In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations ...in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays.
This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (
) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing.
Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding
, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of
mutations (64%, 7/11) and minority of
mutations (4%, 2/50) were clonal hematopoiesis.
Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care.
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Background Gene expression profiling of breast cancer has identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors ...have higher proliferation and poorer prognosis than luminal A tumors. In this study, we developed a clinically practical immunohistochemistry assay to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to breast cancer recurrence-free and disease-specific survival. Methods Tumors from a cohort of 357 patients with invasive breast carcinomas were subtyped by gene expression profile. Hormone receptor status, HER2 status, and the Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. Receiver operating characteristic curves were used to determine the Ki67 cut point to distinguish luminal B from luminal A tumors. The prognostic value of the immunohistochemical assignment for breast cancer recurrence-free and disease-specific survival was investigated with an independent tissue microarray series of 4046 breast cancers by use of Kaplan–Meier curves and multivariable Cox regression. Results Gene expression profiling classified 101 (28%) of the 357 tumors as luminal A and 69 (19%) as luminal B. The best Ki67 index cut point to distinguish luminal B from luminal A tumors was 13.25%. In an independent cohort of 4046 patients with breast cancer, 2847 had hormone receptor–positive tumors. When HER2 immunohistochemistry and the Ki67 index were used to subtype these 2847 tumors, we classified 1530 (59%, 95% confidence interval CI = 57% to 61%) as luminal A, 846 (33%, 95% CI = 31% to 34%) as luminal B, and 222 (9%, 95% CI = 7% to 10%) as luminal–HER2 positive. Luminal B and luminal–HER2-positive breast cancers were statistically significantly associated with poor breast cancer recurrence-free and disease-specific survival in all adjuvant systemic treatment categories. Of particular relevance are women who received tamoxifen as their sole adjuvant systemic therapy, among whom the 10-year breast cancer–specific survival was 79% (95% CI = 76% to 83%) for luminal A, 64% (95% CI = 59% to 70%) for luminal B, and 57% (95% CI = 47% to 69%) for luminal–HER2 subtypes. Conclusion Expression of ER, progesterone receptor, and HER2 proteins and the Ki67 index appear to distinguish luminal A from luminal B breast cancer subtypes.
The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is ...especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we utilize an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We systematically probe the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we construct chromatin interaction maps that enable the annotation of direct target genes for these super-enhancers and confirm their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach examines a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.
biochemical approach to identifying microRNA targets Karginov, Fedor V; Conaco, Cecilia; Xuan, Zhenyu ...
Proceedings of the National Academy of Sciences - PNAS,
12/2007, Volume:
104, Issue:
49
Journal Article
Peer reviewed
Open access
Identifying the downstream targets of microRNAs (miRNAs) is essential to understanding cellular regulatory networks. We devised a direct biochemical method for miRNA target discovery that combined ...RNA-induced silencing complex (RISC) purification with microarray analysis of bound mRNAs. Because targets of miR-124a have been analyzed, we chose it as our model. We honed our approach both by examining the determinants of stable binding between RISC and synthetic target RNAs in vitro and by determining the dependency of both repression and RISC coimmunoprecipitation on miR-124a seed sites in two of its well characterized targets in vivo. Examining the complete spectrum of miR-124 targets in 293 cells yielded both a set that were down-regulated at the mRNA level, as previously observed, and a set whose mRNA levels were unaffected by miR-124a. Reporter assays validated both classes, extending the spectrum of mRNA targets that can be experimentally linked to the miRNA pathway.
CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) -targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, ...we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival.
Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples.
One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71;
= .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94;
= .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS.
In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.
Lymphocytic infiltration of tumors predicts improved survival in patients with breast cancer. Previous studies have suggested that this survival benefit is confined predominantly to the basal-like ...subtype. Immune infiltration in ovarian tumors is also associated with improved prognosis. Currently, it is unclear what aspects of the immune response mediate this improved outcome.
Using The Cancer Genome Atlas mRNA-seq data and a large microarray dataset, we evaluated adaptive immune gene expression by genomic subtype in breast and ovarian cancer. To investigate B-cells observed to be prognostic within specific subtypes, we developed methods to analyze B-cell population diversity and degree of somatic hypermutation (SHM) from B-cell receptor (BCR) sequences in mRNA-seq data.
Improved metastasis-free/progression-free survival was correlated with B-cell gene expression signatures, which were restricted mainly to the basal-like and HER2-enriched breast cancer subtypes and the immunoreactive ovarian cancer subtype. Consistent with a restricted epitope-driven response, a subset of basal-like and HER2-enriched breast tumors and immunoreactive ovarian tumors showed high expression of a low-diversity population of BCR gene segments. More BCR segments showed improved prognosis with increased expression in basal-like breast tumors and immunoreactive ovarian tumors compared with other subtypes. Basal-like and HER2-enriched tumors exhibited more BCR sequence variants in regions consistent with SHM.
Taken together, these data suggest the presence of a productive and potentially restricted antitumor B-cell response in basal-like breast and immunoreactive ovarian cancers. Immunomodulatory therapies that support B-cell responses may be a promising therapeutic approach to targeting these B-cell infiltrated tumors.
Eosinophilic esophagitis (EoE) is an allergic inflammatory condition of the esophagus, often diagnosed late because of its challenging symptoms and costly and invasive diagnostic methods.
To address ...the need for more accessible biomarkers in EoE,
we aimed to investigate the potential of whole-blood RNA expression as a noninvasive biomarker for diagnosing and monitoring EoE, hypothesizing that genetic signatures in blood could distinguish EoE cases, correlate with disease activity, and predict treatment responses.
Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and ...differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c-/-; Ptch1+/- and Ink4c-/-; p53-/-. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17~92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17~92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17~92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c-/-; Ptch1+/- mice. These, but not similarly engineered cells from Ink4c-/-; p53-/- mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17~92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17~92 cluster and the SHH signaling pathway in the development of MBs in mouse and man.
Liquid biopsy has proven valuable in identifying individual genetic alterations; however, the ability of plasma ctDNA to capture complex tumor phenotypes with clinical value is unknown. To address ...this question, we have performed 0.5X shallow whole-genome sequencing in plasma from 459 patients with metastatic breast cancer, including 245 patients treated with endocrine therapy and a CDK4/6 inhibitor (ET + CDK4/6i) from 2 independent cohorts. We demonstrate that machine learning multi-gene signatures, obtained from ctDNA, identify complex biological features, including measures of tumor proliferation and estrogen receptor signaling, similar to what is accomplished using direct tumor tissue DNA or RNA profiling. More importantly, 4 DNA-based subtypes, and a ctDNA-based genomic signature tracking retinoblastoma loss-of-heterozygosity, are significantly associated with poor response and survival outcome following ET + CDK4/6i, independently of plasma tumor fraction. Our approach opens opportunities for the discovery of additional multi-feature genomic predictors coming from ctDNA in breast cancer and other cancer-types.
Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune ...rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC
), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC
in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC
that were nestin
and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC
rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC
delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSC
thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC
-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSC
-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC
is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.
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