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PDF file - 110K, 14 formalin-fixed paraffin-embedded PDCCEs were arrayed, RMA normalized and base on a 3D principal components analysis, CRC057 was omitted from further analysis.

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An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRASG12C-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action.Significance:MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45–58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1

PDF file - 63K, Oxaliplatin sensitivity data to the NCI-60 cell lines.

PDF file - 61K, Gene List of Oxaliplatin Sensitivity Signature.

Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationship between MECP2 amplification and KRAS mutation in uterine cancer subtypes.

Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2 Gene on the Active X Chromosome is Preferentially Amplified, Related to Figure 1. Supplementary Figure S2. MECP2 Overexpression Allows Soft Agar Growth of N minus RAS Cells in Two Different Types of Human Breast Epithelial Cells, and MECP2 Splicing Isoforms Differ In Their Ability to Confer Anchorage Independent Growth, Related to Figure 2A. Supplementary Figure S3: MECP2 Overexpression Allows Two Different Types of N minus RAS Breast Epithelial Cells To Grow As Tumor Xenograft In Nude Mice, Related to Figure 2E, 2F. Supplementary Figure S4. The MECP2 e2 Short Splicing Isoform Allows Sustained Activation of the MAPK Pathway after Prolonged Starvation in Minimal Medium without Growth Factors, Related to Figure 4A-C. Supplementary Figure S5. Both MECP2 Isoforms Activate the PI3K Pathway, Related to Figure 4D. Supplementary Figure S6. Additional human cancer cell lines with high level of MECP2 are growth-inhibited upon inhibition of MECP2 expression, Related to Figure 4E.

Supplementary Table S1. List of validated hits from N minus RAS screen. Supplementary Table S2. Amplicons in Human Cancer (from Broad Institute TCGA Copy Number Portal). Supplementary Table S3. Cancer cell lines used in current study with their MECP2 expression and RAS/MAPK/PI3K pathways mutation status (based on COSMIC, canSAR, ATCC, and CCLE databases).

Despite advances in contemporary chemotherapeutic strategies, long-term survival still remains elusive for patients with metastatic colorectal cancer. A better understanding of the molecular markers of drug sensitivity to match therapy with patient is needed to improve clinical outcomes. In this study, we used in vitro drug sensitivity data from the NCI-60 cell lines together with their Affymetrix microarray data to develop a gene expression signature to predict sensitivity to oxaliplatin. To validate our oxaliplatin sensitivity signature, patient-derived colorectal cancer explants (PDCCE) were developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice from resected human colorectal tumors. Analysis of gene expression profiles found similarities between the PDCCEs and their parental human tumors, suggesting their utility to study drug sensitivity in vivo. The oxaliplatin sensitivity signature was then validated in vivo with response data from 14 PDCCEs treated with oxaliplatin and was found to have an accuracy of 92.9% (sensitivity = 87.5%; specificity = 100%). Our findings suggest that PDCCEs can be a novel source to study drug sensitivity in colorectal cancer. Furthermore, genomic-based analysis has the potential to be incorporated into future strategies to optimize individual therapy for patients with metastatic colorectal cancer. Mol Cancer Ther; 11(7); 1500–9. ©2012 AACR.

Supplementary Methods from MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

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PDF file - 39K, Fifty-nine samples from the NCI-60 cell panel were RMA normalized and base on 3D principal components analysis, MCF-7 was ommitted from further analysis.

PDF file - 39K, Tumor growth inhibition of patient derived colorectal cancer explant to oxaliplatin.

Purpose:In contrast to recurrence after initial diagnosis of stage I–III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS).Experimental Design:Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC.Results:When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2− tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041).Conclusions:Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.

Sensitivity Analyses For the Median-Center and Proliferation Normalization of Microarray Data.

Figures S1-S14

Supplementary Methods

Supplemental Figure Legends

QC analysis

PDF file, 73K, Extended Toxicity Information.

Tables S1-S12

Supplementary Figure 1. Development, optimization and performance of the gene expression assay. Supplementary Figure 2. Evaluation of assay performance using cancer cell line spike-in models. Supplementary Figure 3. Sample processing and quality control measures Supplementary Figure 4. Expression analysis of CTCs and matched normal blood samples. Supplementary Figure 5. Subtype and proliferation status. Supplementary Figure 6. Serial analysis. Supplementary Figure 7. Clustering analysis of copy number data. Supplementary Figure 8. ESR1 and ERBB2 status in CTCs and patient outcome Supplementary Figure 9. CTC molecular subtype and patient outcome.

Figure S1. Summary of HRD testing in PrECOG and combined cisplatin cohorts; Figure S2. Distribution of HRD scores in PrECOG and combined cisplatin cohorts; Figure S3. Performance of HRD in predicting response in PrECOG and combined cisplatin cohorts; Figure S4. Logistic regression models to predict response in PrECOG and combined cisplatin cohorts; Table S1. Study cohorts for threshold training set; Table S2. Summary statistics of HRD scores in BRCA1/2 deficient samples by tissue in training; Table S3. Patient clinical and demographic data; Table S4. HRD score and association with therapy response in entire cohorts; Table S5. Univariate associations between clinical variables and RCB 0/1 or HR deficiency status; Table S6. Analysis of RCB 0/1 and pCR in combined PrECOG 0105 and Cisplatin Trials cohorts (logistic regression models adjusted for cohort)

Gene Expression Signatures

Purpose: We conducted a phase I study of dasatinib, an oral SRC-family tyrosine kinase inhibitor, in combination with paclitaxel and carboplatin in the treatment of advanced and recurrent epithelial ovarian cancer.Experimental Design: The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included defining toxicity, response rate (RR), pharmacokinetics, and pharmacodynamics. Using a “3+3” design, cohorts of three to six patients received paclitaxel (175 mg/m2) and carboplatin (AUC 6) every 3 weeks with escalating doses of dasatinib (100, 120, and 150 mg daily), followed by an eight-patient expansion cohort.Results: Twenty patients were enrolled between June 2007 and December 2009. The median age was 61 years (range: 42–82) with a median of 2 prior regimens (range: 0–6), and 71% had platinum-sensitive disease. There were three to six patients in each cohort, and eight in the expansion cohort. Pharmacokinetics were observed over the first two cycles of therapy. One DLT was observed in the 100 mg dasatinib cohort (grade 3 myalgia). Other toxicities in all cycles included neutropenia (95% grade 3–4; 91% in the 150 mg dosing cohort), thrombocytopenia (35% grade 3–4), and fatigue (10% grade 3). The RR was 40% [three complete responses, (15%); five partial responses, (25%)],10 patients (50%) had stable disease, and two were not evaluable. The PFS6-month actuarial estimate was 86%. The median PFS and OS were 7.8 and 16.2 months, respectively.Conclusions: Due to the high incidence of myelosuppression with subsequent cycles, the recommended phase II dose of dasatinib is 150 mg daily in combination with paclitaxel and carboplatin. The combination was safe with evidence of clinical activity. Clin Cancer Res; 18(19); 5489–98. ©2012 AACR.

Purpose: We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).Methods: CTCs were isolated from 105 patients with MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2–5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (n = 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH; n = 49).Results: Combined transcriptional and genomic profiling showed that CTCs were 26% ESR1−ERBB2−, 48% ESR1+ERBB2−, and 27% ERBB2+. Serial testing showed that ERBB2 status was more stable over time compared with ESR1 and proliferation (MKI67) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression “fingerprints” were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in ESR1/ER (27%) and ERBB2/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (MKI67) status had significantly reduced progression-free survival (P = 0.0011) and overall survival (P = 0.0095) compared with patients with low proliferative CTCs.Conclusions: We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time. Clin Cancer Res; 24(6); 1486–99. ©2018 AACR.

Cox proportional hazards analyses

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Purpose:BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST).Experimental Design: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy.Results: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials.Conclusions: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764–73. ©2016 AACR.

Supplementary Data from A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia

PDF file, 130K, Heatmaps and Safe Plots.

PFS and OS of PAM50 and OncotypeDx risk categories

Purpose: To provide further insight into the role of proliferation and other cellular processes in chemosensitivity and resistance, we evaluated the association of a diverse set of gene expression signatures with response to neoadjuvant chemotherapy (NAC) in breast cancer.Experimental Design: Expression data from primary breast cancer biopsies for 1,419 patients in 17 studies prior to NAC were identified and aggregated using common normalization procedures. Clinicopathologic characteristics, including response to NAC, were collected. Scores for 125 previously published breast cancer–related gene expression signatures were calculated for each tumor.Results: Within each receptor-based subgroup or PAM50 subtype, breast tumors with high proliferation signature scores were significantly more likely to achieve pathologic complete response to NAC. To distinguish “proliferation-associated” from “proliferation-independent” signatures, we used correlation and linear modeling approaches. Most signatures associated with response to NAC were proliferation associated: 90.5% (38/42) in ER+/HER2− and 63.3% (38/60) in triple-negative breast cancer (TNBC). Proliferation-independent signatures predictive of response to NAC in ER+/HER2− breast cancer were related to immune activity, while those in TNBC comprised a diverse set of signatures, including immune, DNA damage, signaling pathways (PI3K, AKT, Ras, and EGFR), and “stemness” phenotypes.Conclusions: Proliferation differences account for the vast majority of predictive capacity of gene expression signatures in neoadjuvant chemosensitivity for ER+/HER2− breast cancers and, to a lesser extent, TNBCs. Immune activation signatures are proliferation-independent predictors of pathologic complete response in ER+/HER2− breast cancers. In TNBCs, significant proliferation-independent signatures include gene sets that represent a diverse set of cellular processes. Clin Cancer Res; 22(24); 6039–50. ©2016 AACR.

Purpose: Fulvestrant is an estrogen receptor (ER) antagonist and an approved treatment for metastatic estrogen receptor–positive (ER+) breast cancer. With the exception of ER levels, there are no established predictive biomarkers of response to single-agent fulvestrant. We attempted to identify a gene signature of response to fulvestrant in advanced breast cancer.Experimental Design: Primary tumor samples from 134 patients enrolled in the phase III CONFIRM study of patients with metastatic ER+ breast cancer comparing treatment with either 250 mg or 500 mg fulvestrant were collected for genome-wide transcriptomic analysis. Gene expression profiling was performed using Affymetrix microarrays. An exploratory analysis was performed to identify biologic pathways and new signatures associated with response to fulvestrant.Results: Pathway analysis demonstrated that increased EGF pathway and FOXA1 transcriptional signaling is associated with decreased response to fulvestrant. Using a multivariate Cox model, we identified a novel set of 37 genes with an expression that is independently associated with progression-free survival (PFS). TFAP2C, a known regulator of ER activity, was ranked second in this gene set, and high expression was associated with a decreased response to fulvestrant. The negative predictive value of TFAP2C expression at the protein level was confirmed by IHC.Conclusions: We identified biologic pathways and a novel gene signature in primary ER+ breast cancers that predicts for response to treatment in the CONFIRM study. These results suggest potential new therapeutic targets and warrant further validation as predictive biomarkers of fulvestrant treatment in metastatic breast cancer. Clin Cancer Res; 22(23); 5755–64. ©2016 AACR.

Purpose: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients.Experimental Design: We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemoimmunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts, representing a total of 301 CLL patients.Results: A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training data set. The progression signature was validated in two independent data sets, showing a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and nonresponding CLL patients.Conclusions: Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients. (Clin Cancer Res 2009;15(22):694755)

Figure S1. REMARK diagram Figure S2. NanoString Tumor Inflammation Signature by breast cancer intrinsic subtype Figure S3. NanoString individual gene expression changes by cohort Table S1. Pre-treatment immune microenvironment and response to preoperative therapy Table S2. Tissue-based immune biomarkers in patients with pathologic complete response Table S3. References for classification of genes as M1-like or M2-like

Purpose:Hormone receptor–positive/HER2-negative (HR+/HER2−) breast cancer is associated with low levels of stromal tumor-infiltrating lymphocytes (sTIL) and PD-L1, and demonstrates poor responses to checkpoint inhibitor therapy. Evaluating the effect of standard chemotherapy on the immune microenvironment may suggest new opportunities for immunotherapy-based approaches to treating HR+/HER2− breast tumors.Experimental Design:HR+/HER2− breast tumors were analyzed before and after neoadjuvant chemotherapy. sTIL were assessed histologically; CD8+ cells, CD68+ cells, and PD-L1 staining were assessed immunohistochemically; whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed.Results:Ninety-six patients were analyzed from two cohorts (n = 55, Dana-Farber cohort; n = 41, MD Anderson cohort). sTIL, CD8, and PD-L1 on tumor cells were higher in tumors with basal PAM50 intrinsic subtype. Higher levels of tissue-based lymphocyte (sTIL, CD8, PD-L1) and macrophage (CD68) markers, as well as gene expression markers of lymphocyte or macrophage phenotypes (NanoString or CIBERSORT), correlated with favorable response to neoadjuvant chemotherapy, but not with improved distant metastasis-free survival in these cohorts or a large gene expression dataset (N = 302). In paired pre-/postchemotherapy samples, sTIL and CD8+ cells were significantly decreased after treatment, whereas expression analyses (NanoString) demonstrated significant increase of multiple myeloid signatures. Single gene expression implicated increased expression of immunosuppressive (M2-like) macrophage-specific genes after chemotherapy.Conclusions:The immune microenvironment of HR+/HER2− tumors differs according to tumor biology. This cohort of paired pre-/postchemotherapy samples suggests a critical role for immunosuppressive macrophage expansion in residual disease. The role of macrophages in chemoresistance should be explored, and further evaluation of macrophage-targeting therapy is warranted.

Correlation between IHC and transcript level by microarray

PFS and RORP classifier

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The percentage of p27+ cells by breast tissue type (S1); The percentage of ER+ cells by breast tissue type (S2); The percentage of Ki67+ cells by breast tissue type (S3); Spearman correlations between markers in normal breast tissue in the Nurses' Health Study and the Nurses' Health Study II (S4).

The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case–control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses' Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67+ cells was positively associated with breast cancer risk among premenopausal women [OR = 10.1, 95% confidence interval (CI) = 2.12–48.0]. Conversely, the frequency of ER+ or p27+ cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER+: OR = 0.70, 95% CI, 0.33–1.50; p27+: OR = 0.89, 95% CI, 0.45–1.75). Notably, high Ki67+/low p27+ and high Ki67+/low ER+ cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared with low Ki67+/low p27+ and low Ki67+/low ER+ cell frequencies, respectively, among premenopausal women (Ki67hi/p27lo: OR = 5.08, 95% CI, 1.43–18.1; Ki67hi/ERlo: OR = 4.68, 95% CI, 1.63–13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. Cancer Res; 76(7); 1926–34. ©2016 AACR.

Image analysis of benign breast lesions (S1); Boxplots of the percentage of p27+, ER+, and Ki67+ cells in benign breast lesions (S2).

Background: Immunotherapy with checkpoint inhibition is active in mTNBC. Both pembrolizumab and atezolizumab are FDA approved for programmed cell death ligand 1 positive (PDL1+) mTNBC. Vaccines may further induce host immune response and enhance therapeutic activity of checkpoint inhibitors. PVX-410 (PVX) (OncoPep, Inc.) is a novel, HLA-A2 restricted, tetra-peptide vaccine, with 3 of its 4 antigens (XBP1[2 splice variants] and CD138) commonly overexpressed in TNBC. We present results from a phase 1b study evaluating the immune response, safety and tolerability, and clinical activity of PVX and pembrolizumab (PEM) in mTNBC. Methods: Eligibility for this phase 1b multi-center, single-arm study included HLA-A2+, PD-L1 unselected female patients (pts) ≥18 years with metastatic or inoperable locally advanced TNBC, measurable disease, and any number of prior therapies, including prior checkpoint inhibitor therapy. Pts received 6 doses of 800µg PVX emulsified in Montanide ISA 720 VG by subcutaneous injection co-administered with intramuscular Hiltonol weekly for 6 weeks (wks) followed by booster vaccine doses at wks 10 and 28, with concurrent intravenous 200 mg PEM every 3 wks starting with the second PVX dose. Therapy was given until progressive disease, unacceptable toxicity or a maximum of 24 months. Blood samples were scheduled for immune response assessment at baseline and at weeks 2, 5, 10, 28, and 52 post-treatment initiation. The primary objective was PVX- specific immune response at week 10. Immune response was defined as a ≥2-fold change over baseline in the proportion of CD3+CD8+ T cells that expressed IFNγ and the proportion of CD3+CD8+ T cells positive for PVX tetramers following an in vitro stimulation of PBMC with PVX peptides using a flow cytometric assay. Secondary objectives were immune response at wk 28, safety and tolerability, and clinical endpoints (RR, CBR, DCR, DoR, PFS, and OS). Results: Between 3/2018 and 8/2020, 19 pts enrolled. Median age was 62 yrs (range 46-79), with median 2 (range 0-9) lines of prior therapy for metastatic disease. Median disease-free interval among 16 pts with prior early TNBC was 3.3 years. Among 19 enrolled patients, 16 were available for analysis at the time of abstract submission. Among the 16, 10 pts were evaluable at week 10 and 7(70%) demonstrated a PVX specific immune response. There were 6 patients who progressed before week 10, of whom 3 (50%) had a positive immune response at the EOT visit. Immune response persisted in all evaluable pts assessed at week 28 (n=4). Immune response data for all evaluable patients will be updated at the presentation. Among 19 patients evaluable for safety analysis, the most common adverse events (AEs) attributable to PVX (grade ≥2) included: fatigue (21%), arthralgia (11%) injection site reaction (5 %) pain (5%) lymphocyte count decreased (5%), maculopapular rash (5%) and skin infection (5%) . There were two grade 3 AEs attributed to PEM (AST elevation, hyponatremia) and one grade 4 AE (ALT elevation). There were no grade 5 AEs. The clinical benefit rate (CR+PR+SD for ≥16 weeks) was 31.6% with no confirmed partial or complete responses. Best overall response was SD in 9 (47%) patients. Analysis of additional clinical endpoints including PFS and OS is ongoing and will be presented at the meeting. Conclusions: PVX plus PEM is safe with manageable toxicity in pts with mTNBC. No new unexpected adverse events were identified. Immune response data show PVX induces antigen-specific T cell expansion as observed by increases in PVX tetramer and IFN positive T cells. Clinical disease control was observed with a CBR of 31.6%. Based on these promising immune response results in this pretreated population, a phase 2 study with PVX+PEM in combination with standard chemotherapy in treatment naïve, PD-L1+ mTNBC is underway (NCT04634747). Citation Format: Steven J Isakoff, Nadine M. Tung, Jun Yin, Nabihah Tayob, Joanne Parker, Julie Rosenberg, Aditya Bardia, Laura Spring, Hannah Park, Maya Collins, William T. Barry, Mariano Severgnini, Doris Peterkin, Sara M. Tolaney. A phase 1b study of PVX-410 vaccine in combination with pembrolizumab in metastatic triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-17.

Purpose: In contrast to recurrence after initial diagnosis of stage I–III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS). Experimental Design: Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC. Results: When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2− tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041). Conclusions: Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.

Objective To assess the safety, mechanism of action, and preliminary efficacy of rituximab followed by belimumab in the treatment of refractory lupus nephritis (LN). Methods In a multicenter, randomized, open-label clinical trial, 43 patients with recurrent or refractory LN were treated with rituximab, cyclophosphamide (CYC), and glucocorticoids followed by weekly belimumab infusions until week 48 (RCB group), or treated with rituximab and CYC but no belimumab infusions (RC group). Patients were followed up until week 96. Percentages of total and autoreactive B cell subsets in the patients' peripheral blood were analyzed by flow cytometry. Results Treatment with belimumab did not increase the incidence of adverse events in patients with refractory LN. At week 48, a complete or partial renal response occurred in 11 (52%) of 21 patients receiving belimumab, compared to 9 (41%) of 22 patients in the RC group who did not receive belimumab (P = 0.452). Lack of improvement in or worsening of LN was the major reason for treatment failure. B cell depletion occurred in both groups, but the percentage of B cells remained lower in those receiving belimumab (geometric mean number of B cells at week 60, 53 cells/μl in the RCB group versus 11 cells/μl in the RC group; P = 0.0012). Percentages of total and autoreactive transitional B cells increased from baseline to week 48 in both groups. However, percentages of total and autoreactive naive B cells decreased from baseline to week 48 in the belimumab group compared to the no belimumab RC group (P = 0.0349), a finding that is consistent with the observed impaired maturation of naive B cells and enhanced censoring of autoreactive B cells. Conclusion The addition of belimumab to a treatment regimen with rituximab and CYC was safe in patients with refractory LN. This regimen diminished maturation of transitional to naive B cells during B cell reconstitution, and enhanced the negative selection of autoreactive B cells. Clinical efficacy was not improved with rituximab and CYC in combination with belimumab when compared to a therapeutic strategy of B cell depletion alone in patients with LN.

INTRODUCTION: Atropine sulfate is an FDA-approved medical countermeasure (MCM) for the treatment of organophosphorus nerve agent and organophosphate pesticide toxicity. Sufficient MCM supplies must be available in an incident involving a mass human exposure either from an accidental chemical release or a terrorist attack. METHODS: We performed a randomized, 3-sequence, 3-period phase I crossover study to assess the bioavailability and pharmacokinetics (PK) of a single dose (0.5 mg and 1.0 mg) of 1% ophthalmic atropine sulfate solution administered sublingually to 15 healthy adult volunteers. The primary endpoint was evaluation of the bioavailability of each of the two sublingual doses against a 1.0 mg reference intravenous (IV) atropine dose. Secondary endpoints included the safety and tolerability (xerostomia scale) of atropine sulfate administered sublingually. RESULTS: Sublingual atropine was safe (no severe AEs or SAEs were reported with either dose) and well tolerated, with a single subject reaching maximum xerostomia on a single dosing day. The geometric mean AUC(∞) was 286.40, 493.81, and 816.47 min*ng/mL for the 0.5 mg and 1.0 mg sublingual doses, and the 1.0 mg IV dose, respectively. Compared to IV administration, the 1.0 mg sublingual dose produced 0.60 (90% CI: 0.55–0.66) of the overall concentration of atropine over time (AUC(∞)). CONCLUSION: Sublingual atropine sulfate 1% ophthalmic solution may be an alternative formulation and route of administration combination which expands the capacity and dosing options of atropine as a nerve agent MCM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13181-021-00873-0.