Background: LCIS has traditionally been recognized as a marker of increased risk for the subsequent development of breast cancer, of either the lobular or ductal phenotype, yet due to the incidental nature of LCIS little is known about its underlying biology. Here we describe the first report of novel mutations in LCIS using targeted exome sequencing of fresh frozen tissue samples. Methods: Fresh frozen tissue samples from patients with a prior history of LCIS undergoing therapeutic or risk-reducing mastectomy from 2003–08 were harvested and systematically reviewed to identify LCIS. Cells from individual LCIS lesions +/− associated cancers were collected by laser capture microdissection. For the purposes of this study, germline DNA (blood) and DNA from 12 unique LCIS lesions were subject to targeted parallel sequencing of exons corresponding to 230 cancer genes using the Illumina HiSeq 2000 platform. DNA from an associated ductal carcinoma in situ (DCIS) and/or an invasive ductal (IDC) or lobular (ILC) lesion was also available for 7 of these cases resulting in 41 samples from 12 pts for mutational analysis. Normalized (RMA) Affymetrix U133A gene expression data were also available. Results: DNA profiling reliably identified 7 somatic mutations in 5/12 LCIS samples (41.7%). Of these, 4/7 mutations were base substitutions (missense mutations); and the others included: 1 deletion; 1 silent and 1 splice-site mutation. Mutations in LCIS were identified in 5/230 cancer genes analyzed, including: PIK3CA, CDH1, NOTCH4, PREX2 and ARAF. PIK3CA and CDH1 mutations were each identified in two samples, representing 4/7 (57.1%) mutations. Specific mutations found in LCIS and their frequencies are listed (table). Among 3 LCIS-ILC pairs, one shared the G914R mutation in PIK3CA, and 1/3 LCIS-IDC pairs exhibited an identical point mutation (R373W) in the NOTCH4 gene. No shared mutations were observed in 3 LCIS-DCIS pairs. Both CDH1 mutated cases were associated with decreased e-cadherin mRNA levels when compared to non-mutated cases (mean 9.88 vs 11.01), as was the NOTCH4 mutation (mean 6.02 vs 6.47). Mutations in ARAF and PREX2 were associated with increased mRNA levels, mean 7.07 vs 6.52 and 4.82 vs 4.22, respectively. The hotspot PIK3CA mutation (E545K) was also associated with increased gene expression (mean 5.15 vs 4.64) whereas the G914R mutant was associated with decreased expression (mean 4.13 vs 4.64). Conclusions: This study represents the first targeted exon sequencing analysis of fresh frozen LCIS. Although LCIS has been regarded as a rather genetically stable lesion, somatic mutations were detected in 41.7% of lesions in this small cohort. While CDH1 mutations are expected in lobular neoplasia, this is the first report of mutations in ARAF, NOTCH4, PIK3CA and PREX2. Given the shared relevance of PIK3CA and PREX2 in the PI3K/AKT pathway, these findings suggest novel mechanisms for new chemoprevention strategies among women with LCIS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-02.
INTRODUCTION: Lobular carcinoma in situ (LCIS) confers one of the greatest risks for subsequent development of invasive breast carcinoma yet historical data suggests that LCIS is not a precursor to invasive disease. Emerging evidence of shared molecular alterations between LCIS and co-existing invasive lesions in a small number of archival samples have reopened the debate regarding the true significance of LCIS and its malignant potential. METHODS: We investigated clonality between LCIS and adjacent malignant lesions in 26 patients using fresh frozen samples harvested from mastectomy specimens. Laser Capture Microdissection was used to isolate pure cell populations and DNA was subject to Affymetrix SNP 6.0 array. Data were converted for copy number analysis and the closeness of identified paired copy number changes was measured to determine the probability of clonality. RESULTS: After excluding cases with low DNA yield/quality and noise signals, 14 LCIS with seven patient matched invasive lobular carcinomas (ILC), 6 matched ductal carcinoma in situ (DCIS) and 3 matched invasive ductal carcinomas with lobular features (IDCLF) were considered adequate for statistical comparison. The immunohistochemical profile of invasive cancer showed: 10 cases ER+/PR+/Her-2(-), 2 cases ER-/PR-/Her-2(+), 1 ER-/PR-/Her2(-) and 1 case non-available for staining (microinvasive carcinoma). The results suggest that the tumor pairs are more likely to be of clonal than independent origin between LCIS and DCIS in 4/6 samples (66.6%), between LCIS and low-grade IDCLF in 3/3 (100%) samples and between LCIS and classic ILC in 5/7 (71.4%) samples. Independent LCIS/DCIS samples were seen in association with Her2+ cancers. In LCIS/DCIS samples favoring a clonal origin the DCIS were low-grade cribriform type associated with ER+ cancers. CONCLUSION: Clonal relatedness was detected in the majority of LCIS and adjacent malignancies, including cases of low-grade DCIS and IDCLF. These results support a true precursor role for LCIS and provide additional evidence to the concept of a low-grade malignancy pathway as opposed to the classic lobular versus ductal dichotomy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-07-01.
Introduction: Lobular carcinoma in situ (LCIS) is considered both a risk factor and non-obligate precursor of invasive breast cancer. We sought to determine the genomic landscape of LCIS and the mutational processes involved in the clonal evolution and progression from LCIS to ductal carcinoma in situ (DCIS) and invasive lobular carcinoma (ILC). Methods: Patients with a history of LCIS undergoing therapeutic or prophylactic mastectomy were prospectively enrolled in an IRB approved protocol. Frozen tissue blocks were collected, screened for lesions of interest (LCIS, DCIS, ILC, invasive ductal carcinomas (IDC)) and subjected to microdissection and DNA/RNA extraction. Matched germline DNA was available for all cases. Whole exome sequencing was performed on a HiSeq2000 and data were aligned to the reference human genome and processed using GATK. Single nucleotide variants (SNVs) and small insertions/deletions were identified using MuTect and Varscan, respectively. Purity and ploidy estimates were calculated using ABSOLUTE. Clonal frequencies were estimated using Pyclone and the clonal structure of each sample was reconstructed using SubcloneSeeker. Shannon index and Simpson index metrics were used to calculate heterogeneity levels. Mutational signatures were defined according to their mutational trinucleotide context, and the expression levels of APOBEC gene family members were assessed by quantitative reverse transcription (qRT)-PCR. Results: 30 LCIS, 10 ILCs, 7 DCIS and 5 IDCs from 15 patients qualified for data analysis. CDH1 was the most frequently mutated gene and found to be targeted by mutations in 26 LCIS samples (23 somatic, 3 germline). The repertoire of somatic mutations in LCIS was similar to that of luminal A breast cancers, with the exception of the significantly higher frequency of CDH1 mutations and the lower prevalence of TP53 mutations. ILCs were clonally related to at least one LCIS in 10 patients, and in 3/7 patients, DCIS was clonally related to at least one LCIS. Clonal composition analysis revealed that the presence of a minor clone(s) in LCIS, and the levels of intra-tumor genetic heterogeneity were significantly higher in LCIS clonally related with DCIS/ILC than in LCIS unrelated to DCIS/ILC. In two cases, a minor LCIS subclone constituted the major clone in the associated DCIS/ILC. A comparative analysis of the mutational signatures in the truncal and branch mutations of these cases revealed that whilst the truncal mutations displayed an aging signature, branch mutations were enriched for the APOBEC signature. qRT-PCR analysis demonstrated that cases displaying the APOBEC signature also harbored significantly higher levels of APOBEC3B expression than samples with the aging signature. Conclusions: LCIS displays intra-lesion genetic heterogeneity, and the progression from LCIS to DCIS or ILC may involve the selection of clones resulting from distinct mutational processes during clonal evolution. Our findings also suggest that cytodine deamination driven by the overexpression of APOBEC3B may drive the progression of LCIS to DCIS/ILC in a subset of cases. Citation Format: Reis-Filho JS, Schizas M, Piscuoglio S, Sakr RA, Ng CKY, Lim RS, Carniello JVS, Towers R, Martelotto L, Giri DD, de Andrade VP, Viale A, Solit DB, Weigelt B, King TA. Lobular carcinoma in situ displays intra-lesion genetic heterogeneity and its progression to invasive disease involves clonal selection and variations in mutational processes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-04.
Background: Recent studies suggest that the percentage of TILs is a predictive factor for response to NAC and a prognostic factor associated with long-term disease control in hormone receptor-negative breast cancer. The TILs working group's current recommendation is to evaluate stromal TILs as the principal parameter in future studies. The term lymphocyte-predominant breast cancer (LPBC) can be used as a descriptive term for tumors that contain more lymphocytes than carcinoma cells. Typically, the threshold of stromal lymphocytes for LPBC is around 50% of the stromal surface area. It is unclear if this cutoff will be used in the future as such an intense TIL infiltration in tumors has been reported to be infrequent (∼10%). Studies with TNBC have demonstrated increasingly better overall survival (OS) and disease-free survival (DFS) associated with continuous scores of TIL in patients treated with adjuvant chemotherapy. In patients treated with NAC, TILs predicted pathological complete response (pCR). Our goal was to evaluate the impact of TIL on OS in TNBC patients treated with NAC. Methods: Data from patients with histologically confirmed TNBC treated with NAC from a single institution (A. C. Camargo Cancer Center - ACCCC), between July 2002 and November 2013, were retrospectively collected using electronic medical records. Patients with metastatic disease or in situ carcinoma at diagnosis were excluded. The density of TILs was evaluated in full-face hematoxylin and eosin-stained (HE) slides. Three blinded pathologists made the assessment of each slide, and a consensus on the TIL percentage was achieved. A cut-off of 10% for TIL percentage was employed for OS and DFS calculations, based on technical statistical maximizing log-rank test. We use this cut-off to test the association with pathological pCR rate as well. For pCR rate, we also used a cut-off of 50% (LPBC). We used Chi-square test to evaluate the association with pCR. A p-value<0,05 was considered statistically significant for all tests. Results: We identified 78 patients that fulfilled all inclusion and exclusion criteria. The median age was 42 years (range 17-70), and the clinical stage distribution was IIA (14%), IIB (22%), IIIA (19%), IIIB (33%) and IIIC (11%). 58 patients had archival FFPE blocks available and suitable for pathological analysis. Median follow-up was 4,1 years. Overall survival in 5 years in this subgroup was 62% (median not reached). 23 (39.7%) tumors had TIL> 10%, however only 10 had TIL > 50%. TIL >10% was associated with improved OS (HR 0.33, 95% CI, 1.0 to 0.11; p = 0.04). The same cut-off was associated with better DFS, although not statistically significant (HR 0.46, 95% CI, 1.1 to 0.18; p = 0.1). The overall pCR rate was 39.6% (48% for patients with TIL > 10% and 34% for patients with TIL <or = 10%; p = 0.3). LPBC had similar pCR rate (40% for LPBC vs. 39% for non-LPBC), probably due to the small number of samples analyzed. pCR was associated with a decreased risk of death (HR 0.06, 95% CI, 0.008 to 0.47; p<0.01). Conclusion: We observed improved OS associated with TIL>10% in TNBC patients treated with NAC. pCR was also associated with better OS. Citation Format: Sampaio CdDAT, de Lima VCC, de Andrade VP, Neotti T, Tavares MC, Sessa VA, Calsavara VF, Zenun GR, Giongo AA, da Costa AABA. Tumor-infiltrating lymphocytes (TILs) is associated with improved overall survival in triple-negative breast cancer (TNBC) patients treated with neoadjuvant chemotherapy (NAC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-44.
Background: The identification of molecular predictors of type of recurrence (non-invasive vs invasive) after local therapy for ductal carcinoma in situ (DCIS) remains a challenge. We hypothesized that gene expression profiling using a panel of 32 breast cancer-related genes could identify a signature predictive of type of recurrence. Methods: From a prospectively maintained database of 1873 pts treated with conservative surgery +/− radiation for DCIS (1991–2006), we identified 190 (10%) patients who recurred. Original DCIS archival blocks were available for 108 (57%) of these cases. Freshly cut sections were obtained for microdissection of pure DCIS lesions, RNA was extraction and mRNA transcript quantification was performed with the Nanostring™ nCounter® system. Data were normalized using the nCounter® digital analyzer software for analysis. Results: Among the 108 studied cases, 66 (61%) recurred as DCIS and 42 (39%) recurred as invasive cancer. Median time to recurrence was 40 months (range 7–156 mo) and did not differ by type of recurrence. Similarly, patient age, grade of DCIS, ER status, use of radiation or tamoxifen therapy did not differ. Unsupervised hierarchical clustering using all 32 genes demonstrated two predominant groups, one enriched for recurrent-as-invasive (RI) tumors, and the other enriched for recurrent-as-DCIS (RD) tumors. Refined analysis by selection of 14 genes with significant differential expression between the RI and the RD tumors identified four main groups. The first cluster was dominated by ERBB2 over-expression and is equally composed of RI and RD tumors. The second cluster was composed almost exclusively of RI tumors and showed high level of COX2 and CCND1 and low level of CDKN2A (p16). The clusters 3 and 4 were enriched for RD tumors, with the fourth cluster completely devoid of RI cases. This RD-only group showed the lowest level of CCND1, but highest level of AKT3, EGFR, CDKN2A, and MKI67, thus displaying molecular traits typical of basal-like tumors. Conclusion: In this cohort of patients who recurred following conservative treatment for DCIS, HER2 expression was not predictive, however gene expression analysis demonstrated that high COX2 and CCND1 and low CDKN2a (p16) levels were strongly associated with invasive recurrences (RI), whereas increased AKT3 and MKI67 were associated with non-invasive recurrences (RD). Although the optimal combination of predictive markers requires validation, these data suggest that molecular alterations associated with RI differ from those associated with RD and warrant further investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD04-05.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.