Pretreatment Specimens Research Articles

Proteogenomic Characterization of Highly Enriched Viable Leukemic Blasts in Acute Myeloid Leukemia: A SWOG Report

Acknowledgements: This work was funded by the following NIH/NCI/NCTN grant awards: RO1CA190661, R01CA160872, U10CA180888, U10CA180819, U24CA196175. The authors wish to gratefully acknowledge the importantcontributions of the late Dr. Stephen H. Petersdorf to SWOG and to the study S0106. The authors would like to acknowledge that a portion of the specimens were obtained from the Fred Hutch/University of Washington Hematopoietic Disorder Repository.AML is a molecularly heterogenous disease that harbors multiple genomic, epigenomic, and transcriptomic abnormalities. Despite the use of newer therapeutic agents and identification of multiple prognostic markers, most patients with AML still relapse or succumb to their disease. Understanding biological factors that determine relapse is of major clinical interest in AML. Proteins are responsible for much of the functional biology of the cells. However, few studies have examined the global proteome in AML, and, to our knowledge, studies have not previously examined the proteome in highly enriched undifferentiated AML blasts. Therefore, we have developed an integrated approach utilizing mass spectrometry-based proteomics and leveraging next generation RNA sequencing (RNAseq) to identify novel protein biomarkers associated with clinical outcome in a homogeneous population of undifferentiated viable leukemic blasts (uVLBs) from AML patients. Cryopreserved specimens from previously untreated de novo AML patients (n=27) were obtained from the SWOG Leukemia Repository. Patients were treated with dose intensive induction and consolidation as part of SWOG-9031, SWOG-9333, S0106, and S0112 studies. uVLBs were isolated from specimens using fluorescence-activated cell sorting (FACS). Targeted genome sequencing examined for genomic mutations in uVLBs, while global RNA sequencing (RNAseq) and tandem mass tag labelling followed by liquid chromatograph with tandem mass spectrometry (TMT-LC-MS/MS) were used to quantify transcript and protein expression, respectively. Analyses identified 6761 unique proteins, with 238 and 460 proteins significantly associated with complete response (CR) and overall survival (OS), respectively. There was modest overlap between the prognostically significant transcript and protein biomarkers (Figure 1). We also were able to identify and quantify aberrant proteins arising from genomic mutations . Examples of the “neoproteins” detected included proteins harboring NPM1 mutations (Figure 2a) and those harboring frameshift insertions/deletions in the terminal region of RUNX1 (Figure 2b).For validation of prognostic associations, we examined the proteome in an independent population of de novo AML patients (N=27) treated at the Fred Hutch and University of Washington. As with the SWOG patients, pre-treatment specimens were obtained from patients receiving intensive induction and consolidation with curative intent; however, OS data was not available for many of these patients. As such, the validation studies focused on CR. Patients were randomized by CR response into 9 pools (CR=6 and no CR=3 pools), and specimens were sorted for uVLBs. TMT-based LC-MS/MS quantified protein expression across pools, and analyses examined the prognostic significance of the 238 proteins from the SWOG analyses associated with CR. Thirteen of the most promising candidates were significantly associated with CR prognosis (Table 1), many of which are associated with cancer biology.Together, these studies show the feasibility and biological importance of examining the proteome in uVLBs. Studies examining for biomarkers in the proteome may be a powerful tool to uncover novel prognostic biomarkers that would otherwise not be identified by examining the genome or transcriptome. Furthermore, the multi-omics approach can be used to confirm the translation of potential neoantigens into actionable protein targets, which may lead to more cost-effective mechanisms for the development of innovative adoptive immunotherapies. [Display omitted] DisclosuresOthus: Biosight: Consultancy; Merck: Consultancy; Celgene: Other: Data safety monitoring board; Glycomimetics: Other: Data safety monitoring board; Daiichi Sankyo: Consultancy. Moseley: BioSight Ltd: Consultancy. Erba: AbbVie Inc; Agios Pharmaceuticals Inc; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Incyte Corporation; Jazz Pharmaceuticals Inc; Novartis: Speakers Bureau; AbbVie Inc; Agios Pharmaceuticals Inc; Astellas; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Daiichi Sankyo Inc; Genentech, a member of the Roche Group; GlycoMimetics Inc; Incyte Corporation; Jazz Pharmaceuticals Inc; Kura Oncology; Nov: Other: Advisory Committee; AbbVie Inc: Other: Independent review committee; AbbVie Inc; Agios Pharmaceuticals Inc; ALX Oncology; Amgen Inc; Daiichi Sankyo Inc; FORMA Therapeutics; Forty Seven Inc; Gilead Sciences Inc; GlycoMimetics Inc; ImmunoGen Inc; Jazz Pharmaceuticals Inc; MacroGenics Inc; Novartis; PTC Therapeutics: Research Funding. Radich: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

TCF-1+ PD-1+ CD8+T cells are associated with the response to PD-1 blockade in non-small cell lung cancer patients.

To determine whether TCF-1+PD-1+CD8+T cells are associated with the response to PD-1 blockade in non-small cell lung cancer (NSCLC) patients. We investigated the expression of TCF-1+PD-1+CD8+T cells and elucidated their predictive role in NSCLC patients. Pretreatment specimens from 20 advanced NSCLC patients who underwent PD-1 immunotherapy or combined with chemotherapy were analyzed. The frequencies of TCF-1+ cells in PD-1+CD8+T cells were determined in these biospecimens using multi-label immunofluorescence staining and multi-spectral acquisition technology. The clinical roles of TCF-1+PD-1+CD8+T cells were assessed via analyzing our cases and human NSCLC data collected from public databases. A high frequency of TCF-1+PD-1+CD8+T cells was identified in responders compared with non-responders (p = 0.0024), and the patients with high expression of this cell subset had durable clinical benefit of anti-PD-1 therapy. There were no significant association between the expression of TCF-1+PD-1+CD8+T cells and patients' age, smoking history, pathologic type, and genetic status. In univariate analysis by the Cox hazard model, high frequency of TCF-1+ PD-1+ CD8+T cells was significantly correlated with patients' benefit of PD-1 blockade (p = 0.024). Our study indicated that TCF-1+PD-1+CD8+T cells are associated with the response to PD-1 blockade, and may be a predictor of anti-PD-1 therapy.

Pathway signatures derived from on-treatment tumor specimens predict response to anti-PD1 blockade in metastatic melanoma

Both genomic and transcriptomic signatures have been developed to predict responses of metastatic melanoma to immune checkpoint blockade (ICB) therapies; however, most of these signatures are derived from pre-treatment biopsy samples. Here, we build pathway-based super signatures in pre-treatment (PASS-PRE) and on-treatment (PASS-ON) tumor specimens based on transcriptomic data and clinical information from a large dataset of metastatic melanoma treated with anti-PD1-based therapies as the training set. Both PASS-PRE and PASS-ON signatures are validated in three independent datasets of metastatic melanoma as the validation set, achieving area under the curve (AUC) values of 0.45–0.69 and 0.85–0.89, respectively. We also combine all test samples and obtain AUCs of 0.65 and 0.88 for PASS-PRE and PASS-ON signatures, respectively. When compared with existing signatures, the PASS-ON signature demonstrates more robust and superior predictive performance across all four datasets. Overall, we provide a framework for building pathway-based signatures that is highly and accurately predictive of response to anti-PD1 therapies based on on-treatment tumor specimens. This work would provide a rationale for applying pathway-based signatures derived from on-treatment tumor samples to predict patients’ therapeutic response to ICB therapies.

Parallel Genomic Alterations of Antigen and Payload Targets Mediate Polyclonal Acquired Clinical Resistance to Sacituzumab Govitecan in Triple-Negative Breast Cancer.

Sacituzumab govitecan (SG), the first antibody-drug conjugate (ADC) approved for triple-negative breast cancer, incorporates the anti-TROP2 antibody hRS7 conjugated to a topoisomerase-1 (TOP1) inhibitor payload. We sought to identify mechanisms of SG resistance through RNA and whole-exome sequencing of pretreatment and postprogression specimens. One patient exhibiting de novo progression lacked TROP2 expression, in contrast to robust TROP2 expression and focal genomic amplification of TACSTD2/TROP2 observed in a patient with a deep, prolonged response to SG. Analysis of acquired genomic resistance in this case revealed one phylogenetic branch harboring a canonical TOP1 E418K resistance mutation and subsequent frameshift TOP1 mutation, whereas a distinct branch exhibited a novel TACSTD2/TROP2 T256R missense mutation. Reconstitution experiments demonstrated that TROP2T256R confers SG resistance via defective plasma membrane localization and reduced cell-surface binding by hRS7. These findings highlight parallel genomic alterations in both antibody and payload targets associated with resistance to SG. SIGNIFICANCE: These findings underscore TROP2 as a response determinant and reveal acquired SG resistance mechanisms involving the direct antibody and drug payload targets in distinct metastatic subclones of an individual patient. This study highlights the specificity of SG and illustrates how such mechanisms will inform therapeutic strategies to overcome ADC resistance.This article is highlighted in the In This Issue feature, p. 2355.

Predictive and Prognostic Biomarker Identification in a Large Cohort of Androgen Receptor-Positive Salivary Duct Carcinoma Patients Scheduled for Combined Androgen Blockade.

Simple SummaryAndrogen receptor signaling seems pivotal in aggressive salivary duct carcinoma. However, androgen deprivation therapy (ADT) frequently fails, emphasizing the need for biomarkers to predict treatment response. Here, the activities of several possible tumor-driving pathways were quantified and related to clinical outcome in a large cohort of SDC patients. Our results indicated that AR pathway activity and SRD5A1 expression levels can be used to predict response to ADT, which could prevent overtreatment in clinical practice.Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). However, CAB frequently fails, resulting in a worse prognosis. Therefore, biomarkers that can predict treatment failure are urgently needed. mRNA from 76 R/M androgen receptor (AR)-positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, MAPK, TGFβ, estrogen receptor (ER), Hedgehog (HH), and PI3K signaling pathway activity scores (PAS) were determined based on the expression levels of target genes. Additionally, 5-alpha reductase type 1 (SRD5A1) expression was determined. These markers were related to clinical benefit (complete/partial response or stable disease ≥6 months) and progression-free and overall survival (PFS/OS). SRD5A1 expression had the highest general predictive value for clinical benefit and positive predictive value (PPV: 85.7%). AR PAS had the highest negative predictive value (NPV: 93.3%). The fitting of a multivariable model led to the identification of SRD5A1, TGFβ, and Notch PAS as the most predictive combination. High AR, high Notch, high ER, low HH PAS, and high SRD5A1 expression were also of prognostic importance regarding PFS and SRD5A1 expression levels for OS. AR, Notch PAS, and SRD5A1 expression have the potential to predict the clinical benefit of CAB treatment in SDC patients. SRD5A1 expression can identify patients that will and AR PAS patients that will not experience clinical benefit (85.7% and 93.3% for PPV and NPV, respectively). The predictive potential of SRD5A1 expression forms a rational basis for including SRD5A1-inhibitors in SDC patients’ treatment.

Molecular Signatures That Predict Response to Azacitidine Treatment for Myelodysplastic Syndromes

Abstract DNA hypomethylating agents (HMAs) constitute standard therapy for higher-risk myelodysplastic syndromes (MDS). However, the lack of reliable genetic markers predicting response to HMAs precludes an optimal use of these agents. Recently, several groups have reported marked efficacy of decitabine for TP53 -mutated myeloid malignancies. However, since decitabine is not approved for MDS in many nations, it is of great interest whether azacitidine (AZA) has similar effects on TP53 -mutated MDS. Thus, we investigated the clonal dynamics and molecular signatures that correlated with response to AZA in MDS. We enrolled a total of 131 cases of MDS patients treated with azacitidine, of which 107 were registered from a prospective multicenter trial of AZA treatment for high-risk MDS patients, while the remaining 24 cases received AZA in off-trial settings. In all patients, a bone marrow specimen was obtained before (pre-therapy samples) and subjected to targeted-capture sequencing, which was designed to detect mutations in 67 known driver genes implicated in myeloid neoplasms as well as genome-wide copy number alterations (CNAs). Mutations and CNAs was also evaluated in samples obtained during and after AZA therapy (post-AZA samples). Analysis of pre-treatment specimens revealed 408 driver mutations in 124 (95%) patients with a mean of 2.7 mutations/sample in 50 genes. At least one CNA was detected in 103 (79%) cases. Most frequently observed lesions included mutations in TP53, ASXL1, RUNX1, TET2, and SRSF2, and other high-risk CNAs, such as -7/7qLOH, 5qLOH, and 17pLOH. Among these TP53 lesions were found in 39 (38%) cases, of which biallelic lesions were suggested for 33 cases (85%), either by multiple mutations (N=11) or accompanying 17p LOH involving the TP53 locus (N=22). TP53 -mutated cases had a significantly lower number of other driver mutations (1.7 vs. 3.1, p 3 years after receiving consolidation with transplant after AZA. Our study revealed a significant positive association of TP53 mutations with favorable responses to AZA in MDS. However, the response was transient, where the development of effective post-AZA therapy after CR should be warranted, such as allogeneic stem cell transplantation to overcome this otherwise intractable disease. Disclosures Chiba: Nippon Shinyaku: Honoraria, Research Funding. Asou: Chugai Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding. Kiyoi: Chugai Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding. Ohyashiki: Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Jansen pharmaceutical KK: Honoraria, Research Funding; Alexion: Honoraria; Fujirebio KK: Honoraria; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Hakko Kirin KK: Honoraria; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Chugai pharmaceutical KK: Research Funding; Phizer KK: Honoraria, Research Funding, Speakers Bureau; MSD KK: Honoraria; Fujirebio KK: Honoraria; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Kyowa Hakko Kirin KK: Honoraria; Taiho pharmaceutical KK: Honoraria; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Taiho pharmaceutical KK: Honoraria; Chugai pharmaceutical KK: Research Funding; Alexion: Honoraria; MSD KK: Honoraria. Miyazaki: Nippon Shinyaku: Honoraria. Naoe: Nippon Shinyaku: Honoraria. Makishima: Yasuda Medical Foundation: Research Funding.

Preliminary efficacy and safety of perioperative treatment of camrelizumab combined with apatinib in resectable hepatocellular carcinoma (HCC): A prospective phase II study.

4082 Background: Although there is no standard perioperative treatment for resectable HCC characterized with high recurrence rate, the strategy of immunotherapy combined with targeted agents is promising in neoadjuvant/adjuvant therapy in various tumors. Methods: In this perspective, single-arm, exploratory phase II trial (NCT04297202), eligible patients (pts) were systemic treatment-naive resectable HCC in intermediate/advanced stage. Preoperative combined treatment of anti-PD-1 antibody camrelizumab (200 mg q2w for 3 cycles) and VEGFR-2 inhibitor apatinib (250 mg qd for 21 days) was started on day 1 cycle 1. On the 7th day after the 3 cycles, radiological imaging was assessed to confirm whether to conduct the hepatectomy. Four weeks after the surgery, combined treatment (camrelizumab 200 mg q3w, apatinib 250 mg qd, 3 weeks per cycle) was resumed for the postoperative 8 cycles. The primary endpoint was major pathologic response (MPR) defined as 50%-99% tumor necrosis in resected tissue. Gene expression profiles (GEPs) using immune-related RNA with pre-treatment specimens were analyzed. The association between immune signatures and pathological response (responders (R) vs. non-responders (NR)) was assessed. Results: A total of 20 pts were enrolled between Dec 5, 2019 and Jan 27, 2021, with a median follow-up of 5.7 months (range 0.7-9.0). All pts were ECOG PS 0-1 and Child-Pugh class A. There were 85% pts with hepatitis B and 10% with hepatitis C, and 55% in BCLC stage B, 35% in stage C and 10% in stage A. In preoperative phase, with 2 withdraw of informed consent form, partial response was reached in 3/18 (16.7%) and 8/18 (44.4%) pts per RECIST 1.1 and mRECIST, respectively, while disease progression was found in 1/18 (5.6%) pts impossible for hepatectomy, which made the resection rate 17/18 (94.4%). After the surgery, one was found to be combined hepatocellular-cholangiocarcinoma by histopathological examination and failed to proceed the postoperative study. The rates of MPR and pathological complete response (pCR) were 5/17 (29.4%) and 1/17 (5.9%), respectively. The preliminary analysis of GEPs (R:NR = 3:4) revealed higher levels of chemokines ( CXCL10 and CXCL11) in responders and higher MS4A4A (marker gene of macrophages ) in non-responders. The most common TEAEs included hypertension (95%), proteinuria (40%), AST elevation (40%), and platelet count decrease (45%). Grade 3 TEAEs were hypertension (20%), rash (10%), and platelet count decrease (10%). No grade 4/5 TEAEs was observed. The most common surgical complications were ALT and AST increase each with the incidence of 70% (all grades) and 45% (grade ≥ 3). Conclusions: This study preliminarily demonstrated that the perioperative treatment of camrelizumab combined with apatinib improved the MPR and pCR with managable safety in intermediate/advanced resectable HCC. Clinical trial information: NCT04297202.

Predictive and prognostic biomarker identification in a large cohort of androgen receptor-positive salivary duct carcinoma patients scheduled for combined androgen blockade.

6071 Background: Patients suffering from recurrent or metastatic (R/M) salivary duct carcinoma (SDC) are often treated with combined androgen blockade (CAB). This treatment however frequently fails (response rates: 18-53%), resulting in a worse prognosis. Therefore, biomarkers that have prognostic value and can predict treatment response are urgently needed. Methods: mRNA from 77 R/M androgen receptor (AR) positive SDC patients treated with leuprorelin acetate combined with bicalutamide was extracted from pre-treatment tumor specimens. AR, Notch, Mitogen-Activated Protein Kinase (MAPK), Transforming Growth Factor beta (TGFβ), Estrogen Receptor (ER), Hedgehog (HH) and the Phosphoinositide 3-Kinase (PI3K) signaling pathway activities were calculated based on expression levels of relevant target genes. Besides this, 5-alpha reductase type 1 ( SRD5A1) expression and Human Epidermal growth factor Receptor 2 (HER2) status were determined. Clinical benefit was defined as complete or partial response or stable disease ≥6 months. Results: Of the 7 signaling pathways, AR pathway activity was the best predictor of clinical benefit (AUC 0.67, 95%-CI 0.54-0.80). At a threshold of 47.8, 21% of the patients tested negative, with a negative predictive value of 93%. SRD5A1 expression outperformed the signaling pathways regarding predictive value (AUC 0.78, 95%-CI 0.67-0.88). Fitting of a multivariable model led to the identification of SRD5A1, Notch and TGFβ as most predictive combination (AUC 0.82, 95%-CI 0.72-0.91). AR, Notch, HH and SRD5A1 were also of prognostic importance regarding progression free survival and SRD5A1 expression levels also for overall survival (median of 175.0 weeks for high versus 96.7 weeks for low expression). Conclusions: Our study revealed predictive and/or prognostic value of AR, HH, Notch and TGFβ signaling activities and SRD5A1 expression in SDC patients treated with CAB. AR pathway activity can be used for identifying non-responders. Further clinical validation is required before implementation of these biomarkers in clinical practice. The observed role of SRD5A1 expression in CAB response forms a rational basis for including SRD5A1-inhibitors in the treatment of SDC patients.[Table: see text]

Whole-exome sequencing in advanced-stage sensitizing EGFR mutation non-small cell lung cancer: Explore resistance biomarkers to EGFR TKI treatment.

9039 Background: Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, 10-20% of de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) in the presence of EGFR mutation remains the issue of concern. Methods: We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 year). Results: Uncommon EGFR mutation subtype was a significant variable in de novo resistance vs. IRs and LTRs with odd ratios of 6.83 ([95% CI 2.36 – 19.80], p-value < 0.001) and 16.84 ([95%CI 1.66 – 171.45, p-value 0.02), respectively. The remaining sensitizing EGFR mutation subtype (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-β, p53 and β-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6×10-4. We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36-73%] compared to IRs/LTRs [range 4-22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4 /6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80-7.31, p-value < 0.001]. Conclusions: The presence of CDK4 /6 amplification in pretreatment specimen serve as a predictive biomarker for de novo resistance in sensitizing EGFR mutation.

Effect of chemoradiotherapy on the proportion of circulating lymphocyte subsets in patients with limited-stage small cell lung cancer.

Chemoradiotherapy (CRT) is the standard treatment for limited-stage small cell lung cancer (LS-SCLC), which can exert anti-tumor effects by regulating immune cells. Different immune cell subsets are associated with a specific sensitivity to CRT. The purpose of this study was to characterize the proportion or composition of peripherallymphocytes in patients with LS-SCLC before and after CRT, and evaluate their prognostic value. A total of 98 patients with LS-SCLC were enrolled. The expression of CD3, CD4, CD8, CD45RA, CD45RO, CD38, CD56, and CD19 on the surface of peripheral blood cells was detected by flow cytometry and retrospectively analyzed. The relationship between the proportion of lymphocyte subsets, progression-free survival (PFS), and overall survival (OS) was evaluated using a log-rank test and Cox regression model. The median PFS was 12.3months and the median OS was 21.7months. Compared with the pre-treatment specimens, post-treatment lymphocytes had increased proportions of CD3+, CD3+CD8+, CD8+CD38+ T cells, and NKT cells, and a decreased proportion of CD3+CD4+ T cells, CD4+CD45RA+ T cells, B cells, NK cells, and CD4/CD8 ratio. Univariate and multivariate analyses showed that prophylactic cranial irradiation, high percentages of CD4+CD45RA+, CD8+CD38+ T cells after CRT independently predicted superior PFS. Male patients with a high baseline CD4+CD45RO+ T cell ratio predicted a poor OS. CRT induced changes in the proportion of circulating lymphocyte subsets in LS-SCLC, which is helpful for designing a regimen of immune drugs to be combined with CRT. The prognostic value of the proportion of lymphocytes aids in understanding the role of peripheral immune profiles in LS-SCLC.

Sex Hormones, Insulin, and Insulin-like Growth Factors in Recurrence of High-Stage Endometrial Cancer.

The influence of sex hormone and insulin/insulin-like growth factor (IGF) axis signaling on endometrial cancer recurrence is unknown. We evaluated these pathways in a prospective cohort of Gynecologic Oncology Group (GOG)0210 trial endometrial adenocarcinoma patients. Stage II-IV patients (N = 816) were included in this study. Pretreatment specimens were tested for tumor mRNA and protein expression of IGF1, IGF2, IGF-binding proteins (IGFBP)-1 and -3, insulin (IR) and IGF-I receptors (IGF1R), phosphorylated IR/IGF1R (pIGF1R/pIR), and estrogen (ER) and progesterone receptors (PR) using qPCR and IHC. Serum concentrations of insulin, IGF-I, IGFBP-3, estradiol, estrone, and sex hormone binding globulin were measured. HRs and 95% confidence intervals (CI) for progression-free survival were calculated from Cox models adjusting for age, stage, and grade. Recurrence occurred in 280 (34%) cases during a median of 4.6 years of follow-up. ER positivity (HR, 0.67; 95% CI, 0.47-0.95), IR positivity (HR, 0.53; 95% CI, 0.29-0.98), and circulating IGF-I (highest vs. lowest quartile: HR, 0.66; 95% CI, 0.47-0.92) were inversely associated with recurrence risk. Circulating estradiol (highest vs. lowest tertile: HR, 1.55; 95% CI, 1.02-2.36) and pIGF1R/pIR positivity (HR, 1.40; 95% CI, 1.02-1.92) were associated with increased recurrence risk. Circulating estradiol and tumor tissue phosphorylated (activated) IGR1R/IR were independently associated with higher risk of recurrence in patients with endometrial cancer. This study may inform future clinical trials of endocrine-targeted adjuvant therapies in patients with endometrial cancer that could include baseline assessment of serum and tissue biomarkers of estradiol and insulin signaling pathways.

Abstract PS5-31: Molecular markers of response to s-equol, a novel oral estrogen receptor (ER) beta agonist for triple negative breast cancer

Abstract Introduction: Patients with TNBC breast cancer have inferior treatment outcomes compared to other breast cancer subtypes and targeted therapies are lacking. S-equol is a novel oral Estrogen Receptor (ER) Beta agonist with preclinical data showing suppression of TNBC cellular proliferation, and recently presented data showed a decrease in Ki-67 in patients treated with S-equol. This abstract will present the molecular markers that correlate with Ki-67 change in patients with TNBC treated with neoadjuvant S-equol. Methods: We conducted a neoadjuvant window trial that enrolled 39 patients with confirmed TNBC on diagnostic core needle biopsy. Cohort A (20 patients) received a daily dose of 50 mg PO twice daily and Cohort B (19 patients) received a higher dose of 150 mg twice daily. Paired biopsies were evaluable for 36 patients. Both cohorts were treated for a duration of 10-21 days. Primary outcome was change from pre- to post-treatment Ki-67 evaluated by paired t-test and that data was previously published. Secondary endpoints include: Tumor infiltrating lymphocytes in intratumoral and peritumoral (stromal) locations using CD3 (pan T cell) and CD8 (cytotoxic T cell) immunohistochemistry, density of lymphocytes per high power field (40x) for both locations in pre-treatment and post-treatment specimens, Cyclin-D1 protein expression in pre-treatment and post-treatment biopsies as percentage of total tumor cells in pre-treatment and post-treatment specimens, and total estrogen receptor-beta protein expression using immunohistochemistry qualitatively and quantitatively using All-red score in pre-treatment and post-treatment specimens.Results: The primary outcome of Ki-67 decrease of at least 20% from baseline was observed in 28% of the patients. This is a placeholder abstract and an updated abstract with the correlative molecular markers as outlined above will be provided. Conclusion: S-equol is a novel well tolerated oral ER-Beta agonist with inhibition of proliferation in patients with TNBC as measured by a decrease in Ki-67. RNA-seq data, also previously presented, supports potential immune activation during this short period of drug exposure. Future studies aim to evaluate S-equol as an immune activating agent for combination with immunotherapies such as checkpoint inhibitors in TNBC. The pending molecular predictors of response will inform future therapeutic clinical trials for this novel therapy. Citation Format: Kate Lathrop, Virginia Kaklamani, Andrew Brenner, Rong Li, Alia Nazarullah, Sarah Hackman, Courtney Thomas, Jonathan Gelfond, Marisa Rodriguez, Richard Elledge. Molecular markers of response to s-equol, a novel oral estrogen receptor (ER) beta agonist for triple negative 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 PS5-31.

Abstract PS17-13: The prognostic role of tumor-infiltrating lymphocytes after treatment with neo-adjuvant chemotherapy in inflammatory breast cancer patients

Abstract Introduction: Inflammatory breast cancer (IBC) is a rare, but aggressive form of breast cancer. Increasing evidence indicates that immune cells in the tumor micro-environment play an important role in IBC progression: infiltration with stromal Tumor Infiltrating Lymphocytes (sTIL) is associated with a better response to neo-adjuvant chemotherapy (NACT) and longer overall survival in IBC patients. However, the prognostic role of sTIL in patients without a complete pathological response (pCR) after NACT remains unclear. In this study we evaluated the effect of NACT on sTIL in IBC and locally advanced non-inflammatory breast cancer (LABC) and the prognostic impact of sTIL in IBC. Methodology: In this retrospective case-control study we evaluated sTIL in patients with IBC (n=60) and LABC (n= 134) that received anthracyclin-taxane based NACT. Tumor tissue was sampled as part of the routine work-up, before (diagnostic biopsy) and after (resection specimen) NACT. sTIL scoring was performed on Haematoxylin & Eosin stained 5-µm sections of formalin-fixed paraffin-embedded tumor tissue by two different researchers according to the recommendations by the International TILs Working Group. In case of discrepancy, the sample was scored by a third observer in order to obtain a consensus score. sTIL difference between pre-treatment and post-treatment specimen was called δsTIL. Cellularity of the residual cancer after NACT was evaluated according to the MD Anderson Residual Cancer Burden (RCB) method. Results: Most of the IBC patients presented with a hormone receptor (HR) positive carcinoma (n=35/60, 58.3%) and 25 patients had pCR after NACT (42%); the latter patients had less than 1% sTIL in the tumor bed area. Besides having histologically more poorly differentiated tumors (P= 0.033), no significant clinicopathological differences between the IBC and LABC cohort were observed. There was no significant difference in the pre-treatment median sTIL score between IBC (12.5%, range: 1% - 80%) and LABC (10%, range: 1% - 85%). Furthermore, both in the IBC (median δsTIL: -4.5%, P= 0.01) and in the LABC (median δsTIL: -1%, P= 0.06) cohort the number of sTIL was lower after NACT. This decrease was significantly greater in the IBC cohort (P= 0.04). In a multivariate model - including HR and HER2 status, histological differentiation grade, nodal status and IBC/LABC phenotype - IBC disease correlated with a stronger decrease of sTILs after NACT (OR: 0.31, 95% CI 0.10 - 0.89, P= 0.04). As we previously demonstrated, higher sTIL score before NACT was associated with better overall survival (OS) (P= 0.006) in IBC. IBC patients without pCR, but with a stronger decrease (> 4.5 %) of the number of sTIL after NACT had a better OS (P= 0.04) and disease-free survival (DFS) (P= 0.04). There was a significant correlation between δsTIL and both higher sTIL score before NACT (P< .001) and lower residual cancer cellularity (P= 0.02). However, in a multivariate model - including sTIL before NACT, δsTIL and residual cancer cellularity - both a strong decrease of sTIL (HR: 0.32; 95% CI 0.10 - 1.00; P= 0.05) and a low residual cancer cellularity (HR: 0.22; 95% CI 0.07 - 0.68; P= 0.009) were independent predictors of a better DFS. Conclusion: Both the IBC and LABC cohort had the same number of sTIL before NACT. However, the IBC phenotype was associated with a stronger decrease of sTIL after NACT independent of molecular subtype and nodal stage. Furthermore, a strong decrease in sTIL was an independent predictor of better prognosis in IBC. Citation Format: Christophe Van Berckelaer, Leonie Vercauteren, Iris Vermeiren, Glenn Broeckx, Steven Van Laere, Luc Dirix, Cecile Colpaert, Peter Van Dam. The prognostic role of tumor-infiltrating lymphocytes after treatment with neo-adjuvant chemotherapy in inflammatory breast cancer patients [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 PS17-13.

Effects of Mycophenolate Mofetil, Methotrexate and Pimecrolimus on cdk4 and p16 in Erosive Oral Lichen Planus.

Background:This study evaluated the effect of mycophenolate mofetil, methotrexate, and pimecrolimus on the expression of cdk4 and p16, important proteins implicated in hyperproliferation and arrest in the G1 phase, in oral lichen planus (OLP).Materials and Methods:In this randomized clinical trial, 60 patients were randomly assigned in three equal groups to receive either pimecrolimus cream, or mycophenolate mofetil or methotrexate, both supplemented with pimecrolimus. Pretreatment and post-treatment specimens were immunohistochemically stained for detecting cdk4 and p16.Results:A significant decrease in cdk4 cytoplasmic positivity was noted in all three treatment groups and was especially more significant in the MTX group (P < 0.0001) than in the other two groups (P < .001). However, a significant decrease in the cdk4 nuclear staining was noted with only systemic treatment groups of MMF (P < 0.05) and MTX (P < 0.01), both supplemented with pimecrolimus. No significant decrease in cytoplasmic p16 levels was noted in the MTX group while a significant decrease in cytoplasmic p16 levels was noted in the other two groups; however, no significant decrease in p16 nuclear staining was noted with any treatment.Conclusion:By decreasing the expression of cdk4 and p16, pimecrolimus, methotrexate, and mycophenolate mofetil decrease the malignant potential of OLP lesions. However, methorexate can be a better alternative in cases showing high cdk4 expression.

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Evaluation of Programmed Death Ligand 1 (PD-L1) Gene Amplification and Response to Nivolumab Monotherapy in Non–small Cell Lung Cancer

Robust predictors for response to anti-programmed death 1 and its ligand (PD-1/PD-L1) immunotherapy in non-small cell lung cancer (NSCLC) are not fully characterized. To evaluate whether PD-L1 (CD274) copy number gains (CNGs), comprising amplification and polysomy, in pretreatment specimens assessed by fluorescence in situ hybridization are associated with response to nivolumab monotherapy in NSCLC. This multicenter cohort study enrolled 200 patients, of whom 194 had assessable tumors, with advanced or recurrent NSCLC who were treated with nivolumab after progression following prior treatment at 14 institutions in Japan between July 2016 and December 2018. Median (interquartile range) duration of follow-up was 12.6 (5.6-20.4) months. Data were analyzed from December 2019 to February 2020. Sequential nivolumab was given on day 1 of a 14-day cycle. Response was assessed every 4 cycles using Response Evaluation Criteria in Solid Tumors version 1.1. Overall response rate (ORR) according to the PD-L1 copy number status. Additional end points were progression-free survival, overall survival, and PD-L1 tumor proportion score (TPS) assessed by immunohistochemistry based on PD-L1 copy number status. A total of 6 of the 200 patients were excluded because of poor-quality tumor specimens for the biomarker study, resulting in 194 assessable patients. Of these, 155 (79.9%) were men, with a median (range) age of 69 (43-83) years. PD-L1 CNGs were identified in 32 patients (16.5%), including 5 (2.6%) with amplification and 27 (13.9%) with polysomy. The ORR among patients with and without PD-L1 CNGs was 28.1% (95% CI, 13.7%-46.7%) and 17.9% (95% CI, 12.3%-24.7%), respectively. Although patients with PD-L1 polysomy did not demonstrate improved ORR (18.5% [95% CI, 6.3%-38.1%]) compared with those without PD-L1 CNGs, 4 of 5 patients (80.0% [95% CI, 28.4%-99.5%]) with PD-L1 amplification showed response, among whom median duration of response was not reached. Patients with PD-L1 amplification showed excellent survival outcomes for progression-free and overall survival. Overall, 3 PD-L1-amplified tumors (60.0%) showed PD-L1 TPS of at least 80%, but 2 (40.0%) had PD-L1 TPS of 15% or less. In this study, tumor PD-L1 amplification but not polysomy was associated with response to nivolumab monotherapy among patients with NSCLC. External validation with a larger sample size is warranted.

Abstract 4331: Nuclear size of circulating tumor cells is associated with prognosis in metastatic, castration-resistant prostate cancer

Abstract Background: Current risk stratification models in prostate cancer (PC) have been based on clinical and pathological variables. Beyond serum prostate-specific antigen (PSA) concentration measurements, there remain few new biomarkers to help identify patients at risk for poor clinical outcomes. Morphological analyses using Gleason scoring along with cell nuclear size and shape remains to be a fundamental pathological practice of PC that have been utilized to identify aggressive diseases and to associate with aggressive metastasis. In particular, changes in nuclear shape and composition have been associated with outcome in early stage disease. Circulating tumor cells (CTCs) have arisen as contemporary noninvasive prognostic biomarkers for PC. Previously, a subgroup of PC CTCs, with prominently small nuclei (&amp;lt; 8.5 μm), were found to be correlated with the presence of visceral metastases. This subgroup was named very-small-nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a putative biomarker of a lethal subtype in metastatic castration resistant PC (mCRPC). Methods: In this study, 76 patients with mCRPC were recruited for overall survival (OS) analysis. Of the 76 patients, 50 had available pre-treatment blood specimens prior to the initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone and enzalutamide) or taxane or tyrosine kinase inhibitor therapy. Using the NanoVelcro CTC Enumeration Assay, CTCs were captured and subjected to immunofluorescence staining. CTCs were identified as DAPI+/CK+/CD45- with a round or oval nucleus. Additionally, CTC nuclear size was measured and defined as the square root of the product of the long axis and the short axis. Kaplan-Meier analysis and Cox proportional hazards model were conducted. Results: Patients with vsnCTC (i.e., vsnCTC+) had a significantly shortened OS compared with patients without vsnCTC (i.e., vsnCTC-). The median OS was 34 (vsnCTC+, n=49) vs. 149 (vsnCTC-, n=27) weeks (log-rank HR=2.6 with 95% CI 1.5 to 4.5, p=0.0006). Progression free survival (PFS) analysis was performed for the 50 patients with pre-treatment blood samples. The median PFS was 12 (vsnCTC+, n=32) vs. 26 (vsnCTC-, n=18) weeks (log-rank HR=2.2 with 95% CI 1.3 to 4.0, p=0.0038). We also found that the hazard ratio of overall survival increased significantly as the CTC nuclear size decreased using the p-spline plot. Conclusions: Our study showed that nuclear size reduction has importance in CTCs in a fashion similar to its utility in tissue. This study points toward the importance of the vsnCTC in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for morality. We posit that the vsnCTC represents a new hallmark of an aggressive subtype of mCRPC. This has potential importance in optimizing therapeutic choices. Citation Format: JASMINE J. WANG, Pai-Chi Teng, Yu Jen Jan, Jie-Fu Chen, Galen Cook-Wiens, Nu Yao, Gina C. Chu, Pin-Jung Chen, Yingying Yang, Yee Hui Yeo, Yi-Te Lee, Leland W. Chung, Sungyong You, Yazhen Zhu, Michael R. Freeman, Andre Rogatko, Ju Dong Yang, Hsian-Rong Tseng, Edwin M. Posadas. Nuclear size of circulating tumor cells is associated with prognosis in metastatic, castration-resistant prostate cancer [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 4331.

Abstract 6161: PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models

Abstract Purpose: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study if SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). Experimental Design: We analyzed HNSCC TCGA data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a Phase I trial investigating olaparib were analyzed. Results: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4 mutant but not SMAD4 positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4 deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared to vehicle treated tumors. In vitro, olaparib or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death and less proliferation in SMAD4 deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a Phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. Conclusion: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs. Citation Format: Christian D. Young, Ariel L. Hernandez, Li Bian, Sana D. Karam, David Raben, Xiao-Jing Wang. PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models [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 6161.

Abstract 6655: Tumor microenvironment evaluation and tumor intrinsic genomic features predict anti-PD-1 response of metastatic gastric cancer: Results from phase II clinical trial and multi-omics data

Abstract Background: Clinical studies support the efficacy of immune checkpoint blockades (ICBs) in a subset of patients with metastatic gastric cancer (mGC). With the aim of identifying determinants of response to ICBs, we performed molecular characterization of tissues from 61 patients with mGC who were treated with pembrolizumab as salvage treatment in a prospective phase II clinical trial (NCT#02589496). Methods: Of 61 patients, 60 patients underwent pretreatment biopsy and 45 specimens were of sufficiently high quality for RNA sequencing. TMEscore, which was previously established to quantify the tumor microenvironment (TME), was used to estimated TME of pretreatment specimens. Additional ACRG and TCGA multi-omics data were applied to validate the aforementioned results. Intrinsic mutations and virus infections correlated to the TMEscore were further identified via analyzation of TCGA and ACRG data. Results: We established a methodology (TMEscore) to evaluate the TME of GC patients, which was previously found to be a robust prognostic and predictive biomarker for patients treated with ICBs. By applying ROC curve analysis, the TMEscore was found to be the best predictive biomarker (TMEscore: AUC = 0.891; CPS: AUC = 0.830; TMB: AUC = 0.672; MSI status: AUC = 0.708; EBV status: AUC = 0.727; respectively). Moreover, TMEscore was the most significant gene signature that correlated with tumor response (TMEscore: P = 1.7e-5; GEPs: P = 0.00035; ImmunoScore: P = 0.29106; CD8+ T cell fraction: P = 0.00011; respectively). A higher TMEscore was significantly associated with EBV+ and MSI-High TCGA molecular subtypes (Kruskal-Wallis test, P = 0.002) which were reported to benefit from ICBs of GC. Moreover, analysis of the TCGA and ACRG data reproductively supported the predictive value of TMEscore and its latent power in identifying GC patients with MSI-High characteristics and EBV+ from other subtypes. Additionally, the study of TCGA dataset revealed that TMEscore was significantly associated with tumor neoantigen load (Spearman test, P = 2.8e-10, r = 0.441). Notably, it highlighted that GC patients with ARID1A and PIK3CA mutations exhibited higher TMEscore (Wilcoxon test, ARID1A: P = 8.4e-10, PIK3CA: P = 4e-10) in both TCGA and ACRG cohorts. Furthermore, TCGA genomic data indicated that patients with ARID1A p.D1850Tfs*33 and p.F2141Sfs*59 mutations exhibited the highest TMEscore than patients with other mutational sites and wild-typed patients (Kruskal-Wallis test, P = 9e-11). Conclusions: These findings indicate that the assessment of TMEscore via high throughput-sequencing and PCA algorithm provides a robust biomarker for the selection of GC patients who may derive greater benefit from pembrolizumab. Our data also suggest that TMEscore may be a more accurate predictive biomarker than TMB, MSI and EBV status, and this resource may help facilitate the development of precision immunotherapy. Citation Format: Dongqiang Zeng, Wangjun Liao, Kyoung Mee Kim, Min Shi, Rui Zhou, Yunfang Yu, Zilan Ye, Jiani Wu. Tumor microenvironment evaluation and tumor intrinsic genomic features predict anti-PD-1 response of metastatic gastric cancer: Results from phase II clinical trial and multi-omics data [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 6655.

Abstract B70: Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens

Abstract Objective: Outcomes for patients with rhabdomyosarcoma (RMS) who have relapsed or refractory disease remain poor. Multiple large-scale tumor genomic profiling efforts have been undertaken; however, little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. Methods: To address this issue, we performed 80x whole-genome sequencing of 23 metastases and 2 pretreatment samples from two patients with lethal rhabdomyosarcoma. Multiregion and spatially separated metastases were available through our research autopsy donation program. RA 17-1 had fusion-positive alveolar RMS, while RA 17-10 had Li-Fraumeni syndrome and anaplastic embryonal RMS. Results: We observed on average 65 and 98 coding single-nucleotide variants (SNV) per sample in RA 17-1 and RA 17-10, respectively. RA 17-1 was found to have the canonical PAX03-FOX01 fusion along with MYC-N amplification, which was truncal to all samples. A somatic TP53 mutation in combination with TP53 loss of heterozygosity (LOH) was present in all metastases sequenced at autopsy. A TP53 mutation was not identified in the pretreatment biopsy sample selected for sequencing; however, TP53 immunohistochemistry demonstrated the presence of a TP53 mutation in at least 2 out of 6 pretreatment biopsy cores. In RA 17-10 each sample exhibited numerous copy number alterations and complex structural variants, including LOH of TP53, a TSC2 mutation with LOH, and MYC amplification, which were truncal to all samples. PTCH1 amplification was private to all relapse specimens in RA 17-10. RA 17-10 had more structural and copy number variations per sample than RA 17-1. Phylogenies were derived based on SNVs and demonstrated a branched evolutionary pattern for each patient. When comparing related samples’ mutational signatures, several dynamics were apparent, including the predominance of signatures 3 and 8 (DNA double-strand break repair pathways) in metastatic samples, consistent with the fact that both patients harbored TP53 mutations. Conclusion: Defining important elements of ITH within metastases remains a goal for genomic study in pediatrics, especially in patients with germline predisposition syndromes. Our findings demonstrate the dynamic nature of genomic instability processes, highlighting the importance of longitudinal sampling at the time of recurrence to define treatment effect and the changing mutational landscape in these tumors. The case of RA 17-1 demonstrates the limitations of targeted sequencing on a single sample at diagnosis, as the subclonal TP53 mutation was not originally identified on the clinical assay. TP53 mutations in fusion-positive RMS have rarely been described and likely contributed to this patient’s refractory disease course. Both RA 17-1 and RA 17-10 had more structural variants per sample then what has previously been reported for fusion-positive and fusion-negative RMS, respectively, likely highlighting the contribution of the TP53 mutation to genomic instability in each patient. Citation Format: Michael D. Kinnaman, Alvin Makohon-Moore, Nancy Bouvier, Dominik Glodzik, Max Levine, Ellie Papaemmanuil, Filemon Dela Cruz, Leonard Wexler, Andrew Kung, Christine Iacobuzio-Donahue. Clonal evolution of chemotherapy-resistant rhabdomyosarcoma via multifocal genomic analysis of pretreatment and treatment-resistant autopsy specimens [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B70.