The methyl CpG-binding protein 2 (MECP2) gene has copy-number gain in a number of human cancers and functions as an oncogene through an unusual epigenetic mechanism. We explored the possibility that MECP2 might be a therapeutic target and whether its epigenetic mode of action is amenable to specific therapy. Constitutively expressed or inducible lentiviral short hairpin RNA directed at MECP2 in human triple-negative breast cancer (TNBC) cell lines with high or low level of MECP2 protein were grown as xenografts to assess oncogene addiction. We next evaluated the effect of DNA methylation inhibitors or histone deacetylase inhibitors on monolayer or soft agar growth of isogenic human mammary epithelial cells with or without MECP2 overexpression and xenograft growth of MECP2-dependent or MECP2-independent human TNBC or lung cancer cell lines. We then investigated the mechanism of MECP2-induced activation of the MAPK pathway and assessed the effect of drug treatment. Human cancer cell lines with MECP2 overexpression show MECP2 dependence, and epigenetic drugs are effective in these models. Activated RAS and other activators of the MAPK pathway caused resistance to these therapies, giving insight into their novel mode of action and demonstrating specificity. The kinase PAK3 is important for MECP2-mediated induction of the MAPK pathway, is modulated by epigenetic drugs affecting MECP2 action as expected, and may itself be a therapeutic target for MECP2-driven cancers. These preclinical studies show that tumors overexpressing MECP2 might benefit from epigenetic therapy targeting MECP2 function and demonstrate a novel mechanism of action for these drugs.

Supplementary Figure S2 shows the effect of MECP2 overexpression on genes that may mediate the oncogenic activity of MECP2 and their properties

Supplementary Figure S1 shows the effect of HDAC inhibitors on MECP2 protein levels

of the study: MECP2 amplification plays a role similar to activated oncogenic RAS in tumorigenesis, activating the MAPK and PI3K signaling pathways. MECP2 amplification and RAS mutations are mutually exclusive across human cancers; solid tumors such as high-grade serous ovarian cancer (HGSOC), triple-negative breast cancer (TNBC) and lung adenocarcinoma have 38%, 33% and 29% rates of MECP2 amplification respectively. The MECP2 protein has DNA- binding activity which is required for oncogenic transformation and binding is dependent on cytosine methylation. Therefore, MECP2 DNA binding may be targeted by inhibiting cytosine methylation with DNMTi. Additionally, since MECP2 protein requires HDAC activity for its stability, treatment with either HDACi and/orDNMTi could be therapeutic strategies in MECP2- dependent tumors. Experimental Procedures and Results: The MDAMB468 (TNBC) cell line was modified stably with a doxycycline-inducible shRNA construct targeting the 3’UTR of MECP2 (shMECP2-3’UTR) and growth was dependent on MECP2 in culture. The cells were then implanted in nude mice subcutaneously and tumors allowed to reach approximately 150 mm3 before shRNA was induced. Tumor growth in doxycycline-treated mice immediately ceased whereas tumors in vehicle-treated mice continued to grow, showing dependence on MECP2 activity in vivo. To verify that this was an on-target effect, the modified cell line was further infected with lentiviruses carrying both splicing isoforms of MECP2 without the 3’UTR shRNA target. This reconstitution allowed growth as a xenograft at a similar rate regardless of shRNA induction, confirming inhibition was on-target. We then assessed the therapeutic efficacy of HDACi and DNMTi alone and in combination in xenograft (for TNBC and lung) or PDX (HGSOC) models of MECP2-dependent and independent tumors. To determine if growth inhibition was an on-target effect of these drugs, MDAMB468 was stably transduced with activated KRAS or GFP as a control and was injected in nude mice. Activated KRAS rescued the epigenetic drug sensitivity of xenografts, demonstrating that these drugs inhibit tumor growth by preventing the ability of MECP2 to induce RAS-activated growth factor pathways. The antitumor effects of the commonly used epigenetic drugs are thought to result from cell reprogramming through re-expression of tumor suppressors that had been silenced by hypermethylation or by repressive chromatin marks. However, in this context, we show that these epigenetic drugs can act directly by interfering with the oncogenic activities of MECP2, culminating in inhibition of key growth factor pathways for transformation. Targeting MECP2 function with epigenetic drugs provides an unanticipated therapeutic opportunity for treating the subset of tumors that overexpress MECP2. Manish Neupane, Mukesh Kumar, Malela M. Werner, Colleen M. Donnelly, Alsu Ibragimova, Brian Curcio, Inna Chervoneva, Irina Vasilevskaya, Daniel P. Silver. Epigenetic drugs inhibit the oncogenic activity of methyl CpG binding protein 2 (MECP2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7187.

Background: Structural variation is a hallmark of breast cancer. Previous single-cell genomic analysis of 10 untreated early-stage primary TNBC tumors identified only one or two subclones per tumor, found that copy number variation (CNV) occurs early in the evolution of the tumor, and asserted that CNV changes do not contribute to genomic variation at later time points of a tumor’s growth. This PCNE model (punctuated copy number evolution) is analogous to the big-bang theory of colorectal cancer. However, cancer exists in a dynamic environment, and systemic chemotherapy provides evolutionary pressure, making stasis quite unlikely. An investigation of late-stage cancers could shed light on CNV evolution. Methods: We performed a longitudinal, prospective observational study of over 130 patients with metastatic breast cancer, with regular follow-up and detailed treatment histories. CTCs were enumerated by CellSearch, and samples with >20 CTCs in 7.5ml of whole blood were further processed for single CTC isolation via DEPArray, single-cell library construction, and whole-genome sequencing. CNV counts for each cell were obtained using previously published binning methods (Ginkgo). Three mathematical models were used to evaluate the evolution of CNVs: a punctuated model, a gradual model, and a gradual-on-punctuated hybrid model. Results: In total, 150 patient-years of data were collected. Among samples with sufficient CTC counts for study inclusion, a total of nine patients and 376 cells were isolated for sequencing. A mean sequencing depth of 0.8× was achieved from each single cell. The degree of CNV heterogeneity varies from patient to patient. CNV changes occurred over time in a gradual manner on a background of punctuated changes. The adjusted R2 value for the punctuated model was 0.46, the adj R2 value for the gradual model was 0.76, and the hybrid model achieved an adj R2 of 0.99. Patient samples with large CNV heterogeneity across single cells tend to be from patients with longer treatment histories and from the last blood draw (i.e., the blood collection closest to patient’s point of death). We also identified multiple whole-genome duplication (WGD) events from the same patient, in contrast to previous findings that WGD events are usually early clonal events in most instances. Finally, X loss events are among the most frequent chromosomal aberrations identified in CTCs (in up to 80% of CTCs in some patients), which supports previous literature on the loss of the Barr body in hastening metastatic spread. Discussion and Conclusion: In contrast to the previous study, our findings support a gradual-on-punctuated hybrid model, and this model can explain how copy number changes can be a source from which tumors gain resistance to systemic therapies. The model explains how CNV heterogeneities are seen in patients with longer treatment histories, suggesting that CNV continues to be a source of genetic variation at the metastatic stage of the disease course. Citation Format: Weelic Chong, Rui Luo, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Ronald Myers, Mariel Becker, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-02.

Background: Although different patterns and changes in DNA methylation play an important role in cancer progression and tumor subtype differentiation, methylation remains relatively less understood in the context of tumor heterogeneity. Intra-tumor heterogeneity influences chemotherapy response, resistance development, and metastatic progression, and can be identified with liquid biopsy, a non-invasive approach to monitor cancer progression and provide predictive information. In this study, we sequenced circulating tumor cells (CTCs) to interrogate whole-genome methylation at single-cell level and single-base resolution. Methods: We enumerated CTCs in 7.5ml of whole blood samples from over 130 metastatic breast cancer patients using CellSearch, and selected blood samples with more than 20 CTCs to be further processed by DEPArray. After single-cell library construction using the Swift Accel-NGS Adaptase Module, whole-genome bisulfite sequencing (WGBS) was performed. The sequencing data were then aligned to the reference genome, and the methylation information was extracted by Bismark. We compared methylation profiles between CTC and WBC samples from each patient to identify differentially methylated regions (DMRs) using Methylkit. Multiple-comparison was conducted by SMART2 to identify DMRs within CTCs. Heatmap was plotted based on the regional methylation rates of DMRs. CpG and genic annotations were performed by annotatr. The pathway and function enrichment analyses (KEGG and GO) were conducted using clusterProfiler. Genomic region-based enrichment was conducted using LOLA. t-SNE clustering was used to investigate intra-patient heterogeneity. Results: A total of 376 cells from nine patients that passed our selection criteria were isolated for WGBS. The mean sequencing depth of all single cells was 0.8×, and an average mapping rate of 54% was achieved. We first compared CTCs and WBCs, and observed a global hypo-methylation pattern in CTCs (average methylation rate 68.8% in CTCs vs. 76.7% in WBCs). Moreover, approximately 2,000 highly differentially methylated regions were found in each patient, with hundreds of hypo- or hyper-methylated genes related to these DMRs. Hypo-methylated DMRs showed genomic region-based enrichment in breast-, prostate-, and fibroblast-related region sets. Then, we investigated methylation heterogeneity within CTCs, where t-SNE clustering identified four different subsets in one representative patient. About 1,500 hypo-methylated DMRs were found, differentiating those four subgroups. KEGG pathway analysis indicated enrichment in the Rap1 signaling pathway and focal adhesion. GO enrichment analysis highlighted the regulation of GTPase activity and membrane potential. Conclusions: Our study identified differentially methylated regions in CTCs of metastatic breast cancer patients, and demonstrated intra-patient heterogeneity based on cellular methylation information. Future studies are warranted to validate our findings and explore their biological mechanisms and clinical relevance. Citation Format: Rui Luo, Weelic Chong, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Kevan Ip, Ronald Myers, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Whole-genome bisulfite sequencing of single circulating tumor cells identifies cellular methylation heterogeneity in metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-05.

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

e18562 Background: We evaluated the utility of an electronic medical record (EMR)-based risk stratification tool of social determinants of health (SDOH), financial toxicity and social barriers to identify metastatic breast cancer (MBC) patients (pts) at risk of inequity in cancer care. Methods: We enrolled 40 female pts within 60 months (mo) of a MBC diagnosis (dx). Pts had an encounter with social work (SW) at baseline that includes: 1) Eleven SDOH domains 2) Financial toxicity 3) CMS validated psychosocial assessment (Ax). A 2 nd Ax within 3 mo was optional. SW documented interventions offered to pt. Retrospective chart review included pt demographics, treatment (Tx) and clinical outcomes. Descriptive statistics were calculated for pt demographics, clinical outcomes, SDOH acuity scores, and interventions offered by SW. Clinical outcomes were summarized with frequency counts and percentages. All analyses were performed with SAS 9.4 (SAS Institute Inc., Cary, NC). Interactions between race and SDOH risks were explored by logistic regression analysis but limited by sample size constraints. Results: Of 40 enrolled pts, 39 completed baseline and 24 completed 2 nd Ax. Mean age 61 y (range 31-85); 20 White (W) and 19 Minority (MIN) [18 Black, 1 Asian]. Median time from MBC dx to baseline Ax was 7.3 mo (2.7-19.5) for MIN pts and 16.2 mo (3.8-28.8) for W pts. Mean time for SW to complete baseline Ax was 32.7 min and 2 nd Ax was 26.7 min. Of 11 SDOH domains, MIN pts were more likely at risk in 6 domains compared to W pts: Financial Resource Strain (MIN 63% v W 45%), Housing Stability (MIN 16 % v W 0%), Depression (MIN 11% v W 5%), Physical Inactivity (MIN 84% v W 65%), Transportation Needs (MIN 26% v W 10%), and Food Insecurity (MIN 42% v W 10%). W pts were at greater risk compared to MIN pts for Tobacco Use (MIN 47% v W 60%) and Lack of Social Connections (MIN 53% v W 65%). Both groups reported similar risk for Stress (MIN 74% v W 70%). Every pt was at risk in at least one SDOH domain. Eleven (58%) MIN v 5 (25%) W pts were at risk in the 5 or more SDOH domains. At the baseline Ax, the most common interventions provided by SW: financial (MIN 36.8% v W 25.0%), food (MIN 42.1% v W 0%), transportation (MIN 21.1% v W 0%), and mental health referral (MIN 5.3% v W 15.0%). MIN pts were more likely to have delay in starting 1 st line Tx > 21 d with a median time of 29 mo (range 20-52) v 19 mo (range 9-54.5) for W pts. Within 3 months of a referral, 13 MIN (68.4%) v 6 W (30%) pts had a palliative care encounter, and 6 MIN (31.6%) v 5 W (40%) pts met with a genetic counselor. On regression analysis, no associations were detected between SDOH risk or SDOH risk-by-race interactions with time-to 1 st line Tx or time to palliative care encounter. Conclusions: In our catchment, MIN pts with a MBC dx were more likely to face multiple SDOH risks compared to W pts. The impact of interventions to address SDOH and social barriers on cancer care outcomes should be evaluated in a larger cohort of MBC patients determined to be at risk.

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).

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)

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.

BACKGROUND The PARP inhibitors (PARPis) olaparib and talazoparib are currently approved for the treatment of deleterious germline BRCA1/2-mutated (gBRCA+) metastatic breast cancer (MBC). These approvals were based on improvements in progression-free survival (PFS) observed in two randomized controlled trials (RCTs). Other PARPis, such as veliparib and niraparib, have also been studied. We conducted this meta-analysis of RCTs to assess the PFS and overall survival (OS) benefits of PARPis in gBRCA + MBC. METHODS We performed a systematic search for RCTs using the Cochrane Library, PubMed, Embase, and Web of Science databases up to March 2021. Only phase II and III RCTs evaluating PFS and OS for PARPis alone or in combination with chemotherapy (CT) and comparing the findings with standard CT were included in this meta-analysis. Pooled analysis of the hazard ratio (HR) was performed with RevMan v5.4 using a random effects method. RESULTS Five RCTs with a total of 1563 BRCA-mutated MBC patients were included in this meta-analysis. Temozolomide was used in the treatment arm in the BROCADE trial. Since temozolomide has limited effects on breast cancer, this arm was excluded from our meta-analysis. A statistically significant increase in PFS was observed in the PARPi group compared to the standard CT group (HR, 0.64; 95% CI, 0.56-0.74; P < 0.00001). However, the differences in OS did not reach statistical significance (HR, 0.89; 95% CI, 0.77-1.02; P = 0.09). Moreover, differences were not observed in the adverse event profile between the two groups (odds ratio, 1.18; 95% CI, 0.84-1.64; P = 0.33). CONCLUSION The results of our meta-analysis confirm the previously reported PFS benefit of PARPis over standard CT. PARPis lead to superior PFS in gBRCA + MBC when used alone or in combination with standard CT. The OS benefit is similar between PARPis and standard CT. Ongoing trials are evaluating the benefits of PARPis in early stage gBRCA + BC.

Previously undescribed molecular mechanisms of resistance will emerge with the increased use of cyclin-dependent kinase 4/6 inhibitors in clinical settings. To identify genomic aberrations in circulating tumor DNA associated with treatment resistance in palbociclib-treated metastatic breast cancer (MBC) patients, we collected 35 pre- and post-treatment blood samples from 16 patients with estrogen receptor-positive (ER+) MBC, including 9 with inflammatory breast cancer (IBC). Circulating cell-free DNAs (cfDNAs) were isolated for sequencing using a targeted panel of 91 genes. Our data showed that FBXW7 and CDK6 were more frequently altered in IBC than in non-IBC, whereas conversely, PIK3CA was more frequently altered in non-IBC than in IBC. The cfDNA samples collected at follow-up harbored more mutations than baseline samples. By analyzing paired samples, we observed a higher percentage of patients with mutations in RB1, CCNE1, FBXW7, EZH2, and ARID1A, but a lower proportion of patients with mutated TSC2 at the post-treatment stage when they developed progression. Moreover, acquisition of CCNE1 mutations or loss of TSC2 mutations after treatment initiation conferred an unfavorable prognosis. These data provide insights into the relevance of novel genomic alterations in cfDNA to palbociclib resistance in MBC patients. Future large-scale prospective studies are warranted to confirm our findings.

Metastases to the gastrointestinal tract (GIT) from breast carcinoma are rare, detected in approximately <5% of all breast cancer patients. Invasive lobular carcinoma (ILC) is the most common histological type of breast cancer to metastasize to the GIT. We report a case of abdominal recurrence of ILC of the breast causing intra-abdominal contracture leading to extrinsic compression of the duodenum and periampullary biliary tree. Four years after the patient’s diagnosis of a left breast pT1c, pN2, cM0 invasive lobular breast cancer, she presented with liver function tests consistent with biliary obstruction, and there was concern for a periampullary malignancy. Definitive diagnosis was achieved at laparotomy. This case demonstrates the importance of considering metastatic breast cancer as a potential cause of GI symptoms and radiological abnormalities affecting any part of the GIT of women with a previous history of lobular breast cancer. This case also highlights the effectiveness of chemotherapy in improving the survival and quality of life of these patients. Early recognition of this scenario enables prompt initiation of systemic therapy and avoids unnecessary surgical treatment. Despite the rarity, such patients will be encountered in clinical practice given the high prevalence of breast cancer. Moreover, the fact that the presenting symptoms of GI metastasis from breast cancer are usually not specific to the origin and mimic a primary intestinal disorder, health-care professionals beyond oncologists, especially gastroenterologists and primary care physicians, should be aware of this entity.

Abstract Background: PARP Inhibitors (inh), Olaparib and Talazoparib, are approved for deleterious germline BRCA mutated (gBRCA+) metastatic breast cancer (MBC). This approval was based on a progression-free survival (PFS) benefit seen in two randomized controlled trials (RCTs). Other PARP inh such as Veliparib and Niraparib have also been studied. We conducted this meta-analysis of RCTs aiming to assess PFS and OS of PARP inh in gBRCA+ MBC. Methods: We performed a systematic search for RCTs using Cochrane Library, PubMed, Embase, and Web of Science up to June 2020. Only phase II and III RCTs evaluating PFS for PARP inh alone or in combination with chemotherapy (CT) to standard CT were eligible for this meta-analysis. The pooled analysis of hazard ratio (HR) was performed with RevMan 5.4 software using random effect model. Results: A total of 5 RCTs including 1563 patients were included in this meta-analysis. Baseline study characteristics are listed in Table 1. The pooled HR for PFS was 0.77 (95%CI 0.55-1.09) and the pooled HR for OS was 0.96 (95%CI 0.80-1.16). The only statistically significant adverse event more common in PARP inh group was anemia (Odds Ratio, 3.01; CI95%, 1.14-7.93, P=0.03). (Table 2) Conclusion: The results of our meta-analysis confirmed the previously reported PFS benefit of PARP inh either alone or with standard CT when compared to standard CT alone in gBRCA+ MBC. PARP inh when combined with temozolomide did not show PFS benefit. OS benefit is not seen with PARP inh alone or with standard CT. Ongoing trials are evaluating the benefit of PARP inh in early stage gBRCA+ BC. Table 1: Baseline study characteristics as experimental group vs. control groupVCP: veliparib with carboplatin/paclitaxelVT: veliparib with temozolomidePCP: placebo plus carboplatin/paclitaxelNA: Not available** Subgroup germline BRCA+ pts (n=37, 13 vs 24) is used for PFS and OS analysis in this study.TrialsSample sizeExperimental groupControl groupECOG PS % (0/1/2)Previous CT no. (%)Previous platinum no. (%)Median PFS (mo)Median OS (mo)OlympiAD302 (205 Vs 97)OlaparibStandard CT (Capecitabine or Eribulin orVinorelbine)72.2/28.8/0 Vs 63.9/36.1/0146 (71.2) Vs 69 (71.1)60 (29.3) Vs 26 (26.8)7.0 Vs 4.219.3 Vs 17.1EMBRACA431 (287 Vs 144)TalazoparibStandard therapy53.3/44.3/2.1 Vs 58.3/39.6/1.4176(61.4) Vs 90(62.5)46 (16.0) Vs 30 (20.8)8.6 Vs 5.619.3 Vs 19.5BROCADE284 (97 Vs 99)VCPPCP92/5 Vs 93/623(23.7) Vs 37(37.4)NA14.1 Vs 12.328.3 Vs 25.9(94 Vs 99)VTPCP91/3 Vs 93/628(30.6) Vs 37 (37.4)NA7.4 Vs 12.319.1 Vs 25.9BROCADE-3509 (337 Vs 172)VCPPCPNA19%8%14.5 Vs 12.633.5 Vs 28.2SWOG S1416 **321Veliparib + CisplatinPlacebo + Cisplatin69/41Vs 57/4351(32) Vs 49(31)14(9) Vs 18(11)6.2 Vs 6.414.2 Vs 14.6BRAVO-NiraparibPhysician choice CTStudy was prematurely closed after an interim analysis showed too many patients were not completing the necessary assessments in the control arm, and it was no longer suitable as a registration trial. Table 2: Side effect profile of PARP inh arm vs. CT armNA: Not available**side effect includes all 154 pts in experimental arm and 149 in control armAdverse Events (%)OlympiAD EMBRACABROCADE(VCP & VT Vs PCP)BROCADE-3SWOG S1416**Odds Ratio for Adverse EventsAnemia Grade ≥333 (16.1) Vs 4 (4.4)112 (39.2) Vs 6 (4.8)16 (17.2) &7 (7.5) Vs 17 (17.7)91(27) Vs 29(17)35(23) vs 11(7)3.01 (1.14,7.93) P=0.03Neutropenia Grade ≥319 (9.3) Vs 24 (26.4)60 (20.9) Vs 44 (34.9)52(55.9) &34(36.6) Vs 53 (55.2)175(52) Vs 86 (50)71(46) vs 29(19)0.80 (0.40, 1.62) P=0.53Leukopenia Grade ≥37(3.4) Vs 9 (9.9)19 (6.6) Vs 11(8.7)15(16.1) &11(11.8) Vs 11 (11.5)NA42 (27) Vs 11 (7)1.19 (0.42,3.38) P=0.74Nausea/Vomiting Grade ≥30 Vs 1 (1.1)8 (2.7) Vs 4(3.2)2(2.2) &5(5.4) Vs 3 (3.1)NA28(18) Vs 16(10)1.45 (0.85,2.46) P=0.17Diarrhea Grade ≥31 (0.5) Vs 02 (0.7) Vs 7 (5.6)4(4.3) & 2(2.2) Vs 7(7.3)NANA0.33 (0.13, 0.85) P=0.02Fatigue Grade ≥36 (2.9) vs 1 (1.1)5 (1.7) Vs 4 (3.2)5(5.4) &3(3.2) Vs 8(8.3)NA8(5) Vs 9(6)0.73 (0.40,1.33) P=0.30Treatment discontinuation due to any AE10 (4.9) Vs 7(7.7)17 (5.9) Vs 11 (8.7)NANA0.72 (0.39,1.34) P=0.30 Citation Format: Ranju Kunwor, Daniel P Silver, Saveri Bhattacharya, Rebecca Jaslow, Frederick Fellin, Ana Maria Lopez, Maysa M Abu-Khalaf. PARP inhibitors for treatment of BRCA positive metastatic breast cancer: A systematic review and meta-analysis [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 PS10-41.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1 , LAMA3 , FLG2 , UGT2B4 , STK33 , BRCA2 , ACP4 , PIK3CA , and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3 , IL12RB2 , and PDE4B may potentially generate ITH in IBC.

Discordance between HER2 expression in tumor tissue (tHER2) and HER2 status on circulating tumor cells (cHER2) has been reported. It remains largely underexplored whether patients with tHER2−/cHER2+ can benefit from anti-HER2 targeted therapies. cHER2 status was determined in 105 advanced-stage patients with tHER2− breast tumors. Association between cHER2 status and progression-free survival (PFS) was analyzed by univariate and multivariate Cox models and survival differences were compared by Kaplan–Meier method. Compared to the patients with low-risk cHER2 (cHER2+  < 2), those with high-risk cHER2 (cHER2+  ≥ 2) had shorter survival time and an increased risk for disease progression (hazard ratio [HR] 2.16, 95% confidence interval [CI] 1.20–3.88, P = 0.010). Among the patients with high-risk cHER2, those who received anti-HER2 targeted therapies had improved PFS compared with those who did not (HR 0.30, 95% CI 0.10–0.92, P = 0.035). In comparison, anti-HER2 targeted therapy did not affect PFS among those with low-risk cHER2 (HR 0.70, 95% CI 0.36–1.38, P = 0.306). Similar results were obtained after adjusting covariates. A longitudinal analysis of 67 patients with cHER2 detected during follow-ups found that those whose cHER2 status changed from high-risk at baseline to low-risk at first follow-up exhibited a significantly improved survival compared to those whose cHER2 remained high-risk (median PFS: 11.7 weeks vs. 2.0 weeks, log-rank P = 0.001). In advanced-stage breast cancer patients with tHER2− tumors, cHER2 status has the potential to guide the use of anti-HER2 targeted therapy in patients with high-risk cHER2.

Background: Palbociclib is the first-in-class cyclin dependent kinase (CDK) 4/6 inhibitor approved for the treatment of patients (pts) with hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer (MBC). Despite an initial response, pts eventually develop resistance to therapy; however, the underlying molecular mechanisms remain elusive. This study aimed to identify genomic aberrations in circulating tumor DNA (ctDNA) associated with treatment resistance and disease progression in MBC pts receiving palbociclib. Methods: Blood samples were collected from pts with HR+/HER2− MBC who were treated with palbociclib plus endocrine therapies (letrozole or fulvestrant). Circulating cell-free DNAs were isolated from plasma and assayed by targeted-capture next-generation sequencing using an in-house developed panel that covers the exons in 92 breast cancer-related genes. Unique molecular index (UMI)-based sequencing was performed using the Illumina HiSeq 4000 system. Progression free survival (PFS) differences between pts with different mutation status were compared using the log-rank test. The Cox proportional hazards model with time-dependent covariates was used to evaluate associations between mutations and PFS. Results: The study included 16 pts with a median age of 54.7 years (range: 32.6-74.8 years). A total of 42 blood samples were collected, including 16 baseline samples before the initiation of palbociclib +/- endocrine therapies (within 1.5 months prior to initiation), 10 samples during treatment (1.0-3.9 months after initiation), and 16 after treatment completion (within 1.2 months after completion). During a median follow-up of 23.7 weeks (range: 6.0-99.6 weeks), 8 pts had disease progression at initial blood collection after treatment initiation, and 11 had disease progression at final blood collection during follow-up. Among the 3 pts without progression, 2 were lost to follow-up and 1 had a change in regimen due to adverse events. Mutations were detected in 77 out of 92 genes. Mutations were identified for PIK3CA, ALK, ERBB2, NOTCH1, IDH1 in the baseline samples of 7, 3, 7, 6, and 12 patients, respectively. A shorter time to progression was observed in the pts with PIK3CA, ALK, ERBB2, or NOTCH1 mutations (log-rank P = 0.011, 0.016, 0.057, and 0.059, respectively), whereas those pts with IDH1 mutations had a prolonged PFS (log-rank P = 0.005). Similar results were obtained when the events and survival time at the final blood collection were used for analyses (log-rank P = 0.007, 0.004, 0.046, 0.023, and 0.001 for PIK3CA, ALK, ERBB2, NOTCH1, and IDH1 mutations, respectively). To analyze the association between a given mutation and disease progression, we separated pts into four risk groups according to the status change of the mutation prior to and after treatment: Group 1, persistent absence of the mutation; Group2, loss of the mutation after treatment; Group 3, acquisition of the mutation after treatment; Group 4, persistent presence of the mutation. Longitudinal analyses showed that PIK3CA E542K, ERBB2 P1035H, and NOTCH1 K1069Q mutations were associated with unfavorable survival. For PIK3CA E542K mutation, pts in Group 2 had an increased risk for progression, as compared with those in Group 1 (hazard ratio [HR] =9.90, P = 0.110). For ERBB2 P1035H and NOTCH1 K1069Q mutations, when compared with pts in Group 1, those in Group 4 had the highest risk of progression (HR = 17.24, P = 0.049; HR = 19.17, P = 0.043, respectively), whereas pts in Group 2 had a non-significant slightly increased risk. Conclusions: Results of this study show that genomic aberrations in ctDNA were associated with progression in MBC pts receiving palbociclib-containing regimens. Further research is needed to validate these findings in larger independent studies. Citation Format: Maysa M Abu-Khalaf, Chun Wang, Zhong Ye, Zhenchao Zhang, Daniel Silver, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Terrence Cescon, AnaMaria Lopez, Ronald Myers, Qiang Wei, Bingshan Li, Hushan Yang. Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-27.