BackgroundClinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood.MethodsThe Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days).ResultsConsistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive.ConclusionsThese findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.
Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy. They have changed the treatment landscape for a range of tumors, particularly those with a high mutational load. To date, however, modest results have been observed in breast cancer, where tumors are rarely hypermutated. Because BRCA1-associated tumors frequently exhibit a triple-negative phenotype with extensive lymphocyte infiltration, we explored their mutational load, immune profile, and response to checkpoint inhibition in a Brca1-deficient tumor model. BRCA1-mutated triple-negative breast cancers (TNBCs) exhibited an increased somatic mutational load and greater numbers of tumor-infiltrating lymphocytes, with increased expression of immunomodulatory genes including PDCD1 (PD-1) and CTLA4, when compared to TNBCs from BRCA1–wild-type patients. Cisplatin treatment combined with dual anti–programmed death-1 and anti–cytotoxic T lymphocyte–associated antigen 4 therapy substantially augmented antitumor immunity in Brca1-deficient mice, resulting in an avid systemic and intratumoral immune response. This response involved enhanced dendritic cell activation, reduced suppressive FOXP3+ regulatory T cells, and concomitant increase in the activation of tumor-infiltrating cytotoxic CD8+ and CD4+ T cells, characterized by the induction of polyfunctional cytokine-producing T cells. Dual (but not single) checkpoint blockade together with cisplatin profoundly attenuated the growth of Brca1-deficient tumors in vivo and improved survival. These findings provide a rationale for clinical studies of combined immune checkpoint blockade in BRCA1-associated TNBC.
Purpose: The immune microenvironment of breast ductal carcinoma in situ (DCIS) has yet to be fully explored, and the relationship of immune cells to genetic features of DCIS is unknown.Experimental Design: We quantified tumor associated lymphocytes (TIL) and evaluated PD-L1 protein levels by immunohistochemistry in a cohort of pure DCIS (138 and 79 cases, respectively), some of which had copy number (n ¼ 55) and mutation data (n ¼ 20).Results: TILs were identified in the stroma surrounding DCIS (119/138, 86%) and present at a median TIL score of 5% (range, 0%-90%). Most DCIS were negative for tumor cell PD-L1 staining (89%), but 25% of cases were positive for immune cell staining. We observed that, as in invasive breast cancer, TILs and PD-L1 positivity were significantly greater in high-grade (P ¼ 0.002/0.035), ER-negative (P ¼ 0.02/0.02), and ERBB2-amplified tumors (P < 0.001/0.048). Comedo necrosis was significantly positively associated with TILs (P < 0.0001) but not with PD-L1. The TILs score was significantly higher in cases with TP53 mutation (P ¼ 0.03) but not with PIK3CA or GATA3 mutation. In the cases with copy number data, both the fraction of the genome altered and the number of telomeric imbalances were significantly positively correlated with TILs (both P < 0.001). This result strongly contrasted with invasive breast cancer data, where aneuploidy was not correlated to TIL levels.Conclusions: Although a small cohort, our data suggest a preliminary model by which the progression of DCIS to invasive carcinoma may involve an altered relationship of tumor copy number with the immune microenvironment, possibly by the immunoediting of the tumor. Clin Cancer Res; 23(17); 5210-7.Ó2017 AACR.
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.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.