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Background: Treatment of estrogen receptor (ER)-positive breast cancer with selective estrogen receptor degraders (SERDs) frequently results in the loss or reduction of ER expression. Whether these changes are due to on-target effects of SERDs degrading ER or arise as a mechanism of tumor resistance with associated changes in cellular phenotypes remains unknown. It is critical to distinguish between these possibilities to accurately assess treatment response and determine the most appropriate subsequent therapy. To this end, we created and conducted molecular analyses on patient-derived organoid cultures from post-treatment tissue in patients receiving neoadjuvant SERD therapy for early-stage ER+ breast cancer in the I-SPY2 Endocrine Optimization Protocol (EOP). Methods: The I-SPY2 EOP study is a prospective, randomized substudy within the I-SPY TRIAL testing the oral SERD amcenestrant alone or in combination with letrozole or abemaciclib in stage 2/3 ER+ Her2-negative breast cancer. Enrollment is ongoing, with patients receiving amcenestrant neoadjuvantly for 6 months until the day before surgery. Tumor tissue is collected at baseline, 3 weeks, and at surgery. Organoids were generated from post-treatment surgical samples. Organoid cultures were optimized based on established methods (Dekkers et al., Nature Protocols, 2021) to assess ER levels and activity. Pre- and post-treatment tissue samples were also assessed for ER, PR, Ki67, and GATA3, a luminal marker and transcription factor that is functionally linked with ER, via immunohistochemistry. Results: In 7 patients with both pre- and post-treatment tissue samples including fresh surgical samples for organoid generation, the ER in baseline tumor tissue was >=90% in all patients, PR ranged from 40-90%, and Ki67 ranged from 5-30%. In post-treatment surgical tissue from these cases, ER ranged from 0-30%, PR from 0-50%, Ki67 from < 1%-10%, and GATA3 was positive in 5 of 5 cases tested to-date. The creation of organoids from residual disease at surgery was attempted for these 7 patients, with organoids successfully propagated in 5 cases thus far. 3 of 5 organoid cultures were ready for analysis and in all cases strong ER and PR expression in organoids was observed after culture for > 1 month in the absence of amcenestrant. Detailed gene expression profiling (including Mammaprint/Blueprint) and gene set enrichment analyses (GSEA) to assess for intrinsic breast cancer subtype and ER activity in each sample and corresponding organoid culture are in progress and will be reported with the full dataset. Conclusion: Patient-derived organoid culturing of residual disease after neoadjuvant endocrine therapy is feasible. Neoadjuvant treatment with a SERD can render ER and PR low or absent at the time of surgical resection, which does not necessarily imply the presence of endocrine therapy resistant disease. The use of organoids and additional IHC markers (GATA3) demonstrate that receptor negativity may be an indicator of the drug hitting its target, suggesting ER signaling is still intact. In general, patient-derived tumor organoid cultures modeling residual disease states can be a useful adjunct to existing methods used to monitor the effects of neoadjuvant endocrine therapy and is being explored in the I-SPY EOP trial. Citation Format: Jennifer Rosenbluth, Christopher J. Schwartz, Tam Binh Bui, Shruti Warhadpande, Pravin Phadatare, Sigal Eini, Michael Bruck, Julissa Molina-Vega, Kami Pullakhandam, Nicole Schindler, Lamorna A. Brown Swigart, Christina Yau, Gillian Hirst, Rita Mukhtar, Karthik V. Giridhar, Olufunmilayo I. Olopade, Kevin Kalinsky, Cheryl A. Ewing, Jasmine M. Wong, Michael D. Alvarado, Laura Van’t Veer, Laura J. Esserman, Jo Chien. Characterization of residual disease after neoadjuvant selective estrogen receptor degrader (SERD) therapy using tumor organoids in the I-SPY Endocrine Optimization Protocol (EOP) [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 P3-09-01.

Background: The IDEAL trial showed no significant benefit of 5 years extended endocrine therapy (EET) using letrozole in postmenopausal patients with hormone receptor positive (HR+) breast cancer (BC) versus 2.5 years. Genomic classifiers may assist with treatment decisions by predicting EET benefit. The 70-gene MammaPrint (MP) test classifies tumors as having a higher or lower risk of distant metastasis in HR+ early-stage BC. A MP lower risk result can be further classified as either Ultra-Low risk or Low risk of distant metastasis. In the NSABP B42 trial, MP predicted a statistically significant absolute benefit from EET in patients with a MP Low Risk result. Here, we aimed to determine the utility of MP in identifying a subgroup of patients enrolled in the IDEAL trial for which 5 years of EET is beneficial compared to 2.5 years. Methods: A total of 869 patients had available primary tumor tissue for testing. MP results were available for 545/869 patients, of which 515 did not have an event at 2.5 year after randomization and were used for our analyses. The MP result for each patient was calculated by Agendia while blinded to patient clinical outcomes. The primary endpoint was distant recurrence (DR). Secondary endpoints were recurrence free interval (RFI) and breast cancer free interval (BCFI) as defined by STEEP criteria. Patients were classified as higher risk (score -1.000 - 0) or lower risk (score 0.001 - 1.000). Lower risk tumors were further classified as either MP Ultra-Low (score > 0.355) or MP Low Risk (score ≥ 0.001, ≤ 0.355). Likelihood ratio test based on stratified Cox proportional hazards (PH) model were used to evaluate treatment by risk group interaction. Differences in endpoints between treatment groups were assessed by stratified log-rank tests. Hazard ratios (HR) and 95% Confidence Intervals (CI) were computed based on the stratified Cox PH model. Results: The clinical characteristics of the 515 IDEAL samples with a MP result were comparable to the whole IDEAL cohort (n=1820). Within the 2.5 year EET group, 50.6% (n=134) were MP higher risk and 49.4% (n=131) MP lower risk, of which 14.5% (n=19/131) were MP Ultra-Low. Within the 5 year EET group, 50.0% (n=125) were MP higher risk and 50.0% (n=125) MP lower risk, of which 11.2% (n=14/125) were MP Ultra-Low. Among patients with MP lower risk tumors, 5 years vs. 2.5 years of EET resulted in a significant absolute benefit of 9.8% for DR (HR=0.42, [95% CI 0.174-0.996]), 9.8% for RFI (HR=0.43, [95% CI 0.198-0.934]), and 8.8% (HR=0.53, [95% CI 0.264-1.055]) for BCFI, whereas patients with MP higher risk tumors did not derive significant benefit (Table 1). Within the MP lower risk group, 5 year vs 2.5 year EET benefit was more pronounced in MP Low tumors, which exhibited a significant benefit of 10.1% for DR (HR=0.32, [95% CI 0.116-0.866]), 11.7% for RFI (HR=0.35, [95% CI 0.147-0.824]), and 9.7% for BCFI (HR=0.48, [95% CI 0.225-1.015]); MP Ultra Low tumors did not derive significant benefit. Treatment-by-risk group interaction was statistically significant for RFI. Conclusion: A significant EET benefit was observed for MammaPrint lower risk tumors but not for MP higher risk tumors. MammaPrint Low tumors exhibited the largest absolute benefit of 5 years of EET compared to 2.5 years. Consistent with the findings in the NSABP B42 trial, the results from this second randomized trial provide clinically meaningful implications in patient selection for extended endocrine therapy. Table 1. IDEAL: 10-year outcome analysis comparing 5 years vs. 2.5 years of EET using letrozole stratified by MP risk. **MammaPrint Lower Risk & Higher Risk (n=515) and *** MammaPrint Low Risk & High Risk (n=482) Citation Format: Gerrit-Jan Liefers, Elma Meershoek-Klein Kranenbarg, Marjolijn Duijm-de Carpentier, Cornelis J.H. van de Velde, Miranda Kleijn, Christa Dreezen, Andrea Menicucci, Laura van’t Veer, William Audeh. Utility of the 70-gene MammaPrint test for prediction of extended endocrine therapy benefit in patients with early-stage breast cancer in the IDEAL Trial [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 GS5-10.

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.

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.

Breast cancer comprises a group of distinct subtypes that despite having similar histologic appearances, have very different metastatic potentials. Being able to identify the biological driving force, even for a subset of patients, is crucially important given the large population of women diagnosed with breast cancer. Here, we show that within a subset of patients characterized by relatively high estrogen receptor expression for their age, the occurrence of metastases is strongly predicted by a homogeneous gene expression pattern almost entirely consisting of cell cycle genes (5-year odds ratio of metastasis, 24.0; 95% confidence interval, 6.0-95.5). Overexpression of this set of genes is clearly associated with an extremely poor outcome, with the 10-year metastasis-free probability being only 24% for the poor group, compared with 85% for the good group. In contrast, this gene expression pattern is much less correlated with the outcome in other patient subpopulations. The methods described here also illustrate the value of combining clinical variables, biological insight, and machine-learning to dissect biological complexity. Our work presented here may contribute a crucial step towards rational design of personalized treatment.

Supplementary Tables S1-S4 from A Cell Proliferation Signature Is a Marker of Extremely Poor Outcome in a Subpopulation of Breast Cancer Patients

Supplementary Figures S1-S10 from A Cell Proliferation Signature Is a Marker of Extremely Poor Outcome in a Subpopulation of Breast Cancer Patients

Methods, Figure legends

Background: In high-risk hormone-receptor (HR)+/HER2- BC patients germline (g) mutations can be found in approximately 14% in BRCA1/2 and in BRCA1/2 and other BC predisposition genes in 20% (Pohl-Rescigno E, et al. JAMA Oncol 2020). In metastatic BC CDK4/6 inhibitors may have greater activity in patients with a BRCA mutation detected in ctDNA (André F, et al. J Clin Oncol 2020). The PENELOPE-B trial did not to show an improved invasive disease-free survival (iDFS) by adding palbociclib to ET in high-risk HR+/HER2- BC (Loibl S, et al. J Clin Oncol 2021). Methods: Blood samples from 898 of 1250 PENELOPE-B patients were available. 445 patients were sampled following a case-cohort design (220 cases defined as patients with any event during follow-up and 225 randomly selected patients without any event [non-cases]) and analyzed for germline variants in BRCA1/2 and 16 non-BRCA1/2 cancer predisposition genes (ATM, BARD1, BRIP1, CDH1, CHEK2, FANCM, MRE11A, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2) by targeted next generation sequencing (NGS). The primary definition of mutational status was the prevalence of a pathogenic mutation (mt) in one or more analyzed BC predisposition genes. Statistical analyses for time-to-event endpoints (iDFS, distant disease-free survival [DDFS], and overall survival [OS]) were based on inverse probability weighting: weighted Cox proportional hazard models and Kaplan-Meier estimates were used. Results: 442 of 445 patients (placebo arm: 104 cases and 105 non-cases; palbociclib arm: 114 cases and 119 non-cases) were successfully analyzed for mutational status. A total of 42 (9.5%) patients (placebo arm: 9.1%; palbociclib arm: 9.9%) carried any mutation. 15 (3.4%) patients had a gBRCA1/2 mt (one of whom carried a gATM mt and one a gCHEK2 mt in addition to gBRCA2 mt) and 29 (6.6%) had mutations in one of the other BC predisposition genes (n=8 CHEK2, n=7 PALB2, n=5 ATM, n=2 RAD50, n=1 for BARD1, FANCM, MRE11A, RAD51C, RAD51D, TP53 and n=1 both RAD51D and BRIP1). The mutational status with respect to all genes analyzed showed no significant correlation to clinical baseline variables. With regard to gBRCA1 and gBRCA2 genes only, the mutational status significantly correlated with age but not with other clinical variables: all 15 (100%) gBRCA mt carriers were younger than 50 years compared to 238 (56%) wildtype (wt) patients (p=0.002). The iDFS rate after 3 years was 80.9% in patients with any mutation and 79.5% in patients without. Mutational status (mt vs. wt) based on all genes analyzed was not prognostic (iDFS: hazard ratio 1.015, 95%CI 0.558-1.784; DDFS: 0.970, 95%CI 0.521-1.758; OS: 0.768, 95%CI 0.274-1.615). Neither the mutated patients had a benefit from palbociclib treatment (palbociclib vs placebo; iDFS: hazard ratio 0.766, 95%CI 0.263-3.022; DDFS: 0.897, 95%CI 0.275-3.489; OS: 0.666, 95%CI 0.063-5.671) nor the wt patients (iDFS: hazard ratio 0.918, 95%CI 0.650-1.303; DDFS: 0.966, 95%CI 0.679-1.393; OS: 0.901, 95%CI 0.573-1.433); interaction tests for treatment arm/mutational status for all time-to-event endpoints were not statistically significant. Analysis in the subgroups of patients by gBRCA1/2 showed similar results but had less statistical power. Conclusions: In this case-cohort analysis of 442 patients enrolled in the PENELOPE-B trial, the detection of BC predisposition genes was lower than expected with 10%. This is probably due to the low rate of gBRCA1/2 carriers (3.4%), which could be influenced by the selection criteria of the trial. Patients with gBRCA1/2 or other BC disposition genes had a comparable outcome to non-carriers in the PENELOPE-B trial. Citation Format: Sibylle Loibl, Jan Hauke, Karen Gelmon, Frederik Marmé, Corinna Ernst, Miguel Martin, Michael Untch, Hervé Bonnefoi, Erik Knudsen, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Sung-Bae Kim, Harry Bear, Nicole McCarthy, Nicholas Turner, Agnieszka Witkiewicz, Federico Rojo, Peter A Fasching, José A García-Sáenz, Catherine M Kelly, Toralf Reimer, Masakazu Toi, Hope S Rugo, Carsten Denkert, Michael Gnant, Andreas Makris, Yuan Liu, Olga Valota, Bärbel Felder, Karsten Weber, Valentina Nekljudova, Eric Hahnen. Germline BRCA1/2 and other predisposition genes in high-risk early-stage HR+/HER2- breast cancer (BC) patients treated with endocrine therapy (ET) with or without palbociclib: A secondary analysis from the PENELOPE-B study [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 P5-13-36.

Background: The CK12 expression signature consists of genes CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, CXCL13 and was previously shown to associate with the presence of T and B cell rich tertiary lymphoid structures in melanoma and other cancers, and with better patient survival independent of tumor staging and treatment. I-SPY 2 is a biomarker-rich, phase II neoadjuvant platform trial for high risk early stage breast cancer. Here we leverage the I-SPY 2 biomarker program to test the hypothesis that this signature associates with sensitivity to neoadjuvant immunotherapies and potentially other classes cancer therapeutics in breast cancer. Methods: Data from 1130 patients across 12 arms of I-SPY2 (control (ctr): 210; veliparib/carboplatin (VC): 71; neratinib (N): 114; MK2206: 93; ganitumab: 106; ganetespib: 93; AMG386: 134; TDM1/pertuzumab(P): 52; H/P: 44; pembrolizumab (pembro): 69; durvalumab/olaparib (durva/olap): 71; pembro/SD101: 72) were available for analysis. Pre-treatment FF (n=987) or FFPE (n=143) biopsies were assayed using Agilent gene expression arrays. Signature scores were calculated as the average expression level across the 12 genes, after z-score normalization. We used logistic modeling to assess association with pCR in each arm in a model adjusting for HR and HER2 (likelihood ratio test, p Citation Format: Hatem Soliman, Denise Wolf, Jo Chien, Christina Yau, Michael Campbell, Mark Magbanua, Ruixiao Lu, Nicholas O'Grady, Lamorna Brown-Swigart, Gillian Hirst, Beverly Parker, Laura Sit, Smita Asare, Doug Yee, Angie DeMichele, Rita Nanda, Lajos Pusztai, Don Berry, Laura Esserman, Laura Van't Veer. Chemokine12 (CK12) tertiary lymphoid gene expression signature as a predictor of response in 3 immunotherapy arms of the neoadjuvant ISPY 2 TRIAL - pembrolizumab with and without SD101, and durvalumab combined with olaparib - and in 9 other arms of the trial including platinum-based and dual-anti-HER2 therapies [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 PD10-07.

Background: Tamoxifen is a standard endocrine therapy for both pre- and postmenopausal ER-positive breast cancer patients. Patients with ER-positive disease have a long-term risk of distant recurrence, thus, long-term follow-up studies are essential to understand true treatment benefit. Clinically used tumor characteristics are prognostic 5-10 years after primary diagnosis, however, whether these characteristics are predictive of long-term tamoxifen benefit is largely unexplored. Therefore, we aimed to determine the long-term tamoxifen therapy benefit by the clinically used tumor characteristics in pre- vs postmenopausal patients in the Stockholm tamoxifen (STO)-trials with 20-years complete follow-up. Methods: Secondary analysis of 1242 ER-positive/HER2-negative patients from the STO-trials, randomized to at least 2 years of 40 mg tamoxifen vs no endocrine therapy (control). Premenopausal lymph node-positive patients were allocated to chemotherapy as standard of care and postmenopausal high-risk patients were further randomized to chemotherapy vs radiotherapy. Tumor immunohistochemical analysis was recently conducted. Complete 20-year follow-up was obtained from Swedish high-quality registries. Long-term distant recurrence-free interval (DRFI) was assessed by multivariable Cox proportional hazard regression and time-varying analysis using flexible parametric modelling. Results: Premenopausal patients showed significantly improved long-term DRFI from tamoxifen vs control if they were lymph node-negative (Hazard Ratio [HR]=0.46; 95% CI, 0.24-0.87), PR-positive (HR=0.61; 95% CI, 0.41-0.91), or of genomic low risk (HR=0.47; 95% CI, 0.26-0.85), see Table. In postmenopausal patients, significantly improved long-term DRFI from tamoxifen vs control was seen for all good prognosis tumor characteristics, i.e. small tumor size (pT≤20mm: HR=0.55; 95% CI, 0.39-0.77), tumor grade 1-2 (HR=0.55; 95% CI, 0.41-0.73), lymph node-negative (HR=0.44; 95% CI, 0.30-0.64), PR-positive (HR=0.60; 95% CI, 0.44-0.80), Ki-67-low (< 15%: HR=0.51; 95% CI, 0.38-0.68), and genomic low risk (HR=0.53; 95% CI, 0.37-0.74), see Table. Also, postmenopausal patients with large tumor size (pT>20mm: HR=0.64; 95% CI, 0.44-0.94) and PR-negative tumors (HR=0.51; 95% CI, 0.32-0.81) showed significant long-term tamoxifen benefit. Time-varying analysis in premenopausal patients indicated that tamoxifen therapy benefit diminished over time. Significant tamoxifen benefit until year 5, 10, and 15 after primary diagnosis was observed for PR-positive, lymph node-negative, and genomic low-risk patients, respectively. Postmenopausal patients had a significant long-term tamoxifen benefit if they had tumors of small or large tumor size, tumor grade 1-2, lymph node-negative status, PR-positive status, low Ki-67 levels, or genomic low risk. Conclusions: This study suggests a differential long-term tamoxifen therapy benefit in pre- vs postmenopausal patients. Clinically defined low-risk postmenopausal patients have long-term tamoxifen benefit, whereas the benefit is absent or diminish over time for premenopausal patients. Improved long-term prognostic and endocrine therapy predictive markers in premenopausal breast cancer patients with poor prognosis and long life-expectancy is needed, which could involve molecular tools. Long-term tamoxifen benefit in premenopausal and postmenopausal breast cancer patients by the clinically used tumor characteristics. Table Multivariable Cox proportional hazard regression analysis of 20-year distant recurrence-free interval (DRFI) for patients with ER-positive/HER2-negative tumors, comparing patients randomized to tamoxifen vs patients randomized to no endocrine therapy (control). Adjusted for age, randomization year, tumor size, tumor grade, lymph node status, PR status, Ki-67 status, chemotherapy, radiotherapy, and type of surgery. Citation Format: Annelie Johansson, Huma Dar, Anna Nordenskjöld, Gizeh Perez-Tenorio, Christina Yau, Christopher C. Benz, Laura J. Esserman, Laura Van’t Veer, Bo Nordenskjöld, Olle Stål, Tommy Fornander, Linda S. Lindström. Differential Long-Term Benefit from Adjuvant Tamoxifen Therapy in Estrogen Receptor (ER)-Positive/Human Epidermal Growth Factor Receptor 2 (HER2)-Negative Premenopausal and Postmenopausal Breast Cancer Patients [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 P2-03-11.

Background: Molecular plasticity of breast cancer is crucial for the development of therapy-resistant disease. In this investigation, we studied changes in molecular signatures between pretherapeutic (pre-Tx) and post-therapeutic (post-NACT) tumor samples from patients included in the PENELOPE-B (NCT01864746) trial. The phase III PENELOPE-B study did not show a significant benefit from palbociclib in women with centrally confirmed HR+, HER2- primary breast cancer without a pathological complete response after taxane-containing neoadjuvant chemotherapy (NACT) and at high-risk of relapse (CPS-EG score ≥3 or 2 and ypN+) (Loibl et al. JCO 2021). However, first translational investigations showed that a small number of patients with a luminal-B tumor subtype, based on absolute intrinsic molecular subtyping (AIMS, Paquet & Hallet, JNCI 2014) subtyping after NACT, had a numerical benefit from post-NACT palbociclib. We have therefore extended the analysis and included a cohort of paired pre-Tx and post-NACT samples. Methods: We investigated gene expression in pre-Tx (n=259) tumor tissue samples using the HTG EdgeSeq Oncology Biomarker Panel including 2549 genes (HTG Molecular Diagnostics Inc.); for the same patients the same panel on post-NACT residual tumor samples were available. The paired samples were selected based on a case-cohort approach. Based on 91 genes of this panel, the AIMS subtype was calculated. In addition, we performed exploratory biomarker analyses to identify genes and gene signatures with prognostic and predictive relevance. After completion of NACT, PENELOPE-B patients were randomized to palbociclib versus placebo in addition to standard endocrine therapy. Results: The prevalence of AIMS subtypes, in particular LumA vs LumB, changed in pre-Tx and post-NACT tumors. In the pre-Tx samples, 115 (44%) and 123 (47%) of tumors had LumA and LumB subtypes, respectively, as expected from a high-risk cohort. However, in the post-NACT samples, LumA tumors were predominant (n=183, 71%) over LumB (n=30; 12%). 78 (30%) and 6 (2%) tumors switched their subtype from LumB to LumA and LumA to LumB, respectively. For further analyses, we compared the groups of low proliferating (LumA and NormL) and high proliferating subtypes (LumB, BasalL and HER2E). In bivariable Cox regression analysis, the grouped pre-Tx and post-NACT AIMS subtypes were independent prognostic factors for iDFS: HR=1.85 (1.16-2.98, p=0.011) for pre-Tx LumB/BasalL/HER2E vs LumA/NormL and HR=2.18 (1.24-3.84, p=0.007) for post-NACT. Similar results were found when adjusted for prognostic clinical factors and for DDFS and OS endpoints although the pre-Tx subtype did not reach significance. These and further Cox models investigating interaction effects show that patients with tumors developing from high (pre-Tx) to low proliferation (post-NACT) had a higher iDFS risk compared to stable low proliferating tumors but a lower iDFS risk compared to stable high proliferating tumors. Neither in the pre-Tx LumB/BasalL/HER2E nor in the pre-Tx LumA/NormL subgroup a benefit from palbociclib was observed. Based on the results of the AIMS subtyping, we extended the exploratory analysis to identify genes that might be involved in the prognostic and predictive effects as well as genes driving the subtype switch. The analysis is ongoing and the relevant genes will be presented at the conference. Conclusions: Our findings show that the switch from high-risk molecular subtypes (in particular LumB) to low-risk subtypes (in particular LumA) is common in neoadjuvant therapy of luminal tumors. The adaptation of luminal high-risk tumors to therapy-induced stress is crucial for the clinical outcome and the results suggest that molecular defined tumor subtypes might not be as stable as originally thought. Citation Format: Carsten Denkert, Frederik Marmé, Miguel Martin, Michael Untch, Hervé Bonnefoi, Sung-Bae Kim, Harry Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Thorsten Stiewe, Karen A. Gelmon, José A. García-Sáenz, Catherine M. Kelly, Toralf Reimer, Erik Knudsen, Nicholas Turner, Federico Rojo, Peter A. Fasching, Julia Teply-Szymanski, Yuan Liu, Masakazu Toi, Hope S. Rugo, Michael Gnant, Andreas Makris, Bärbel Felder, Karsten Weber, Sibylle Loibl. Molecular plasticity of luminal breast cancer and response to CDK 4/6 inhibition - The biomarker program of the PENELOPE-B trial investigating post-neoadjuvant palbociclib [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 PD2-04.

Patients with metastatic breast cancer (MBC) can develop malignant effusions (MEs) when excess fluid and tumor cells accumulate in the pleural or peritoneal space. MEs are associated with significant morbidity and mortality. Tropism of tumor cells in these spaces is poorly understood and tumor cells residing in these fluids are difficult to study with traditional methods due to low abundance. To overcome this limitation and understand the pathobiology of MEs in MBCs, we applied a label-free method to isolate tumor cells based on morphology using an artificial intelligence (AI) model This method enabled isolation of live cells for downstream molecular analysis and characterization of morphological heterogeneity. We collected ME samples from 9 MBC patients with cytology-confirmed MEs, including 3 triple negative and 6 hormone receptor-positive/HER2-negative breast cancers. ME cells were imaged and isolated using a Deepcell AI model trained on a separate cohort of MEs to isolate and enrich tumor cells based on deep learning representations of morphological features. Quantitative morphological features were then extracted from cell images to perform cluster analysis and visualized by dimensionality reduction projections. In parallel, flow cytometry (FC) was used to quantify % EpCAM+/Claudin4+ tumor cells in ME samples. Copy number (CN) analysis was performed before and after enrichment with as few as 200 cells from each sample. Tumor cell fraction (TCF) estimates were calculated from the AI classifier, FC, and CN analyses. CN profiling of malignant cells sorted by the AI classifier revealed genomic alterations common in breast cancer in 7 of 9 patients. We observed increased amplitude of genomic alterations in tumor-enriched vs. unenriched samples, indicating successful tumor cell enrichment in 14 out of 16 samples. We observed a significant correlation in TCF between the AI classifier and FC with a Spearman test (rho=0.568, p0.008). In some cases, the AI classifier reported higher TCF than FC. This could indicate that the AI model detected tumor cells missed by antibody labeling or false positives from morphologically similar cell types, such as macrophages and mesothelial cells. Remarkably, analysis of serial ME samples from the same patient revealed morphological shifts in cell populations which were consistent with changes in TCF by CN analysis. We demonstrate the feasibility of label-free identification and isolation of tumor cells in MEs using an AI classifier. CN profiling of isolated tumor cells verified enrichment from low initial frequencies. Future work will include expression analysis of ME tumor and immune cells, and analysis of organoids derived from MEs. Combining molecular and morphological profiling of MEs will enable new insights into their pathobiology and inform targeted treatment strategies for patients with this condition. Citation Format: Mark J. Magbanua, Laura Huppert, Janifer Cruz, Jackson Goudreau, Shaopu Zhou, Keyi Yin, Pavel Stejskal, Ryan Carelli, Julie Kim, Linda Hsie, Maggie Wang, Zhouyang Lian, Michael Phelan, Manisha Ray, Chassidy Johnson, Amy L. Delson, Ron Balassanian, Jennifer Rosenbluth, Laura van't Veer, Michelle Melisko, Hope S. Rugo, Mahyar Salek, Maddison Masaeli, Hani Goodarzi. Artificial intelligence-assisted morphology-based detection and enrichment of malignant effusion tumor cells as a method for molecular profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB170.

Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes defined by hormone-receptor (HR), HER2, and MammaPrint (MP) status to evaluate novel agents as neoadjuvant therapy for high-risk breast cancer. The primary endpoint is pathologic complete response (pCR). Cemiplimab is an anti-PD-1 inhibitor approved for the treatment of NSCLC and cutaneous basal and squamous cell CA. Lymphocyte activation gene 3 (LAG-3) binds MHC class II leading to inhibition of T-cell proliferation and activation and is often co-expressed with PD-1. REGN3767 is a fully humanized mAb that binds to LAG-3 and blocks inhibitory T-cell signaling. Concurrent blockade of LAG-3 with an anti-PD-1 may enhance efficacy of an anti-PD-1. Methods: Women with tumors ≥ 2.5cm were eligible for screening. Only HER2 negative (HER2-) patients were eligible for this treatment; HR positive (HR+) patients had to be MP high risk. Treatment included Paclitaxel 80 mg/m2 IV weekly x 12 and Cemiplimab 350 mg and REGN3767 1600 mg both given q3weeks x 4, followed by doxorubicin/cyclophosphamide (AC) every 2 weeks x 4. The control arm was weekly paclitaxel x 12 followed by AC every 2-3 weeks x 4. Cemiplimab/REGN3767 was eligible to graduate in 3 of 10 pre-defined signatures: HER2-, HR-HER2-, and HR+HER2-. The statistical methods for evaluating I-SPY 2 agents has been previously described. To adapt to changing standard of care, we constructed “dynamic controls” comprising ‘best’ alternative therapies using I-SPY 2 and external data and estimated the probability of Cemiplimab/REGN3767 being superior to the dynamic control. Response predictive subtypes (Immune+ vs Immune-) were assessed using pre-treatment gene expression data and the ImPrint signature. Results: 73 HER2- patients (40 HR+ and 33 HR-) received Cemiplimab/REGN3767 treatment. The control group included [357 patients with HER2- tumors (201 HR+ and 156 HR-) enrolled since March 2010. Cemiplimab/REGN3767 graduated in both HR-/HER2- and HR+/HER2- groups; estimated pCR rates (as of June 2022) are summarized in the table. Safety events of note for Cemiplimab/REGN3767 include hypothyroidism 30.8%, adrenal insufficiency (AI) 19.2%, hyperthyroidism 14.1%, pneumonitis 1.3%, and hepatitis 3.8%. All were G1/2 except for 6 (7.7%) G3 AI and 3 (3.8%) G3 colitis. Rash occurred in 62.8%, 9% G3 and 2 pts (2.6%) had pulmonary embolism. X% of adrenal insufficiency cases required replacement therapy. 40 patients (11 HR+ and 29 HR-) in Cemiplimab/REGN3767 were predicted Immune+; 32 (29 HR+ and 3 HR-) were predicted Immune-. In the HR+ group pCR was achieved in 10/11 (91%) patients with Immune+ subtype compared with 8/29 (28%) with Immune- subtype. Additional biomarker analyses are ongoing and will be presented at the meeting. Conclusion: The I-SPY 2 study aims to assess the probability that investigational regimens will be successful in a phase 3 neoadjuvant trial. Dual immune blockade with a LAG-3 inhibitor and anti-PD1 therapy resulted in a high predicted pCR rate both in HR-/HER2- (60%) and HR+/HER2- (37%) disease. The novel Imprint signature identified a group of HR+ patients most likely to benefit from this active regimen. Table 1: Estimated pCR rates Citation Format: Claudine Isaacs, Rita Nanda, Jo Chien, Meghna S. Trivedi, Erica Stringer-Reasor, Christos Vaklavas, Judy C. Boughey, Amy Sanford, Anne Wallace, Amy S. Clark, Alexandra Thomas, Kathy S. Albain, Laura C. Kennedy, Tara B. Sanft, Kevin Kalinsky, Hyo S. Han, Nicole Williams, Mili Arora, Anthony Elias, Carla Falkson, Smita Asare, Ruixiao Lu, Maria Pitsouni, Amy Wilson, Jane Perlmutter, Hope Rugo, Richard Schwab, W. Fraser Symmans, Nola M. Hylton, Laura Van’t Veer, Douglas Yee, Angela DeMichele, Donald Berry, Laura J. Esserman, I-SPY Investigators. Evaluation of anti-PD-1 Cemiplimab plus anti-LAG-3 REGN3767 in early-stage, high-risk HER2-negative breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL [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 GS5-03.

Background: The Penelope-B trial did not show improvement in invasive disease-free survival (iDFS) with the addition of palbociclib to endocrine therapy (ET) in patients with high-risk early breast cancer (BC) after neoadjuvant chemotherapy (NACT). Biomarkers may be able to identify subgroups of patients deriving benefit from Palbociclib and guide future studies. Estrogen-receptor (ER), progesterone-receptor (PgR) and Ki-67 might be helpful in identifying patients benefiting from palbociclib. Concordantly, tumors with elevated expression of Cyclin D1 and phosphorylated retinoblastoma protein (phospho-RB) may harbor more dependency on CDK4/6 and thus higher sensitivity to palbociclib. Methods: The percentage of positive ER and PgR cells and Ki-67 assessed in surgical specimens after NACT were combined to obtain the immunohistochemical score 3 (IHC3, Cuzick et al JCO 2011, low vs high based on the median IHC3 value). Cyclin D1 and phospho-RB Ser 807/811 immunoreactive (phospho-RB) scores were analyzed in residual tumors after NACT (range 0-12 each). Proportional hazard regression model was used to assess the predictive and prognostic value of IHC3 and treatment on iDFS. Subgroup analysis was performed according to BC intrinsic subtypes (luminal-A/normal-like, luminal-B/HER2-enriched/basal) and HER2-status (HER2 0, HER2 low). Cox/Fine-Gray regression was used to define the predictive and prognostic value of CyclinD1 (≤1, >1), phospho-RB (≤2, >2) as dichotomized and continuous variables on iDFS, distant DFS (DDFS), locoregional invasive recurrence-free interval (LRRFI) and overall survival (OS). Multivariate analyses (MVA) were adjusted for age (≤50 vs >50), Ki-67 (≤15 vs >15), region (non-Asian vs Asian), ypN (ypN0-1 vs ypN2-3), risk status (CPS-EG=2 ypN+ vs ≥3), cT (cT1-2 vs cT3-4), ypT (ypT0-2 vs ypT3-4), and grade (G1-2 vs G3). The MVA for IHC3 includes all the covariates above except Ki-67. p< 0.05 was defined as statistically significant. Results: Data for ER, PgR, Ki-67, HER2, Cyclin D1 and phospho-RB were available for 1250 patients. Overall, 98.9% of the patients had ER+ tumors, 75.0% PgR+, 52.2% had HER2 low, 25.5% Ki-67>15, 50% had IHC3 score higher than median, 93.9% had Cyclin D1 >1, 57.8% had phospho-RB >2. Patients with IHC3 score high had a worse iDFS compared to patients with IHC3 score low (MVA HR 2.28 95%CI (1.78-2.91), p< 0.0001). Patients with luminal-A/normal-like tumors and IHC3 low had an improved iDFS with the addition of palbociclib to ET (MVA HR 0.35 95%CI (0.14-0.90), test for interaction p=0.01). No difference was observed according to HER2 status. Cyclin D1>1 has no predictive value but is prognostic for better iDFS (MVA HR 0.62 95%CI (0.41-0.94), p=0.023), LRRFI (MVA HR 0.30 95%CI (0.15-0.63), p=0.001) and OS (MVA HR 0.50 95%CI (0.28-0.89), p=0.019). Similar results were obtained when Cyclin D1 was analysed as a continuous variable. Phospho-RB had neither predictive nor prognostic value. Phospho-RB highly correlates with Ki-67 (p< 0.001, Spearman correlation 0.248). Conclusions: Patients with high Cyclin D1 expression had a favorable prognosis independent of treatment arm, but patients with luminal-A/normal-like tumors and IHC3 low after NACT had an improved outcome when receiving palbociclib in addition to adjuvant ET. Theses exploratory studies suggest specific signatures/phenotypes could predict benefit from Palbociclib in high-risk early breast cancer. Citation Format: Erik S. Knudsen, Sivaramakrishna Rachakonda, Frederik Marmé, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Wolfgang D. Schmitt, Sung-Bae Kim, Harry D. Bear, Agnieszka Witkiewicz, Seock-Ah Im, Angela DeMichele, Laura Van’t Veer, Nicole McCarthy, Bruno V. Sinn, Karen Gelmon, José Ángel García-Sáenz, Catherine M. Kelly, Toralf Reimer, Nicholas Turner, Federico Rojo, Martin Filipits, Peter A. Fasching, Christian Schem, Lesley-Ann Martin, Yuan Liu, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Jenny Furlanetto, Karsten Weber, Carsten Denkert, Sibylle Loibl. Immunohistochemical markers and determinants of clinical response in the Penelope-B trial [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 PD17-06.

Background: The SET2,3 index combines an accurate measure of transcription related to both estrogen and progesterone receptors (SETER/PR index) with a baseline prognostic index (BPI) derived from c-T Stage, c-N status and molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). SET2,3 index was translated from a microarray-based signature to a customized hybridization assay that yields highly reproducible results from routine pathology tissue blocks. Methods: The SET2,3 index was calculated from microarray data prepared from baseline biopsies of HR+/HER2- cancers prior to neoadjuvant taxane-anthracycline chemotherapy followed by adjuvant endocrine therapy in: 1) a test set of 307 samples from the MDACC cohort with 8 years median follow-up (Affymetrix U133A microarrays, Symmans et al JAMA 2011), and 2) an independent blinded validation set of 268 high-risk Mammaprint (MP) samples from the I-SPY2 clinical trial with 3.8 years median follow-up (Agilent 44K microarrays, Agendia). The cut-point for high versus low SET2,3 was defined in the MDACC cohort and then tested in the I-SPY2 cohort. Association between the SET2,3 index and distant relapse-free survival (DRFS) was evaluated in each cohort using multivariate Cox regression models. One model adjusted for residual cancer burden (RCB) after neoadjuvant chemotherapy and its interaction with the SET2,3 index, also including treatment arm (experimental vs. control) in the I-SPY 2 trial. Another model adjusted for other prognostic gene expression signatures (GES) for recurrence score (RS), MP, or endopredict (EP) in the MDACC cohort, and actual MP in the I-SPY2 trial. A third model tested the ability of prognostic GES to substitute for RNA4 subtype (part of BPI) or SETER/PR index as components of the SET2,3 index. Results: Predicted sensitivity to endocrine therapy (SET2,3) and response to neoadjuvant chemotherapy (RCB) were independently prognostic, with nonsignificant (NS) interaction term, in multivariate analyses of the MDACC study: SET2,3 HR 0.26 (p=0.016); RCB HR 2.04 (p Conclusions: SET2,3 index of endocrine transcriptional activity in a pre-treatment core biopsy added independent significant prognostic information to any baseline prognostic score and response to neoadjuvant chemotherapy. Approximately 40% of clinical or genomic (MP) high-risk HR+/HER2- cancers had high SET2,3 index, and their response to neoadjuvant chemotherapy (RCB class) was not prognostic. This suggests that SET2,3 index might be used to select appropriate candidates for endocrine-based neoadjuvant treatments in clinical trials. Citation Format: Lili Du, Christina Yau, Lamorna Brown-Swigart, Rebekah Gould, Gillian L Hirst, I-SPY2 Consortium, Marieke van der Noordaa, Isabelle Bedrosian, Rachel M Layman, Vicente Valero, Laura van't Veer, Laura Esserman, W. Fraser Symmans. Prognostic contribution of predicted sensitivity to endocrine therapy (SET) prior to neoadjuvant chemotherapy for stage II-III hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer [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 P2-11-04.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected., SCOPUS: er.j, info:eu-repo/semantics/published

Background: Traditionally, new drug combinations are first tested for safety and efficacy in the stage 4 setting. The I-SPY 2 TRIAL has focused on bringing promising new combinations into the phase 2/3 high-risk setting once safety is established. I-SPY 2 is an adaptive neoadjuvant platform trial for women with stage 2/3 breast cancer at high risk for early recurrence. In this setting, complete pathologic response (pCR) is the primary endpoint. We and others have demonstrated that pCR is highly predictive of distant recurrence free survival at 3 years. In I-SPY 2 the hazard rate is 0.2 (Confidence Interval 0.11-0.37), regardless of subtypes or agent. Agents that extend progression free survival in the metastatic setting, can be lifesaving when given at an earlier stage setting. Methods: The health economic benefits of achieving pCR were investigated using a Markov model describing the risk of recurrence, progression and death. The risk of recurrence was informed by I-SPY 2 and the risk of cancer progression and the associated treatment costs were based on literature values. We assumed constant recurrence rate without accounting for subtype specific rates in this first version of the model. Results: For each additional patient with pathologic complete response a net monetary benefit of $170,000 (Credible Interval 95% 100,000—250,000) and a gain of 4 life-years (CrI 95% 2—6) was predicted. The ICER for pCR is -$45,000. This represents the aggregate benefit across all I-SPY cancers. Modeling accounting for cancer subtype is in process and will be presented also. Conclusion: A short term endpoint, response to chemotherapy in the neoadjuvant setting, which is highly correlated with long term outcome, provides the opportunity to focus drug development on improving that endpoint. Enabling more patients to achieve a pCR would provide enormous benefit in terms of lives and resources saved. The economic modeling, along with the findings from I-SPY 2 suggests that we should shift drug development to the high risk early setting and evolve drug designs to improve every patient’s outcome. In I-SPY 2, we are shifting our platform trial design to increase every patient’s chance to achieve pCR. Platform adaptive trials in the early stage neoadjuvant setting are ideal for advancing the field and maximizing benefit for patients. Citation Format: Andreas Karlsson, Yiwey Shieh, Andre Dempsey, Christina Yau, Angela Dmichele, Doug Yes, Laura van't Veer, Nola Hylton, Martin Eklund, Laura Esserman. A prescription for new trial designs for drug development focused on the neoadjuvant setting: Save lives, resources, and time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3361.

International audience; Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Item does not contain fulltext We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations.

International audience; The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumors from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of over 25,000 cancer genomes at the genomic, epigenomic, and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically-relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

N-Acetyl-L-cysteine (NAC) has been shown to exert cancer-protective mechanisms and effects in experimental models. We report here the results of a randomized, double-blind, placebo-controlled, Phase II chemoprevention trial with NAC in healthy smoking volunteers. The subjects were supplemented daily with 2 x 600 mg of oral tablets of NAC (n = 20) or placebo (n = 21) for a period of 6 months, and internal dose markers [plasma and bronchoalveolar lavage (BAL) fluid cotinine, urine mutagenicity], biologically effective dose markers [smoking-related DNA adducts and hemoglobin (Hb) adducts], and biological response markers (micronuclei frequency and antioxidants scavenging capacity) were assessed at both pre- and postsupplementation times (T(0) and T(1), respectively). Overall, the internal dose markers remained unchanged at T(1) as compared with T(0) in both NAC and placebo groups. When quantifying the biologically effective dose markers, we observed an inhibitory effect of NAC toward the formation of lipophilic-DNA adducts (5.18 +/- 0.73 versus 4.08 +/- 1.03/10(8) nucleotides; mean +/- SE; P = 0.05) as well as of 7,8-dihydro-8-oxo-2'-deoxyguanosine adducts in BAL cells (3.9 +/- 0.6 versus 2.3 +/- 0.2/10(5) nucleotides; P = 0.003). There was no effect of NAC on the formation of lipophilic-DNA adducts in peripheral blood lymphocytes or polycyclic aromatic hydrocarbon-DNA adducts in mouth floor/buccal mucosa cells or 4-aminobiphenyl-Hb adducts. Likewise, quantification of the biological response markers showed an inhibitory effect of NAC on the frequency of micronuclei in mouth floor and in soft palate cells (1.3 +/- 0.2 versus 0.9 +/- 0.2; P = 0.001) and a stimulating effect of NAC on plasma antioxidant scavenging capacity (393 +/- 14 versus 473 +/- 19 microM Trolox; P = 0.1) but not on BAL fluid antioxidant scavenging capacity. We conclude that NAC has the potential to impact upon tobacco smoke carcinogenicity in humans because it can modulate certain cancer-associated biomarkers in specific organs.