<p>Differences in infiltrating immune cell populations as determined by multiplex immunofluorescence among subjects with and without clinical benefit</p>
2560 Background: NCI-MATCH was the largest precision medicine initiative conducted to date. Arm Z1D was a study of nivolumab in patients (pts) whose tumors were mismatch repair deficient (MMRd) by immunohistochemistry (IHC). Plasma was available for analysis of circulating tumor DNA (ctDNA), a component of cell-free DNA (cfDNA), in 39 of 47 pts enrolled to treatment. Large ctDNA gene panels that interrogate microsatellite instability (MSI), tumor mutational burden (TMB) and other complex biomarkers may provide an important complement to tumor-based assessments. Methods: 10 – 30 ng of cfDNA extracted from Streck tubes was tested with a modified version of the 523-gene TruSightä Oncology 500 ctDNA RUO assay (Illumina, San Diego, CA). Blood MSI (bMSI) status was calculated by interrogating ~2400 microsatellite sites and applying an experimentally validated cut-off. Blood TMB (bTMB) was calculated as total number of nonsynonymous and synonymous SNVs and Indels identified per Mb. Tumor sequencing was performed with the 143-gene Oncomine Comprehensive Assay v2. Overlap of tumor and plasma gene panels was evaluated for concordance. MMRd was evaluated by MLH1 and MSH2 IHC expression. MSI PCR testing was performed on 7 microsatellite sites. Clinical response was assessed by RECIST v1.1 criteria. Results: ORR was 31% (12/39) for 39 MMRd patients with plasma samples. Among these, 34 pts (87%) were classified as MSI-high (MSI-H) by ctDNA at baseline. The 5 MSS cases included 1 CR, 2 PRs and 2 unevaluable for response. The median bTMB was 56.4 mut/Mb (range: 1.65 – 435 mut/Mb). All pts had at least one oncogenic/likely oncogenic alteration identified in plasma. Of 136 of these mutations identified in tumor, 107 (78.6%) were detected in plasma. The most frequently mutated genes in plasma included TP53 (n = 26 pts), RNF43 (n = 16 pts, all G659Vfs*41) and PIK3CA (n = 16 pts). MLH1 (n = 5) and MSH2 (n = 5) mutations were also identified. RNF43 G659Vfs*41, which was not interrogated by the targeted tumor panel, is known to be strongly associated with MSI-H status. In this heavily pre-treated cohort (median prior therapies = 3), variants in genes commonly associated with clonal hematopoiesis of indeterminant potential were identified in all pts. Changes in cfDNA tumor fraction tracked with clinical response in 4 of 4 pts where on-treatment blood was collected for TSO 500 assessment. Conclusions: 78.6% of oncogenic/likely oncogenic alterations found in tumor where also identified in plasma. 87% of MMRd pts by tumor IHC were defined as MSI-H by baseline ctDNA, and median bTMB was 56.4 mut/Mb. These data suggest blood-based MSI and TMB should be further explored as biomarkers for the non-invasive identification of patients who may be candidates for immune checkpoint blockade, particularly when tissue is limiting.
<p>Differences in infiltrating immune cell populations as determined by multiplex immunofluorescence among subjects with and without clinical benefit</p>
<div>AbstractPurpose:<p>Mismatch repair–deficient (dMMR) tumors have demonstrated favorable responses to immune checkpoint inhibition targeting PD-1. However, more in-depth identification of predictors of response could further refine patient selection for immunotherapy treatment.</p>Patients and Methods:<p>We undertook integrated evaluation performed on samples collected from 28 of 42 patients enrolled on the NCI–Molecular Analysis for Therapy Choice arm Z1D trial that evaluated PD-1 inhibition treatment with nivolumab in patients with noncolorectal dMMR tumors. Genomic analyses were performed using next-generation sequencing (NGS), whole-exome sequencing, and RNA sequencing and supplemented by multiplex immunofluorescence performed on tissue samples.</p>Results:<p>In this dMMR population, more extensive alterations of microsatellites as assessed by measures of NGS were associated with clinical benefit and tumor mutational burden. RNA sequencing further revealed associations between clinical benefit and immune infiltration index. Gene sets enriched in patients with clinical benefit included IFN signaling, antigen processing, and PI3K–AKT–mTOR signaling, whereas hedgehog signaling was found to be enriched in subjects lacking clinical benefit.</p>Conclusions:<p>These genomic data highlight the importance of immune infiltration and antigen presentation in dMMR tumors that respond to immune checkpoint blockade. In addition, they suggest that, even within a dMMR population, NGS-based measures of microsatellite instability could serve as biomarkers of immunotherapy response.</p></div>
Supplementary Appendix 1 from High Expression of Lymphocyte-Associated Genes in Node-Negative HER2+ Breast Cancers Correlates with Lower Recurrence Rates
3506 Background: The NCI-MATCH (EAY131) is a platform trial that enrolls patients (pts) with solid tumors, lymphomas, or multiple myeloma to targeted therapies based on matching genomic alterations of interest (NCT02465060). Arm Z1F evaluated copanlisib, a highly selective, pan-Class 1 PI3K inhibitor with predominant activity against both the δ and α isoforms in pts with PIK3CA mutations. Methods: Pts received copanlisib (60 mg IV) on days 1, 8, and 15 in 28-day cycles until progression/toxicity. Tumor assessment was every 2 cycles. The primary endpoint was objective response rate (ORR); secondary endpoints were PFS, 6-month PFS, and predictive biomarkers. Pts with KRAS mutations, HER2+ve breast cancers, lymphomas were excluded. Results: 35 pts were enrolled (from 8/2/18 to 12/27/18), of which, 28 pts were available for analysis (7 patients, not eligible or did not start therapy). Multiple histologies were enrolled with gynecologic (n = 7), gastrointestinal (n = 6), and genitourinary (n = 5) the most common tumors. Median age 61 (range 42-78). 75% of pts had ≥ 3 lines of prior therapy. 54% of PIK3CA mutations were located in the helical domain, 32% in kinase domain and 14% in other domains. Twenty-six pts had co-occurring gene alterations (median 3; range 1-9), with 9 patients having 4 or more gene alterations. The ORR was 11% (3/28, 90% CI: 3%-25%). Partial responses were seen in uterine cancer, clear cell carcinoma of anterior abdominal wall, and liposarcoma. 6 pts had > 6 months of stable disease and clinical benefit rate was 32% (9/28). Two pts are still on treatment. The most common reason for protocol discontinuation was disease progression (n = 18, 69%). Thirty pts were included for toxicity analysis. Ten pts (33%) had grade 1 or 2 toxicities, 16 pts (53%) had grade 3 toxicities, and one patient (3%) had grade 4 toxicity (CTCAE v5.0). Most common toxicities include hyperglycemia (n = 19), fatigue (n = 11), hypertension (n = 10), diarrhea (n = 10), and nausea (n = 9). Total of 5 deaths were reported, none related to treatment. Conclusions: Copanlisib showed meaningful clinical activity across various tumors with PIK3CA mutation in the late-line refractory setting. Further study either alone or in combinations in select tumors is warranted. G3/4 toxicities observed were consistent with reported toxicities for PI3K pathway inhibition. Clinical trial information: NCT02465060 .
5027 HER2 amplification is found in 20-25% of breast cancers and is associated with a worse prognosis. Trastuzumab (Herceptin®) is a valuable therapy but patients do not always benefit and mechanisms of resistance are unclear. We have generated eight Herceptin resistant HER2 amplified breast cancer cell lines by serial passage through increasing Herceptin (10-100 μg/mL) over 3-6 months. All resistant cell lines are maintained in 100μg/mL Herceptin. Characterization of changes in growth, morphology and signaling pathways are presented. Methods: HER-2 amplified breast cancer cell lines (BT474, MDA-361, T47D, MD453, UACC812, UACC893 and SKBr3) were used. Cell proliferation for parent and Herceptin-resistant lines was measured by WST-1 cell proliferation assay (Promega). Akt/MAPK signaling was evaluated using commercially available antibodies and Proteome Profiler Array (Human Phospho-MAPK Array Kit, BD Biosciences, USA). Morphology was assessed by confocal microscopy. Results: HER2 amplified cell lines were divided into primary Herceptin-sensitive (SKBr3 and BT474) and de novo resistant lines (T47D, UACC812, UACC893, MD361, MD453) based on IC50. Proliferation studies demonsrate that cells that acquire Herceptin resistance grow 20-30% faster than parental counterparts. Interestingly, BT474, MD361 and T47D cells that were passaged in 100 ug/ml Herceptin have 10-25% increase in proliferation with increased Herceptin in media (150 to 750μg/mL). In addition, resistant cells exhibit distinct cell morphology and growth characteristics. Cell morphology changes include increased cell size and pseudopodia formation. BT474 resistant cells now grow as monolayer compared to clumped growth pattern in parental lines. Preliminary analysis using western blot and Proteome Profiler Array suggests increased pAKt and decrease in pErk1/Erk2 level in Herceptin resistant lines. Completed signaling pathway analysis will be presented. Conclusions: HER2 amplified Herceptin resistant cell lines develop a more aggressive phenotype whereby Herceptin appears to behave as an agonist.
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