Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer ...large sequences. Methods for sequencing mid-sized sequences of 600-5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000-300,000 reads with an accuracy per base pair of 90-99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.
Levels of circulating tumor cells (CTCs) in blood have prognostic value in early and metastatic breast cancer. CTCs also show varying degrees of concordance with molecular markers of primary tumors ...they originate from. It is expected that individual cells reflect the heterogeneity and evolution of tumor cells as they acquire new functions and differential responses to chemotherapy. However, a degree of commonality is also plausible, highlighting alterations that allow tumor cells to perform CTC‐defining activities such as invasion and intravasation. Using a matched tumor‐normal approach, we performed high‐resolution copy number profiling of CTCs from breast cancer to identify occult changes occurring during progression to metastasis. We identified a signature of recurrent gain in CTCs, consisting of 90 minimal common regions (MCRs) of copy number gain. These were predominantly found across chromosome 19 and were identified at low frequencies (3–4%) in 787 primary breast carcinomas examined. CTC genomic signatures clustered into two groups independent of subtype: a dormancy‐related signature with 16 MCRs (AKT2, PTEN, CADM2); and a tumor‐aggressiveness related signature with 358 MCRs (ANGPTL4, BSG, MIR‐373). There were two MCRs in common between the groups on 19q13 and 21q21, containing genes involved in resistance to anoikis, TGFβ‐signaling and metastasis (TFF3, LTBP4, NUMBL). Furthermore, a region harboring the ERBB2 gene was gained in a majority of patients. Regions 20q13 and 15q24 were associated with distant metastasis. The distinctiveness of CTC signatures highlights cell populations with different functional or metastatic potential. Such novel targets could help to specifically identify and block dissemination.
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People die of breast cancer not from the primary tumors, but because of uncontrolled metastasis, and circulating tumor cells (CTCs) could be the key to detecting it. In this paper, the authors examined the DNA of CTCs looking for identifying features that could help spot these harbingers of metastasis. They found certain copy number changes, predominantly on chromosome 19 that may indicate the cell's ability to travel. The changes fell into two categories: one associated with dormancy, the other with tumor aggressiveness. Detection of these genetic signatures could help target the most mobile cells and thwart metastasis.
A child had been followed since infancy by our multi-disciplinary neuro-oncology clinic with annual magnetic resonance imaging (MRI) under the presumed diagnosis of encephalocraniocutaneous ...lipomatosis (ECCL), with clinical features including nevus psiloliparus, scalp lipoma, nodular skin tag on and coloboma of the eyelid, cortical atrophy and meningeal angiomatosis. At the age of 4, she was found to have a large temporoparietal lesion causing elevated intracranial pressure requiring surgical resection. Histopathological exam of the tumor was suggestive of an intracranial sarcoma. Sequencing analysis of the tumor revealed mutations in
DICER1
,
KRAS
and
TP53
. Subsequent germline testing confirmed
DICER1
syndrome and revealed an insignificant
FGFR1
variant at a low frequency. Methylation profile of the tumor showed the tumor clustered most closely with sarcoma (rhabdomyosarcoma-like), confirming this tumor to be a primary
DICER1
-sarcoma. Compared to the previously reported cases, our unique case of primary
DICER1
-sarcoma also demonstrated neurofilament and chromogranin positivity, and genomic instability with loss of chromosome 4p, 4q, 8p, 11p, and 19p, as well as gains in chromosome 7p, 9p, 9q, 13q, and 15q on copy variant analysis. The detailed sequencing and methylation information discovered in this unique case of
DICER1
-sarcoma will hopefully help further our understanding of this rare and emerging entity.
Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this ...amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling.
In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo.
CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ).
CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling.
Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.
Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial ...cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development.
There is a paucity of data regarding the impact of mutations on outcomes in accelerated-phase (AP) and blast-phase (BP) myeloproliferative neoplasms (MPNs). Moreover, it is unknown whether mutational ...status affects survival, as seen in chronic-phase MPNs. Therefore, we performed a retrospective analysis of all patients treated at our institution with AP/BP MPNs (N = 122; AP = 14; BP = 108) to comprehensively describe the mutational profile and correlate with clinical outcomes. Targeted sequencing with a 54-gene panel was performed. Forty-four patients were treated with intensive therapy, 27 with nonintensive therapy, and 51 with best supportive care (BSC). The most common mutation was JAK2V617F, occurring in 55% of subjects; CALR was found in 13% of patients and MPL in 6%. Thirty-two (26%) patients were triple negative. Other frequently mutated genes were ASXL1 (30%), TET2 (25%), SRSF2 (22%), RUNX1 (20%), and TP53 (17%). Mutations in 1, 2, 3, and ≥4 genes were seen in 15%, 13%, 25%, and 46% of patients, respectively. There was no difference in survival between patients treated with intensive vs nonintensive therapy, and the benefit of intensive therapy was limited to patients who were able to undergo transplantation. TP53 was the only individual mutation to correlate with shorter overall survival (hazard ratio, 1.89; P = .03). In the multivariate analysis, mutated TP53, ≥4 mutations, low albumin, increased peripheral blood blasts, ≥3 cytogenetic abnormalities, and BSC were associated with shorter survival. In conclusion, mutational data enhance the understanding of patients with AP/BP MPN who are likely to benefit from current therapeutic options.
•Mutant TP53 and ≥4 mutations predict dismal outcomes with current therapeutic options in patients with accelerated/blast phase of MPNs.•The benefit of intensive therapy is only seen in patients who are able to undergo transplant.
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Although next‐generation sequencing (NGS) has helped characterize the complex genomic landscape of myeloid malignancies, its clinical utility ...remains undefined. This has resulted in variable funding for NGS testing, limiting its accessibility. At our center, targeted sequencing (TAR‐SEQ) using a 54‐gene NGS myeloid panel is offered to all new patients referred for myeloid malignancies, as part of a prospective observational study. Here, we evaluated the diagnostic, prognostic, and potential therapeutic utility of clinical grade TAR‐SEQ in the routine workflow of 179 patients with myeloproliferative neoplasms (MPN).
Of 13 patients with triple negative (TN) MPN, who lacked driver mutations in JAK2, CALR, and MPL, TAR‐SEQ confirmed clonal hematopoiesis in 8 patients. In patients with intermediate‐risk myelofibrosis (MF), TAR‐SEQ helped optimize clinical decisions in hematopoietic cell transplant (HCT)‐eligible patients through identifying a high molecular risk (HMR) mutation profile. The presence of an HMR profile favored HCT in 9 patients with intermediate‐1 risk MF. Absence of an HMR profile resulted in a delayed HCT strategy in 10 patients with intermediate‐2 risk MF, 7 of which were stable at the last follow‐up. Finally, TAR‐SEQ identified patients with various targetable mutations in IDH1/2 (4%), spliceosome genes (28%), and EZH2 (7%). Some of these patients can be potential candidates for future targeted therapy trials.
In conclusion, we have demonstrated that TAR‐SEQ improves the characterization of TN MPN, can be integrated in clinical practice as an additional tool to refine decision making in HCT, and has the potential to identify candidates for future targeted therapy trials.
The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from ...a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature's concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature's successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.
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