whether involution is mediated by wound healing-like programs. Methods: Adjacent normal breast tissues from pre-menopausal women (n = 140), aged 20-45 years, were grouped by reproductive categories of never-been-pregnant (NBP), pregnant, lactating, and by time since last delivery, and evaluated histologically and by special stain for epithelial area, lobular subtype composition, apoptosis, immune cell infiltration, and collagen deposition, using computer assisted quantitative methods. Statistical comparisons between multiple categories were done using one way ANOVA. Results: Dramatic increases in breast epithelial area and lobule differentiation were observed, with 5-8 fold increases for pregnancy and 10-13 fold for lactation, over nulliparous controls. By 12 months postpartum, epithelial content and lobular differentiation were indistinguishable from nulliparous controls, consistent with complete regression of the lobular structures developed in preparation for lactation. Analyses of apoptosis, immune cell infiltration, and collagen deposition confirmed human postpartum breast involution is characterized by wound healing-like, tissue remodeling programs. Conclusion: Human postpartum breast involution is a dominant tissue-remodeling process that returns the gland to a morphological state largely indistinguishable from the never-been-pregnant gland. Further, involution occurs within a defined window of time. Our data implicate postpartum breast involution as window of risk for breast cancer progression and suggest a rational window for intervention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-05-01.

Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC. Cancer Res; 72(16); 4154–64. ©2012 AACR.