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Preferred first-line treatment for patients with metastatic EGFR-mutant lung cancer is osimertinib, yet it is not known if patient outcomes may be improved by identifying and intervening upon molecular markers associated with therapeutic resistance.All patients with metastatic EGFR-mutant lung cancer treated with first-line osimertinib at Memorial Sloan Kettering Cancer Center (n=327) were identified. Available pre-treatment and post-progression tumor samples underwent targeted gene panel sequencing and mutational signature analysis using SigMA algorithm. Progression-free and overall survival were estimated using Kaplan Meier methods.Using multivariate analysis, baseline atypical EGFR (mPFS 5.8 mo, p0.001) and concurrent TP53/RB1 alterations (mPFS 10.5 mo, p=0.015) were associated with shorter progression-free survival on first-line osimertinib. Of 95 patients with post-progression biopsies, acquired resistance mechanisms were identified in 52% (off-target n=24, histological transformation n=14, on-target n=12), with MET amplification (n=9), small cell lung transformation (n=7) and acquired EGFR amplification (n=7) the most frequently identified mechanisms. While there was no difference in post-progression survival based on identified resistance (p=0.07), patients with subsequent second-line therapy tailored to post-progression biopsy results had improved post-progression survival (HR 0.09, p=0.006). Paired post-progression tumors had higher tumor mutational burden (p=0.008) and further dominant APOBEC mutational signatures (p=0.07) compared to pre-treatment samples.Patients with EGFR-mutant lung cancer treated with first-line osimertinib have improved survival with treatment adaptation based on identified mechanisms of resistance at time of progression using tissue-based genomic analysis. Further survival gains may be achieved using risk-based treatment adaptation of pre-treatment genomic alterations.

Background: Varying levels of HER2 expression without ERBB2 gene amplification can be detected by immunohistochemistry (IHC) in approximately 60% of all invasive breast cancers (BCs). HER2-low-expressing BCs have recently been shown to respond to novel anti-HER2 antibody drug conjugates. Several studies have demonstrated that HER2-low BCs do not seem to constitute a distinct clinical and transcriptomic subtype as compared to HER2-0 BCs. Here we sought to define the clinicopathologic features and repertoire of somatic genetic alterations in HER2-low BCs. Methods: We retrieved clinical, pathological, and genomic data of BCs that were subjected to targeted sequencing using the FDA-cleared MSK-IMPACT assay from April 2014 to December 2021. After removing cases where any available biopsy had HER2 3+ and/or positive ERBB2 fluorescence in situ hybridization (FISH), 3608 samples (primary=1347; post-treatment/metastatic=2261) were included. Tumors were classified as HER2-low if they had an HER2 IHC score of 1+ or 2+ with a non-amplified FISH assay and HER2-0 if they had an HER2 IHC score of 0. Somatic mutations and DNA copy number alterations from MSK-IMPACT were analyzed. Multiple testing correction using the Benjamini-Hochberg method was applied to control for the false discovery rate (q). Q values < 0.1 were considered significant. Results: Among 3608 HER2- BCs, 1460 (40%) and 2148 (60%) were HER2-0 and HER2-low, respectively. Hormone receptor (HR) expression was significantly higher in HER2-low than HER2-0 tumors in both primary (781 [68.3%] vs 362 [31.7%]; p< 0.001) and metastatic (1031 [60.5%] vs 673 [39.5%]; p< 0.001) samples. A higher proportion of HER2-low tumors was found in metastatic than primary samples (59% vs 41%; p< 0.001) in this cohort. No difference in histology subtype, tumor grade, disease stage (among primary tumors), mutational signatures, and tumor mutational burden was found overall and when cases were stratified by HR expression. In HR-positive BCs, HER2-0 BCs harbored higher frequency of TP53 (33% vs 25%; odds ratio [OR] 1.49, 95% confidence interval [CI] 1.25-1.78, q< 0.001) and CDKN1A (1% vs 0%; OR 17.47, 95% CI 2.48-756.37, q=0.02) alterations than HER2-low BCs. Similar findings were observed in metastatic but not in primary HR-positive BCs. No differences were detected in HR-negative BCs stratified into HER2-low and HER2-0. Given the potential misclassification that exists between IHC HER2-0 and HER2-1+, we then conducted an exploratory analysis splitting the HER2-low group into HER2 1+ and 2+. TP53 alterations remained significantly enriched in HER2-0 compared to HER2-1+ HR-positive tumors (33% vs 24%; OR 1.55, 95% CI 1.28-1.87, q< 0.001). In HR-positive BCs, HER2-2+ displayed a higher frequency of genetic alterations in genes encoding for transcription factors, such as MYC (14.2% vs 7.3%; OR 2.09, 95% CI 1.44-3.04, q=0.02) and YAP1 (2% vs 0.3%; OR 6.86, 95% CI 1.7-39.57, q=0.1), and DNA damage response, such as FAM175A (1.6% vs 0%; OR 18.23, 95% CI 2.43-807.73, q=0.03) and BRCA2 (4% vs 1%; OR 3.09, 95% CI 1.49-6.55, q=0.1), than HER2-0 tumors. In HR-negative HER2-2+ tumors, a higher frequency of PIK3CA mutations was observed in comparison to HER2-0 (36.9% vs 19.5%; OR 2.41, 1.4-4.1, q=0.1), overall and in the metastatic setting. Conclusions: HER2-low BCs seem not to represent a distinct pathologic subtype. At the genomic level, however, some differences were identified and these became more conspicuous upon subclassification of HER2 IHC expression into 1+ and 2+. Further investigation into methods that more accurately detect and quantify low levels of HER2 expression in BC samples as well as better characterize the biology behind the HER2-low/ultralow expression is warranted. Citation Format: Antonio Marra, Anton Safonov, Joshua Drago, Emanuela Ferraro, Pier Selenica, Andrea Gazzo, Giuseppe Curigliano, Shanu Modi, Pedram Razavi, Jorge Reis-Filho, Sarat Chandarlapaty. HER2-07 Genomic Characterization of Primary and Metastatic HER2-low Breast Cancers [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 HER2-07.

Background: The HER2-specific tyrosine kinase inhibitor (TKI) tucatinib (Tuca) recently approved for advanced HER2+ breast cancer is making a move towards the early setting. Given its growing use, resistance is inevitable, as observed in the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on Tuca. Driven by the prevailing lack of knowledge about the mechanisms of resistance, in this study, we sought to define these mechanisms and identify treatment strategies to overcome them. We previously reported (SABCS 2021) that our BT474 TucaR models acquired EGFR amplification and showed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6. Since the HER pathway is activated by ligands, here we aim to assess if hyperactivation of EGFR via high levels of its ligands is an alternative mechanism of Tuca resistance. Materials and Methods: Our recently developed HER2+ BT474 (ATCC and AZ) cell models with acquired resistance to Tuca (TucaR) developed through long-term exposure to gradually increasing doses of Tuca and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (western blot) characterization were performed. Changes in cell growth and migration were assessed by methylene blue and Incucyte wound healing assays, respectively. Results: RNA-seq analysis demonstrated that the levels of TGFα was significantly higher in our BT474 TucaR models compared to P cells. Our results now demonstrate that exogenous supplementation of EGF to BT474-P cells rescues the Tuca-mediated inhibition of pEGFR, pHER2, and the downstream pAKT, pERK, and pS6 levels. Exogenous EGF was also found to reduce the levels of apoptosis, as assessed by cleaved PARP, mitigating the Tuca-induced cell death. Exogenous EGF or TGFα rendered naïve BT474 and SKBR3 cells resistant to Tuca while neratinib, a pan-HER TKI, effectively inhibited this ligand-driven cell growth. We previously showed that the HER signaling reactivation observed in our EGFR-amplified TucaR cells was inhibited by the EGFR-specific TKI gefitinib (Gef) (SABCS 2021) and that the TucaR cells displayed enhanced migratory capabilities (AACR 2022). Here, we demonstrate that in addition to curbing the growth of TucaR cells, Gef, either alone or together with Tuca, also markedly reverts the migration of the TucaR cells. Knockdown (KD) of EGFR but not HER2 selectively and substantially inhibited the migration of the TucaR cells. KD of EGFR also had a marked cell killing effect on only the TucaR cells, whereas HER2 KD inhibited the growth of P but not TucaR cells. Our findings are consistent with the notion that while the P cells are functionally dependent on HER2, in TucaR cells the survival dependence could be rewired to rely primarily on the hyperactive EGFR signaling. Genomic analysis further revealed that in addition to EGFR amplification, the AZ TucaR cells also acquired a gain of YES1, a src family receptor tyrosine kinase implicated in cancer cell growth, invasion, and metastasis. Functional studies using 2 siRNAs, however, showed that YES1 KD had no effect on the growth of TucaR cells, and the migration of both TucaR and P cells was equally affected by YES1 KD, precluding the potential role of YES1 in driving the resistant and enhanced migratory phenotypes. Conclusions: Hyperactivation of the EGFR pathway via amplification of EGFR or increased expression of its ligands confers resistance to Tuca, which may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Given the rapidly evolving treatment landscape of HER2+ breast cancer and biomarkers of resistance, our novel findings have potentially crucial therapeutic implications and suggest that rationally sequencing the currently available TKIs may be clinically important. Citation Format: Fu-Tien Liao, Tia Gordon, Chia Chia Liu, Pier Selenica, Yingjie Zhu, Juber Patel, Sarmistha Nanda, Lanfang Qin, Xiaoyong Fu, Andrea Gazzo, Antonio Marra, Juan Blanco-Heredia, Britta Weigelt, Jorge Reis-Filho, C. Kent Osborne, Mothaffar Rimawi, Rachel Schiff, Jamunarani Veeraraghavan. Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models [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 P1-13-17.

Background: Cyclin E1 (CCNE1) plays a critical role in cell cycle regulation. CCNE1 overexpression and/or gene amplification (amp) have been associated with poor outcome in several tumors, including breast cancer (BC). CCNE1 amp has recently been identified as a potential therapeutic target for novel synthetic lethality-based therapies. Here we sought to define the clinical and genomic features of BCs carrying CCNE1 amp. Methods: Genomic and clinical data from all consecutive BCs, which had been subjected to targeted sequencing using the FDA-authorized MSK-IMPACT assay from April 2014 to December 2021, were retrieved. Allele-specific copy number and fraction genome altered (FGA) were assessed using FACETS. Whole genome doubling (WGD) status was inferred from MSK-IMPACT sequencing data. Samples were categorized as CCNE1 amp or non-amp based on copy number profile assessed by FACETS. Mutual exclusivity and co-occurrence analyses between CCNE1 amp and other genetic alterations were performed using CoMEt. Multiple testing correction using the Benjamini–Hochberg procedure was applied to control for the false discovery rate. Progression-free survival (PFS) was assessed by Kaplan Meier method and Cox proportional-hazards models. Survival analyses were restricted to only patients with available pre-treatment samples. Results: Of 3,753 BCs with full clinical and genomic data, 125 (3.3%) harbored CCNE1 amp. A significant difference in the proportion of CCNE1 amp between treatment-naïve and post-treatment/metastatic samples was observed (2.4% vs 4%, p=0.007). CCNE1 amp was significantly less frequently detected in hormone receptor (HR)+/HER2- BCs than in HR-/HER2+ and HR-/HER2- subtypes (2% vs 7.6% and 7.2%, respectively, p< 0.001), and was particularly rare in invasive lobular BCs (1/452 cases). BCs with CCNE1 amp displayed a higher frequency of WGD (p< 0.001) and higher median FGA (p< 0.001) than non-amp tumors, overall and in different subtypes, suggesting increased genomic instability. No difference in tumor mutational burden (TMB) between CCNE1 amp and non-amp was found. In primary BC (n=1,385), a higher proportion of TP53 alterations was found in cases with CCNE1 amp (odds ratio [OR] 6.0, 95% confidence interval [CI] 2.5-16.6, q< 0.001). Conversely, CCNE1 amp was mutually exclusive with CDH1 alterations (q< 0.001). Comparable results were found in the analysis of post-treatment/metastatic samples (n=2,368). A subset analysis on HR+/HER2- BCs confirmed that TP53 (OR 4.2, 95%CI 2.28-8.11, q< 0.001) and CDH1 (OR 0.09, 95%CI 0.002-0.57, q< 0.1) alterations co-occurred and were mutually exclusive, respectively, with CCNE1 amp. ARID2 alterations were also enriched in HR+/HER2- tumors harboring CCNE1 amp (OR 10.6, 95%CI 2.54-33.93, q< 0.1). CCNE1 amp was significantly associated with reduced median PFS (8.8 vs 15.2 months in CCNE1 amp [n=9] vs CCNE1 non-amp [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75, p=0.004) on first line treatment with CDK4/6 inhibitor plus endocrine therapy (ET) in HR+/HER2- metastatic BCs, regardless of the ET partner, FGA and TMB. CCNE1 amp was also associated with numerically inferior median PFS (7.3 vs 20.8 months in CCNE1 amp [n=5] vs CCNE1 non-amp [n=106]; HR 3.1, 95% CI 1.24-7.87, p=0.01) on first line trastuzumab/pertuzumab/taxane treatment in HER2+ metastatic BCs, with a trend toward significance after adjusting for FGA and TMB (p=0.09). Conclusions: CCNE1 amp is associated with specific clinicopathological and genomic features in BCs and linked to an increased genomic instability. CCNE1 amp defines a subset of metastatic BCs with marked poor clinical response to available standard-of-care treatments. Further studies testing novel therapeutic approaches, including synthetic lethality-based strategies targeting CCNE1 amp and CDK2-selective inhibition, are warranted. Citation Format: Antonio Marra, Pier Selenica, Yingjie Zhu, Pedram Razavi, Anton Safonov, Emanuela Ferraro, Sarat Chandarlapaty, Jorge Reis-Filho. Clinical and Genomic Landscape of Breast Cancers Carrying CCNE1 Amplification [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 P4-08-12.

Background: Invasive lobular carcinomas (ILCs) are the most frequent special histologic subtype of breast cancer, accounting for up to 15% of all breast cancer cases. ILCs are characterized by a distinctive discohesive growth pattern, with cells arranged in single cell infiltrative file and dispersed throughout the stroma, which stems from the loss of E-cadherin expression due to bi-allelic inactivation of the CDH1 gene. A subset of breast cancers display a similar single cell infiltrative growth pattern but, in contrast to classic ILC, display diffuse strong membranous E-cadherin reactivity and membranous p120 expression. We refer to such cases as “lobular-like invasive ductal carcinoma” (LLIDC), but it is unclear if this terminology is appropriate and if such cases show biallelic inactivation of CDH1, similarly to ILCs. Here, we sought to define whether LLIDCs would harbor bi-allelic alterations of CDH1 and to perform an exploratory, hypothesis generating analysis of the repertoire of somatic genetic alterations of LLIDCs and classic ILCs. Materials and methods: Representative H&Es, as well as sections subjected to E-cadherin and p120 immunohistochemistry from seven classic ILCs and seven bona fide “lobular-like invasive ductal carcinomas” were retrieved and independently reviewed by two pathologists with experience and expertise in breast pathology. DNA samples were extracted from representative sections from tumor and normal breast tissue from each patient and subjected to an FDA-approved targeted sequencing assay comprising the coding regions and selected regulatory elements of 515 genes. Somatic single nucleotide variants (SNVs) were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet. All mutations were manually inspected using the Integrative Genomics Viewer (IGV). The cancer cell fraction (CCF) of each mutation was inferred, as well as clonal probability, using ABSOLUTE. Copy number alterations and loss of heterozygosity were determined using FACETS. Mutational signatures were inferred using SigMA based on all synonymous and nonsynonymous somatic mutations. Results: Based on the histopathologic evaluation, of the 14 cases analyzed, seven were classified as ILC, and the other seven were classified as LLIDC. Sequencing analysis revealed that the classic ILCs harbored 16q LOH and CDH1 mutations (7/7), of which five were frameshift indel and two were splice site mutations consistently coupled with loss-of-heterozygosity (LOH) of the wild-type allele. Conversely, five of the seven LLIDCs did not harbor CDH1 mutations or genomic rearrangements. CDH1 mutations were identified in 2 LLIDCs: one harbored a subclonal CDH1 in-frame indel mutation coupled with LOH. This case displayed membranous E-cadherin and p120 expression with areas of aberrant expression. The other CDH1-mutated LLIDC harbored a complex in-frame indel with subclonal LOH. This case displayed membranous E-cadherin and p120 expression. The comparative analysis of the repertoire of somatic genetic alterations and mutational signatures present in LLIDCs and classic ILCs did not reveal any significant differences. Conclusion: Despite the histologic similarities, LLIDCs differ from classic lobular carcinomas based on the lack of CDH1 bi-allelic inactivation and the patterns of expression of E-cadherin and p120 catenin. Further whole-genome sequencing analyses are warranted to define the molecular basis of the discohesive cancer cells of LLIDC display. Citation Format: Edaise M. da Silva, Thais Basili, Jing Yu, Juan Blanco-Heredia, Pier Selenica, Qiqi Ye, Arnaud da Cruz Paula, Higinio Dopeso, Antonio Marra, Steffi Oesterreich, Jorge Reis-Filho, Rohit Bhargava. Histologic, immunohistochemical and genomic comparison between classic Invasive lobular carcinomas and lobular-like invasive ductal carcinomas [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 P2-23-15.

Background We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features. Methods Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests. Results Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P Conclusions Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention.

Background: Clinical trials reported 25-30% pathologic complete response (pCR) rates in HER2+ breast cancer (BC) patients treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2 “addicted” tumors who may benefit from a chemotherapy-sparing strategy. Patients and Methods: Baseline HER2+ BC specimens from TBCRC023 and PAMELA trials of neoadjuvant lapatinib+trastuzumab (plus endocrine therapy in ER+ tumors) were used. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. Results: In TBCRC023, 72 BCs had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio≥4.6 and %3+ IHC-staining≥97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (wt). For clinical implementation, the classifier was locked as HER2 ratio≥4.5 and %3+ IHC-staining≥90% and PIK3CA-wt and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier’s high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment de-escalation. Conclusions: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable to chemotherapy plus dual anti-HER2 therapy in unselected patients.

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers.Significance:Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring.See related commentary by Liggett and Sankaran, p. 889.This article is highlighted in the In This Issue feature, p. 873

Supplementary Data from Cancer-Causative Mutations Occurring in Early Embryogenesis

Supplementary Data from Microsatellite Instability–High Endometrial Cancers with MLH1 Promoter Hypermethylation Have Distinct Molecular and Clinical Profiles

Purpose:Microsatellite instability–high (MSI-H) endometrial carcinomas are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H endometrial cancers harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph).Experimental Design:Of > 1,100 patients with endometrial cancer that underwent clinical tumor-normal sequencing, 184 had MSI-H endometrial cancers due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor-infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using nonparametric tests. Log-rank tests were used for categorical associations; Kaplan–Meier method and Wald test based on Cox proportional hazards models were employed for continuous variables and survival analyses.Results:Compared with patients with germline (n = 25) and somatic (n = 39) mutations, patients with MLH1ph endometrial cancers (n = 120) were older (P < 0.001), more obese (P = 0.001) and had more advanced disease at diagnosis (P = 0.025). MLH1ph endometrial cancers were enriched for JAK1 somatic mutations as opposed to germline MMR-D endometrial cancers which showed enrichment for pathogenic ERBB2 mutations. MLH1ph endometrial cancers exhibited lower tumor mutational burden and TIL scores compared with endometrial cancers harboring germline or somatic MMR mutations (P < 0.01). MLH1ph endometrial cancer patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models, stage at diagnosis remained the only predictor of survival. For stage I/II endometrial cancer, two-year PFS was inferior for patients with MLH1ph endometrial cancers compared with germline and somatic MMR groups (70% vs. 100%, respectively).Conclusions:MLH1ph endometrial cancers likely constitute a distinct clinicopathologic entity compared with germline and somatic MMR-D ECs with potential treatment implications.

Supplementary Methods and Figure Legends

Repertoire of non-synonymous somatic mutations in tumors and plasma DNA derived from ovarian cancer patients with BRCA1/2 germline mutations resistant/ refractory to platinum-based chemotherapy.

Supplementary Figure Legends

Supplementary Table S2: Comparison of clonal relatedness of the lesions analyzed between Begg et al. (2), Sakr et al. (45) and this study.

Purpose:Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease.Experimental Design:Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods.Results:DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed.Conclusions:Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

List of 143 genes included in the targeted capture massively parallel sequencing assay.

Supplementary Figure 1. Comparison of tumor mutational burden, dominant mutational signatures and MSISensor scores of Lynch syndrome BC cohort to control-matched BC cohort using MSK-IMPACT targeted sequencing.

Supplementary Figure S3 shows the copy number profiles of ductal carcinomas in situ (DCIS) and invasive ductal carcinomas of no special type (IDC-NST), and frequency of most commonly mutated genes in DCIS, compared to luminal B IDC-NSTs, and according to ER/HER2 status.

Supplementary Legend

Purpose:Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers.Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods.Results:WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases.Conclusions:Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

Supplementary Fig. S1: Repertoire of somatic mutations, clonal composition and phylogenetic tree analysis of cases 1-3 and cases 5-9.

Excel file containing Supplementary Data File 1: Non-synonymous somatic mutations identified by whole-exome sequencing and amplicon sequencing in the LCIS, DCIS, ILCs and IDCs studied.

Supplementary Data 1-8