Pretreatment Tumor Biopsy Samples Research Articles

Dynamic cytokines signature predicts survival outcome from severe Immune-related hepatitis with PD-1/PD-L1 blockade in lung cancer

BackgroundImmune-related adverse events (irAEs), particularly immune-related hepatitis (IRH) is a potentially serious complication of immune checkpoint inhibitor (ICI) therapy. This retrospective cohort study investigated potential prognostic and predictive biomarkers for IRH. MethodThis study included 37 patients with advanced lung cancer who received ICIs and were divided into two groups: ≥Grade 3 (G3)-IRH group (n = 17) and without irAE (no-irAE) group (n = 20). Blood samples collected at three different time points and pre-treatment tumor biopsy samples were analyzed using multi-omics assays. ResultsThe IL-1B RNA expression was significantly increased (limma, fold = 1.94) in the ≥ G3-IRH group than the no-irAE group. Compared with no-irAE group, ≥G3-IRH group had higher monocyte and eosinophil infiltration and lower macrophage infiltration, particularly macrophage M2. Transcriptomics analyses of pre-treatment tumor samples revealed significant upregulation of various inflammation-related genes in the ≥ G3-IRH group (False discovery rate < 0.05). Moreover, various proinflammatory cytokines and chemokines were significantly lower in the plasma of the ≥ G3-IRH group than in the no-irAE group. Subgroup analyses of the ≥ G3-IRH group revealed that plasma IL-1A was significantly higher among those whose IRH resolved than those who had IRH-related death. Patients who died had a greater increase in immune score and Euclidean distance from the baseline to the seventh day of IRH onset, with a dramatic increase in Euclidean distance after immunosuppression, suggesting overstimulated immune status. ConclusionOur study demonstrated the association between IL-1B overexpression and IRH susceptibility. Immune score and Euclidean distance of inflammatory cytokines may provide predictive value on the survival outcome from ≥ G3 IRH.

Transcriptomic Analyses of Pretreatment Tumor Biopsy Samples, Response to Neoadjuvant Chemoradiotherapy, and Survival in Patients With Advanced Rectal Cancer

Neoadjuvant chemoradiotherapy (CRT) is the standard of care for advanced rectal cancer. Yet, estimating response to CRT remains an unmet clinical challenge. To investigate and better understand the transcriptomic factors associated with response to neoadjuvant CRT and survival in patients with advanced rectal cancer. A single-center, retrospective, case series was conducted at a comprehensive cancer center. Pretreatment biopsies from 298 patients with rectal cancer who were later treated with neoadjuvant CRT between April 1, 2004, and September 30, 2020, were analyzed by RNA sequencing. Data analysis was performed from July 1, 2021, to May 31, 2022. Chemoradiotherapy followed by total mesorectal excision or watch-and-wait management. Transcriptional subtyping was performed by consensus molecular subtype (CMS) classification. Immune cell infiltration was assessed using microenvironment cell populations-counter (MCP-counter) scores and single-sample gene set enrichment analysis (ssGSEA). Patients with surgical specimens of tumor regression grade 3 to 4 or whose care was managed by the watch-and-wait approach for more than 3 years were defined as good responders. Of the 298 patients in the study, 205 patients (68.8%) were men, and the median age was 61 (IQR, 52-67) years. Patients classified as CMS1 (6.4%) had a significantly higher rate of good response, albeit survival was comparable among the 4 subtypes. Good responders exhibited an enrichment in various immune-related pathways, as determined by ssGSEA. Microenvironment cell populations-counter scores for cytotoxic lymphocytes were significantly higher for good responders than nonresponders (median, 0.76 [IQR, 0.53-1.01] vs 0.58 [IQR, 0.43-0.83]; P < .001). Cytotoxic lymphocyte MCP-counter score was independently associated with response to CRT, as determined in the multivariable analysis (odds ratio, 3.81; 95% CI, 1.82-7.97; P < .001). Multivariable Cox proportional hazards regression analysis, including postoperative pathologic factors, revealed the cytotoxic lymphocyte MCP-counter score to be independently associated with recurrence-free survival (hazard ratio [HR], 0.38; 95% CI, 0.16-0.92; P = .03) and overall survival (HR, 0.16; 95% CI, 0.03-0.83; P = .03). In this case series of patients with rectal cancer treated with neoadjuvant CRT, the cytotoxic lymphocyte score in pretreatment biopsy samples, as computed by RNA sequencing, was associated with response to CRT and survival. This finding suggests that the cytotoxic lymphocyte score might serve as a biomarker in personalized multimodal rectal cancer treatment.

Pharmacodynamics and molecular correlates of response to glofitamab in relapsed/refractory non-Hodgkin lymphoma

AbstractGlofitamab, a novel CD20xCD3, T-cell–engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.

A Phase 1, Open-Label Study of MGD013, a Bispecific DART® Molecule Binding PD-1 and LAG-3 in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: MGD013 is an investigational, first-in-class, Fc-bearing bispecific tetravalent DART molecule designed to bind PD-1 and LAG-3 and sustain/restore the function of exhausted T cells (1). MGD013 demonstrates in vitro ligand blocking properties and improved T-cell responses beyond that observed with anti-PD-1 and anti-LAG-3 benchmark antibodies alone or in combination. PD-1 targeted therapy with nivolumab in patients (pts) with relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL) has yielded modest efficacy (2). LAG-3, highly expressed in DLBCL (3), has emerged as another therapeutic target of interest in this population with continued unmet need. Methods: This study characterizes the safety, tolerability, dose-limiting toxicities, maximum tolerated dose, PK/PD, and antitumor activity of MGD013 in pts with advanced solid and hematologic malignancies. R/R DLBCL pts are being treated at the recommended Phase 2 dose of 600 mg every two weeks in Cohort Expansion. Results: At data-cutoff, 17 DLBCL pts received MGD013 (2.5 median prior lines of therapy, 41.2% with prior CAR-T therapy). Treatment-related adverse events (TRAEs) occurred in 11/17 (64.7%) pts, with no same TRAE occurring in &amp;gt; 1 patient except pyrexia (n=3). One grade ≥ 3 TRAE occurred (pneumonia), and no events of tumor lysis syndrome were observed. Among 11 response-evaluable pts (i.e. received at least one on-treatment scan), 1 complete response (CR) and 3 partial responses (PRs) per the Lugano Classification were observed. Analyses of available pre-treatment tumor biopsy samples corresponding to responding pts demonstrated relatively high levels of LAG-3, PD-1, and PD-L1 by IHC. More comprehensive translational analyses were undertaken for the pt with CR, observed after a single MGD013 infusion in a 28-year-old male in relapse 6 months after CD19-directed CAR T-cell therapy. In contrast to a pre-CAR T biopsy specimen demonstrating no LAG-3 or PD-1 expression, IHC analysis of a lymph node specimen biopsied post-CAR T and prior to MGD013 treatment revealed high levels of both LAG-3 and PD-1 on both infiltrating T-cells and malignant B-cells. Consistent with CD19 CAR T resistance, no CD19 expression was evident. MHC class II and PD-L1 expression were observed, which when bound to LAG-3 and PD-1, respectively, form immune checkpoints that can decrease T cell function. Following MGD013 treatment, serum IFN-γ markedly increased to &amp;gt;140-fold above baseline. No significant changes were observed for IL-6 or IL-2. Expansion of circulating CD3+CD8+ and CD3+CD4-CD8- T-cell subsets and associated cytolytic markers (i.e., perforin, granzyme B) were observed following MGD013 treatment. The patient underwent allogeneic stem cell transplant (allo-SCT) and remains in remission 14 months post-MGD013 and 12 months post-allo-SCT. Further correlative biomarker analyses are underway in the ongoing clinical trial. Conclusion: MGD013, a novel molecule designed to coordinately block PD-1 and LAG-3, has preliminarily demonstrated an acceptable safety profile and encouraging early evidence of anti-tumor activity in R/R DLBCL pts with and without prior treatment with CAR T. Biomarker analyses confirmed expression of both PD-1 and LAG-3 axes in responding pts with evidence of pharmacodynamic responses consistent with the ability of MGD013 to enhance T-cell function.

NRF2 mRNA Expression Signature Associated with Regional Nodal Recurrence in Early-stage Non-small Cell Lung Cancer (NSCLC)

Lung stereotactic body radiation therapy (SBRT) achieves high local control in the treatment of early-stage non-small cell lung cancer (NSCLC). Nevertheless, up to 15-20% of patients may have regional nodal or distant recurrence after treatment. Molecular profiling may allow for personalized identification of patients at highest risk for recurrence. To this end, we conducted a transcriptomic analysis of lung SBRT samples with focus on the NRF2 pathway, which is involved in antioxidant regulation and has been associated with recurrence and survival in a variety of cancers. We studied the association of a NRF2 pathway mRNA expression profile with recurrence and survival. We identified patients with T1-3N0 NSCLC treated with SBRT at our institution. We isolated total RNA from formalin fixed paraffin embedded pretreatment tumor biopsy samples using an RNA Purification kit. Expression of mRNAs was analyzed using an assay. We used a published 20-mRNA risk score based on NRF2 pathway genes and dichotomized patients into high- and low-NRF2 groups using the median risk score value of the dataset. Kaplan-Meier analysis was used to study the association of the high NRF2 score with recurrence and survival. Cox proportional hazards multivariable analysis was used to account for histology, age, T-stage, and ECOG performance status. We identified 92 patients treated with lung SBRT with available mRNA data. There were no significant differences in age, sex, race, performance status, T-stage, histology, biologically effective dose, or central vs peripheral tumor location between high NRF2 (n = 46) and low NRF2 (n = 46) cohorts. There was significantly worse regional nodal recurrence in high- vs low-NRF2 patients (HR = 1.69; 95% CI 10.13 – 25.8; p = 0.035). There was no statistically significant difference in local recurrence (HR = 1.78; 95% CI 0.42 – 7.56; p = 0.43), distant recurrence (HR = 1.19; 95% CI 0.74 – 1.93; p = 0.48), or overall survival (HR = 1.67; 95% CI -.88 – 3.15; p = 0.12). On multivariable analysis, the association of high NRF2 score with regional nodal recurrence persisted (HR = 4.5; 95% CI 1.02 – 26.6; p = 0.046). There was no significant association of histology, age, T-stage, or ECOG performance status with regional nodal recurrence on multivariable analysis. We validated a published NRF2 mRNA expression signature, and found a high NRF2 signature was associated with regional nodal recurrence in patients with early-stage NSCLC treated with lung SBRT. This risk score may help to identify patients who would benefit from more aggressive mediastinal staging. These findings should be validated in an independent cohort.

Tumor microenvironment associated with increased pretreatment density of activated PD-1+ LAG-3+/− TIM-3− CD8+ T cells facilitates clinical response to axicabtagene ciloleucel (axi-cel) in patients (pts) with large B-cell lymphoma.

3022 Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after ≥ 2 prior therapies. In ZUMA-1 (NCT02348216), the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019). T cell-related biology (Immunosign 21; Immunoscore) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel (Rossi et al. AACR 2018. #LB-016; Rossi et al. AACR 2019. #CT153). This expanded analysis characterized the pretreatment TME immune contexture and examined associations between immune cell subsets and response. Methods: In ZUMA-1, pts received axi-cel at a target dose of 2.0 × 106 CAR T cells/kg. Archival pretreatment tumor biopsy samples were analyzed by multiplex immunohistochemistry (Brightplex). Two panels were developed and applied to assess T cell (CD3, CD8, FoxP3, PD-1, LAG-3, TIM-3) and myeloid cell (CD11b, CD14, CD15, LOX1, S100A9, CD68) subsets (n = 14 total). The association between T cell and myeloid cell subset density, prespecified immune scores (Immunosign 21; Immunoscore), and objective response was evaluated. T test values were based on Brightplex analysis. Results: Pretreatment tumor biopsy samples from 18 pts were analyzed (14 objective responders and 4 nonresponders). The pretreatment TME comprised all major myeloid and T cell subsets, with diverse distribution across samples analyzed. The median TME density of monocytes (CD11b+ CD15− CD14+; 1215 cells/mm2) and macrophages (CD68+; 530 cells/mm2) was greater than that of the total CD8+ T cell subset (312 cells/mm2). The pretreatment Immunosign 21 and Immunoscore scores associated positively with the density of all major T cell subsets and some myeloid subsets. The density of activated CD8+ T cells (PD-1+ LAG-3+/− TIM-3−) was most significantly associated with clinical response versus other T cell subsets. The density of nonactivated CD8+ T cells (PD-1− LAG-3− TIM-3−) and exhausted CD8+ T cells (PD-1+ LAG-3+ TIM-3+) were not significantly associated with response. Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusions: These results suggest that a TME associated with increased density of activated PD-1+ LAG-3+/− TIM-3− CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post–axi-cel.

Correlation of MYC gene signature variations with clinical response to fimepinostat in patients with aggressive B-cell lymphoma.

e15654 Background: Fimepinostat (F), a small molecule dual inhibitor of PI3K α, β, and δ isoforms and Class I and II histone deacetylases (HDACs), is currently under investigation for the treatment of aggressive hematological cancers. Preclinical studies demonstrated that F both decreases transcription of myc and MYC-targeted genes (via HDAC inhibition) and increases destruction of MYC protein (via PI3K inhibition). This study sought to identify differences in transcript-level signatures of gene networks in pre-treatment tumor samples associated with future clinical response to F. Methods: Among 105 patients (pts) with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) treated in phase 1/2 clinical trials of F (± rituximab), 33 pts had pre-treatment tumor biopsy samples available for molecular testing. All pts had measurable disease at baseline and had at least 1 post-baseline tumor assessment. To be included in the analysis set, pts must have been on study for ≥42 days. RNA libraries were created and sequenced on the Illumina HiSeq platform. Gene Set Enrichment Analysis (GSEA; Subramanian et al., 2005) was used to study changes in transcript expression patterns that correlate with responder (complete/partial response) or non-responder (progressive disease) pt populations. ~20,000 genes were rank-ordered by log2-fold change and tested against the MSigDB (Liberzon et al., 2011) collection of gene expression signatures. Results: &gt; 1500 Gene Sets with False Discovery Rate (FDR) &lt; = 0.05 and Normalized Enrichment Score (NES) &gt; 1.3 for genes upregulated in the F-responder population were identified. These Gene Sets represent transcriptional activity associated with well characterized biological pathways or gene expression signatures determined empirically. The top 20 signatures by NES were associated with general transcription functions (E2F, RNA polymerase), chromosomal structure (meiotic recombination, telomere maintenance) and specifically, MYC transcription targets (NES: 2.7; FDR q-val: 0). Additional signatures consistent with PI3K (GSK3b) and HDAC activity in the responder population were also identified, as would be expected for a dual inhibitor of PI3Ks and HDACs. Conclusions: GSEA analysis of genes upregulated in the F-treated responder DLBCL pts are consistent with preclinical data showing that F functions to suppress MYC in tumor cells. F is currently undergoing active clinical study in combination with venetoclax for the treatment of R/R DLBCL or high grade B-cell lymphoma (HGBL): NCT01742988.

Abstract C009: Clinical pharmacokinetic and pharmacodynamic analyses support biological effectivity of the PARP inhibitor olaparib as radiosensitizer at low doses

Abstract Introduction: PARP inhibitors are promising radiosensitizers. Currently identified recommended phase II doses of PARP inhibitors in combination with radiotherapy are 10-fold lower than the EMA and FDA approved dose as single agent. The purpose of this study is to investigate the biologically effective dose range of olaparib for its use as radiosensitizer by assessing pharmacokinetics (PK) and pharmacodynamics (PD). Both the inhibition of pre-treatment PAR levels and the inhibition of radiation induced PARylation therefore have to be determined. Methods: We included PK and PD samples of all NSCLC and breast cancer patients treated at olaparib doses up to 50mg bi-daily (BID) in two phase 1 trials combining radical radiotherapy with olaparib. Plasma samples were collected for PK analysis (ranging from steady state pre-dose olaparib until +12/24 hrs after intake) and peripheral blood mononuclear cells (PBMCs) for PD analysis (pre-treatment, +3 hrs and +12/24 hrs after olaparib intake). Breast cancer patients with the primary tumor in situ had biopsies taken pre-treatment and during olaparib treatment (+3 hrs after intake) before the first fraction of radiotherapy. Olaparib concentrations were determined by HPLC-MS/MS. PAR levels were determined by commercially available ELISA, following both the NCI protocol and REP assay (de Haan et al, 2017) that includes ex vivo irradiation of intact cells to activate PARylation by PARP. PAR levels determined by the REP assay were used to calculate PAR level inhibition during treatment. Results: PK/PD data from blood samples were available from 28 NSCLC patients and 7 breast cancer patients treated with radical radiotherapy and olaparib (17 at 25mg once daily (QD), 14 at 25mg BID and 4 at 50mg BID). Repeat biopsies were available from six out of seven breast cancer patients. The plasma olaparib concentrations increased with the dose levels and showed an expected wide intra-patient variation. In tumors the olaparib concentration varied between 178 and 1441 ng/g with a median tumor to plasma ratio of 0.47. PD analyses demonstrate a significant reduction in PAR levels in PBMCs during olaparib treatment (&amp;gt;95% at +3 hrs after olaparib intake for all dose levels, &amp;gt;90% at +12 hrs in the 25mg BID dose level and 66-99% at +24 hrs in the 25mg QD dose level; all p&amp;lt;0.0001). In pre-treatment samples, ex vivo radiation induced PAR levels by 66-fold (range 23 to 174). This radiation induced PARylation was abolished during olaparib treatment. Compared to corresponding pre-treatment PBMC samples, PAR levels in pre-treatment tumor biopsy samples were higher in all but one patient (median 7-fold higher, range 0.6-60), and increased by only 1.5-fold after ex vivo radiation. Also in tumors, olaparib abolished radiation induced PARylation. PAR levels were reduced by 89% (range 83-99.7%, p&amp;lt;0.0001). Conclusion: Olaparib doses as low as 25mg once and twice daily inhibit PARP activation by irradiation and reduce PAR levels &amp;gt;95% in PBMCs and &amp;gt;83% in tumors, thereby showing biological effectivity. The tumor olaparib concentrations determined in this study were all within a range that has been shown to radiosensitize in preclinical models. Together this supports further development of PARP inhibitors as radiosensitizer at low doses. Citation Format: Rosemarie de Haan, Dick Pluim, Manon Verwijs, Gabe Sonke, Michel van den Heuvel, Baukelien van Triest, Marcel Verheij, Conchita Vens. Clinical pharmacokinetic and pharmacodynamic analyses support biological effectivity of the PARP inhibitor olaparib as radiosensitizer at low doses [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C009. doi:10.1158/1535-7163.TARG-19-C009

Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors

ObjectivesLow-frequency epidermal growth factor receptor (EGFR) T790M mutation could be detected by ultrasensitive methods in EGFR tyrosine kinase inhibitor (TKI)-naïve non-small cell lung cancer (NSCLC). However, the impact of pretreatment T790M (preT790M) on the efficacy of EGFR-TKIs and on resistance remains unclear. Materials and methodsTwo independent cohorts consisting of advanced EGFR-mutated NSCLC patients treated with first-line EGFR-TKIs, a derivation cohort that started treatment between August 2013 and July 2016 (cohort A, n = 44) and a validation cohort between August 2016 and December 2017 (cohort B, n = 22), were examined in this study. Among these, 28 patients underwent re-biopsy at disease progression. DNAs from pretreatment tumor biopsy samples and re-biopsy samples were assessed to detect T790M by the Cobas EGFR Mutation Test v2 (Cobas) and for quantitating T790M by droplet digital polymerase chain reaction (ddPCR). ResultsDetection rates of preT790M were 40.9% (18/44) in cohort A and 45.5% (10/22) in cohort B by ddPCR, and none by Cobas. A cutoff value of 0.3% for dividing into high- vs. low-preT790M allele frequency was determined by receiver operating characteristic curve analysis in cohort A. Progression-free survival (PFS) was significantly shorter in the high- preT790M group (n = 12) than in the low-preT790M (n = 6) and negative (n = 26) groups (combined low-preT790M) (median: 6.9 vs. 13.8 months, P = 0.00073). These observations were validated in cohort B [median: 6.2 (n = 5) vs. 15.3 months (n = 17), P = 0.0029]. In 28 paired biopsies, Cobas detected post-progression T790M in 60% (3/5) of the high-preT790M, in 57% (4/7) of the low-preT790M, and in 56% (9/16) of the negative-preT790M groups. ConclusionEGFR-mutated NSCLC with high preT790M had significantly shorter PFS on EGFR-TKIs. However, preT790M abundance may not necessarily confer post-TKI T790M resistance.

A Pilot Study of the PD-1 Targeting Agent AMP-224 Used With Low-Dose Cyclophosphamide and Stereotactic Body Radiation Therapy in Patients With Metastatic Colorectal Cancer

BackgroundThe prognosis of metastatic colorectal cancer (mCRC) is poor. We assessed the feasibility, safety, and efficacy of the anti-programmed cell death 1 fusion protein AMP-224 in combination with low-dose cyclophosphamide and stereotactic body radiation (SBRT) treatment in patients with mCRC refractory to standard chemotherapy. Patients and MethodsFifteen patients were enrolled. Six received SBRT 8 Gy on day 0 (dose level 1), whereas 9 received 8 Gy on days −2 to day 0. All received cyclophosphamide 200 mg/m2 intravenously (I.V.) on day 0. On day 1, both groups received AMP-224 10 mg/kg I.V., repeated every 2 weeks for a total of 6 doses. Primary end points were feasibility and safety. ResultsTen (67%) patients completed 6 doses of AMP-224; 5 patients (33%) discontinued treatment because of disease progression. No dose-limiting toxicity was observed; 9 patients (60%) experienced treatment-related adverse events, all Grade 1 or 2. No objective response was noted; 3 patients (20%) had stable disease. Median progression-free survival and overall survival were 2.8 months (95% confidence interval [CI], 1.2-2.8 months) and 6.0 months (95% CI, 2.8-9.6 months), respectively. M2 macrophage polarization was present in the pretreatment tumor biopsy samples, but not post-treatment samples. ConclusionAMP-224 in combination with SBRT and low-dose cyclophosphamide was well tolerated, however, no significant clinical benefit was observed in patients with mCRC.

Abstract CT153: Pretreatment immunoscore and an inflamed tumor microenvironment (TME) are associated with efficacy in patients (Pts) with refractory large B cell lymphoma treated with axicabtagene ciloleucel (Axi-Cel) in ZUMA-1

Abstract Background: Axi-cel is an FDA-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after ≥ 2 prior therapies. In Phase 2 of ZUMA-1, the objective response rate was 83% (58% complete responses [CRs]) (Locke et al. Lancet Oncol. 2018). Previous work showed that pre-existing expression of immune-related genes (Immunosign® 21 with NanoString®) may be associated with response to axi-cel (Rossi et al. AACR 2018. #LB-016). This expanded post-hoc analysis includes novel analysis associating T cell density with efficacy. Methods: Pts received a target dose of 2.0 × 106 CAR T cells/kg in ZUMA-1 (NCT02348216). Pretreatment (before conditioning) tumor tissues were analyzed by immunohistochemistry for density of T cell subsets (CD3+, CD8+). A prespecified bioinformatics algorithm was used to generate a numerical index (Immunoscore®; Galon et al. J Pathol. 2014) and analyses cutoffs. Higher Immunoscore® represents increased intra-tumor T cell infiltration. TME was evaluated for Immunosign® 21 and for additional myeloid- and dendritic cell-related genes. The association between Immunoscore®, CD3+, or CD8+ T cell density and CR was evaluated, as was the association of Immunoscore® with Immunosign® 21. Results: Pretreatment tumor biopsy samples from 25 pts were analyzed (data cutoff Aug 11, 2017). Of pts with high Immunoscore®, 77% (7/9) had CR and 11% (1/9) were nonresponders. Of those with low Immunoscore®, 38% (6/16) were nonresponders and 50% (8/16) had CR. The pts in this category who achieved CR had a lower median pretreatment tumor burden by sum of product diameters. Overall, higher pretreatment Immunoscore® was associated with achievement of CR (P = .048). Pretreatment intra-tumor densities of CD3+ and CD8+ T cells separately quantified, were also positively associated with achievement of CR (P = .02 and .036 by t test). In addition, higher pretreatment Immunosign® 21 was associated with clinical efficacy. Values for Immunosign® 21 and Immunoscore® demonstrated 82% concordance (95% CI, 65-93; r2 = 0.451). Finally, TME gene expression analysis indicated a pan-inflammatory profile, including myeloid- and dendritic cell-related gene expression, in pts who achieved CR with higher Immunosign® 21 and Immunoscore®. Conclusions: These results link pre-existing T cell involved lymphoma TME with clinical outcome in patients post axi-cel. They also suggest that factors intrinsic to tumor biology may influence CAR T cell efficacy through the immune microenvironment. In addition, this treatment modality may overcome an unfavorable TME in a subset of patients with low disease burden. Finally, these results support CAR T cell treatment optimizations designed to overcome an immune detrimental TME. John Rossi and Jérôme Galon contributed equally. Citation Format: John M. Rossi, Jérôme Galon, Edmund Chang, Regis Perbost, Nathalie Scholler, Sarah Turcan, Corinne Danan, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Francesca Milletti, William Y. Go, Adrian Bot. Pretreatment immunoscore and an inflamed tumor microenvironment (TME) are associated with efficacy in patients (Pts) with refractory large B cell lymphoma treated with axicabtagene ciloleucel (Axi-Cel) in ZUMA-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT153.

Clonal expansion of tumor infiltrating lymphocytes (TILs) in the peripheral blood of metastatic melanoma patients is significantly associated with response to CTLA4 blockade-based immunotherapy.

2541 Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.

Abstract 1991: Vulnerability of platinum-resistant ovarian cancer to FAK inhibition

Abstract Platinum (CP)-resistant ovarian cancer (OC) has few effective treatment options. Adaptive chemotherapy resistance occurs in part through cancer stem cell (CSC) generation. Wnt/beta-catenin signaling is a driver of CSC survival via induction of gene expression, including aldehyde dehydrogenase (ALDH) enzymes. The gene for focal adhesion kinase (FAK) is commonly amplified in advance-stage OC and this is associated with decreased patient survival. How FAK is activated in OC and whether this is connected to CSC survival is unknown. Here, we find that FAK Y397 phosphorylation (a marker of FAK activation) is increased in non-necrotic Pax8-positive OC tumor tissue after neo-adjuvant chemotherapy compared to paired pre-treatment tumor biopsy samples. FAK activation occurs after CP plus paclitaxel treatment of xenograft tumors, within 60 min of CP treatment of OC cells, and FAK Y397 levels are constitutively-elevated in OC cells pre-adapted to exhibit elevated CP resistance. Pharmacological FAK inhibition (VS-4718, 0.1 µM) selectively prevented CP-resistant tumorsphere formation in vitro and combined with CP to promote cell apoptosis. Combinatorial VS-4718, CP, and paclitaxel chemotherapy exhibited additive inhibitory effects in preventing CP-resistant tumor growth in mice. VS-4718 monotherapy of tumor-bearing mice reduced tumor-associated ALDH activity and resulted in an 85-fold reduction in secondary tumors formed in limiting dilution assays. CRISPR-mediated FAK knockout in OVCAR3 cells combined with stable re-expression of FAK wildtype or a kinase-inactive FAK (K454R) mutant revealed that intrinsic FAK activity was essential for beta-catenin activation, ALDH-1A1 expression, and OVCAR3 tumorsphere growth. As activated constructs of beta-catenin but not YAP1 rescued FAK KO OVCAR3 phenotypes, these studies provide important insights into a FAK signaling linkage to beta-catenin in promoting CSC survival and adaptive resistance to CP chemotherapy. These studies provide the foundational support for a Phase I-II clinical trial for treatment of recurrent platinum-resistant ovarian cancer (NCT03287271) termed ROCKIF: Re-sensitization of carboplatin-resistant Ovarian Cancer by Kinase Inhibition of FAK. As FAK is activated in tumor cells surviving carboplatin-paclitaxel chemotherapy, co-targeting of this FAK/beta-catenin adaptive resistance pathway may expose a vulnerability of CP-resistant tumors. Citation Format: Carlos J. Diaz Osterman, Lisa M. Bean, Florian J. Sulzmaier, Kristin N. Taylor, Shulin A. Jiang, Isabelle Tancioni, Kristen Anderson, Christine Jean, Xiao Lei Chen, Elizabeth G. Kleinschmidt, Vihren N. Kolev, David T. Weaver, Jonathan A. Pachter, Denise C. Connolly, Alfredo Molinolo, David D. Schlaepfer. Vulnerability of platinum-resistant ovarian cancer to FAK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1991.

Oncogene mutation profile predicts tumor regression and survival in locally advanced rectal cancer patients treated with preoperative chemoradiotherapy and radical surgery.

Tumor response to preoperative chemoradiotherapy and postoperative survival differs among patients with locally advanced rectal cancer. The objective was to find correlations of mutated oncogenes and clinical outcomes in locally advanced rectal cancer. A total of 70 patients with preoperative preoperative chemoradiotherapy followed by radical surgery at a single cancer center between 2006 and 2012 were enrolled. Pretreatment tumor biopsy samples were assayed for 238 mutation hotspots harboring 19 oncogenes by time-of-flight mass spectrometry and OncoCarta Array. Oncogene mutations were found in 48.6% of patients (34/70). KRAS was the most frequent driver mutation, found in 35.7% of patients (25/70), followed by PIK3CA (14.3%), NRAS (5.7%), FLT3 (2.9%), and BRAF (1.4%). Multiple gene mutations were observed in eight patients (11.4%). Tumors with KRAS mutations responded poorly to preoperative chemoradiotherapy (p = 0.044). Patients with oncogene mutations had worse 3-year disease-free survival than those without mutations (67.2% vs 94.2%, p = 0.010). Patients with KRAS or RAS mutations had lower 3-year disease-free survival (68% vs 88.3%, p = 0.016; 65.5% vs 92.3%, p = 0.004, respectively) and 3-year overall survival (88% vs 95.4%, p = 0.020; 89.7% vs 94.9%, p = 0.036, respectively) than those without KRAS or RAS mutations. Oncogene mutation status affected tumor response to treatment and long-term survival in locally advanced rectal cancer.

Predictive value of ex vivo biodynamic imaging in determining response to chemotherapy in dogs with spontaneous non-Hodgkin's lymphomas: a preliminary study.

Biodynamic imaging (BDI) is a novel phenotypic cancer profiling technology which optically characterizes changes in subcellular motion within living tumor tissue samples in response to ex vivo treatment with cancer chemotherapy drugs. The purpose of this preliminary study was to assess the ability of ex vivo BDI to predict in vivo clinical response to chemotherapy in ten dogs with naturally-occurring non-Hodgkin's lymphomas. Pre-treatment tumor biopsy samples were obtained from all dogs and treated ex vivo with doxorubicin (10 μM). BDI measured six dynamic biomarkers of subcellular motion from all biopsy samples at baseline and at regular intervals for 9 h following drug application. All dogs subsequently received doxorubicin to treat their lymphomas. Best overall response to and progression-free survival time following chemotherapy were recorded for all dogs. Receiver operating characteristic (ROC) curves were used to determine accuracy and identify possible cut-off values for the BDI-measured biomarkers which could accurately predict those dogs' cancers that would and would not respond to doxorubicin chemotherapy. One biomarker (designated 'MEM') showed 100% discriminative capability for predicting clinical response to doxorubicin (area under the ROC curve = 1.00, 95% CI 0.692-1.000), while other biomarkers also showed promising predictive capability. These preliminary findings suggest that ex vivo BDI can accurately predict treatment outcome following doxorubicin chemotherapy in a spontaneous animal cancer model, and is worthy of further investigation as a technology for personalized cancer medicine.

22 Prognostic impact of tumor associated macrophages and tumor infiltrating lymphocytes in head and neck cancer treated with primary chemoradiotherapy

We investigated the prognostic role of tumor infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT). The expression of CD3+, CD4+, CD8+, FOXP3+, CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n = 106 pre-treatment tumor biopsy samples and was correlated with clinicopathological characteristics, including HPV-p16-status, T-stage, N-stage, grading, tumor localization, age and sex as well as local failure free- (LFFS), distant metastases free- (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n = 12 available early local recurrence samples and compared to their matched primary tumors. All local recurrences occurred in the high radiation dose region (⩾70 Gy). With a median follow-up of 40 months, OS at 2 years was 60.5%. Low CD3 and CD8 as well as high CD68 and CD163 expression in primary tumors were associated with decreased OS, PFS, LPFS and DMFS. After multivariate analysis all of the above markers remained significant or marginally significant for these endpoints (such as for OS: p = 0.009, p = 0.011, p = 0.002 and p = 0.052 for CD3, CD8, CD68 and CD163 respectively). On the contrary CD4, FOXP3 and CD11b did not have any prognostic impact. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (p = 0.0097) but decreased CD31-positive vessel density (p = 0.0004) compared to their matched primary samples. Altogether, baseline numbers of M2-TAMs and cytotoxic lymphocytes seem to be important prognostic parameters in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of the immune system in mediating response to CRT in patients with HNSCC and need further evaluation in a prospective trial.

Selective activity over a constitutively active RET-variant of the oral multikinase inhibitor dovitinib: Results of the CNIO-BR002 phase I-trial

Background: Given our preclinical data showing synergy between dovitinib and paclitaxel in preclinical models we conducted this phase I trial aiming to define the recommended phase II-dose (RP2D) on the basis of toxicity and pharmacodynamic criteria while searching for genetic variants that could sensitize patients to the regimen under study. Patients and methodsA 3+3 escalation schedule was adopted. Seriated FGF23 and dovitinib and paclitaxel pharmacokinetic profiles were determined along a single-agent dovitinib “priming-phase” followed by a dovitinib + paclitaxel combination phase. RECIST 1.1 criteria and NCI CTCAE V.4.0 were used. In fresh pre-treatment tumor biopsy samples, FGFR1, 2 and 3 amplifications were revealed by FISH probes; 32 missense variants were genotyped in tumors and peripheral blood mononuclear cells with Taqman genotyping assays (FGFR1-3 and RET). Constructs encoding for wild-type and variant genes associated with clinical benefit were transfected into HEK-293 cells for preclinical experiments checking constitutive activation and dovitinib sensitivity of the variants. Resultstwelve patients were recruited in three dose-levels. At level 1B (200 mg dovitinib 5-days-on/2-days-off plus 60 mg/m 2-week of paclitaxel) more than 50% FGF23 upregulation was observed and no dose-limiting-toxicities (DLTs) occurred. The most frequent toxicities were asthenia, neutropenia, nausea/vomiting and transaminitis. Two patients with progressive disease prior to trial inclusion achieved prolonged disease stabilization. Both had the germline variant G2071A in the RET gene, which led to constitutive activation of the protein product and Y-905 phosphorylation, both in transfectants and in patients with the alteration. This variant was sensitive to dovitinib; in addition both patients experienced progression upon medication withdrawal. ConclusionsLevel 1B was the RP2D as it provided adequate pharmacodynamic exposure to dovitinib. The G2071A germline variant act as a genetic modifier that renders different tumors sensitive to dovitinib.

HER2/neu expression in locally advanced rectal cancer cUICC II/III and its correlation with long-term prognosis.

493 Background: With the implementation of multimodal treatment strategies including neoadjuvant radiochemotherapy (RCT), the prognosis of locally advanced rectal cancer has been improved over the last two decades. Most actual clinical trials aim to further develop therapy by intensification of agents administered. We examined Her2/neu in rectal cancer patients to evaluate its expression as a potential drug target as well as its capacity as predictive and prognostic biomarker. Methods: Two-hundred-seventy-seven patients (205 male, 72 female; median age 63.5 years) with locally advanced rectal cancer (cUICC-II/III) were included in this study. Preoperative RCT (50.4 Gy and concomitant either 5-FU or 5-FU and oxaliplatin) was administered in 236 patients followed by surgical resection with total mesorectal excision (TME). Another 41 patients received postoperative RCT. All patients were treated within phase-II/-III-trials of the German Rectal Cancer Study Group or analogous to these standardized protocols. Her2-/neu expression from pre-treatment biopsies and corresponding resection specimens were assessed by immunohistochemical staining and SISH-analysis. Results: A positive Her-2/neu expression was found in 12.3% of all pre-treatment tumor biopsy samples and in 28.7% of the resection specimens. The correlation of the pre-treatment Her-2/neu status did not show a significant correlation with the grade of tumor regression. However, patients with a higher Her-2/neu protein expression as measured in the resection specimen showed a significant survival benefit (p=0.0277) compared with patients having low intratumoral Her-2/neu expression during the follow-up (median 43 months). The 10-year survival rate was 73.2% (Her-2/neu positive) versus 60.1% (Her-2/neu negative). Conclusions: The proportion of patients with a positive Her-2/neu status is relevant in rectal cancer. Her-2/neu might potentially serve as a promising drug target in rectal cancer and should be further assessed within prospective clinical trials. Furthermore the Her-2/neu status seems to be a valuable prognostic marker within the multimodal treatment of advanced rectal cancer.