ABSTRACT
A 66-year-old male presented with hypereosinophilia, thrombocytosis, extensive thrombosis refractory to direct oral anticoagulant therapy, and evidence of end-organ damage, including rash, splenic infarcts, and pulmonary infiltrates. Bone marrow biopsy revealed myeloid malignancy consistent with both chronic eosinophilic leukemia and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with SF3B1 mutation and thrombocytosis. Next-generation sequencing of the patient's eosinophils and neutrophil compartments revealed pathologic variants in EZH2 and SF3B1 in addition to a noncanonical JAK2 R683S mutation that has not been previously described in myeloproliferative disorders or other chronic myeloid neoplasms. These mutations were not present in the patient's lymphoid cell fraction, suggesting that the hematopoietic malignancy arose in a myeloid-committed progenitor cell. Based on this case and previous work from our group, we propose that noncanonical JAK2 mutations may permit signal transduction that biases toward eosinophilic differentiation in chronic myeloid neoplasms. Although the patient's blood counts initially responded to ruxolitinib and hydroxyurea, the response was not durable. Early referral for allogenic bone marrow transplant appears necessary to prevent long-term complications and disease progression in myeloid neoplasms with clonal hypereosinophilia driven by noncanonical JAK2 mutations.
Subject(s)
Eosinophilia , Leukemia , Myelodysplastic Syndromes , Myeloproliferative Disorders , Thrombocytosis , Male , Humans , Aged , Diagnosis, Dual (Psychiatry) , Myelodysplastic Syndromes/genetics , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/therapy , Thrombocytosis/diagnosis , Thrombocytosis/genetics , Thrombocytosis/pathology , Mutation , Janus Kinase 2/geneticsABSTRACT
BACKGROUND: A majority of patients undergoing curative intent surgery for pancreatic ductal adenocarcinoma (PDAC) will unfortunately develop recurrent disease. Treatment outcomes for patients with metastatic disease remain suboptimal. In this study, we evaluated clinical outcomes of patients with recurrent PDAC who received systemic therapy and compared outcomes to patients with de novo metastatic PDAC undergoing systemic therapy. METHODS: Patients diagnosed with metastatic PDAC between 2014 and 2019 were included using a real-world database. Patients were characterized as either de novo or recurrent based on the date of metastatic diagnosis and history of surgical resection. Overall survival (OS) was summarized within groups via Kaplan-Meier survival estimates and compared using Cox proportional hazards models. RESULTS: We included 5170 patients with metastatic PDAC, of which 1101 (21.3%) were classified as having recurrent disease. Median OS for the recurrent group was significantly greater at 10.8 m (95% CI 9.9-11.7) than in the de novo group at 7.3 m (95% CI 7.0-7.7, p < 0.001). We did not observe a significant difference in OS based on when patients recurred after surgery: 10.0 m (95% CI 8.7-11) within six months of surgery versus 11.6 m (95% CI 10-12, p = 0.256) greater than six months from surgery. CONCLUSIONS: These data support the inclusion of patients with recurrent PDAC in clinical trials for advanced disease, including those who develop recurrent disease within six months of surgery. Due to observed differences in survival, randomization should be stratified by disease presentation (recurrent vs de novo).
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Adenocarcinoma/surgery , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/surgery , Humans , Neoplasm Recurrence, Local/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/surgery , Prognosis , Retrospective Studies , Treatment Outcome , Pancreatic NeoplasmsABSTRACT
Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.
Subject(s)
Circulating Tumor DNA , MicroRNAs , Nucleic Acids , Biomarkers , Humans , MicroRNAs/genetics , Nucleic Acid HybridizationABSTRACT
New approaches for genomic DNA/RNA detection are in high demand in order to provide controls for existing enzymatic technologies and to create alternatives for emerging applications. In particular, there is an unmet need in rapid, reliable detection of short RNA regions which could open up new opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, a viral RNA sequence can be detected in less than 2 h without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity of <10 pM target RNA.
Subject(s)
Ebolavirus/genetics , Oligonucleotide Probes/metabolism , RNA, Viral/metabolism , Carbocyanines/chemistry , Click Chemistry , Discriminant Analysis , Nucleic Acid Hybridization , Oligonucleotide Probes/genetics , Oligonucleotides/chemistry , Polymorphism, Single Nucleotide , RNA, Viral/analysisABSTRACT
In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.
Subject(s)
DNA Copy Number Variations , Polymorphism, Single Nucleotide , Base Sequence , DNA PrimersABSTRACT
ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is one of the main causes of cancer death in well-developed countries. Therapeutic advances in PDAC to date have been modest. Recent progress to understand the molecular landscape of the disease has opened new treatment opportunities for a small subset of patients, frequently those with KRAS wild-type disease. Novel treatment strategies in PDAC include, among others, the use of nanotechnology and metabolic reprogramming. In addition, new strategies are being investigated, which are designed to overcome the resistance to checkpoint inhibitors, targeting DNA repair pathways including mismatch repair, increasing antigen presentation through the use of vaccines, targeting various signaling pathways, and reprogramming the tumor microenvironment. Here, we review the landscape of PDAC treatment strategies and some of these new agents.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Pancreatic Ductal/therapy , Immunotherapy, Adoptive/methods , Immunotherapy/methods , Molecular Targeted Therapy/methods , Pancreatic Neoplasms/therapy , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Clinical Trials as Topic , DNA Repair/genetics , Disease-Free Survival , Humans , Mutation , Outcome Assessment, Health Care/methods , Outcome Assessment, Health Care/statistics & numerical data , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
We developed a sensitive sequencing approach that simultaneously profiles microsatellite instability, chromosomal instability, and subclonal structure in cancer. We assessed diverse repeat motifs across 225 microsatellites on colorectal carcinomas. Our study identified elevated alterations at both selected tetranucleotide and conventional mononucleotide repeats. Many colorectal carcinomas had a mix of genomic instability states that are normally considered exclusive. An MSH3 mutation may have contributed to the mixed states. Increased copy number of chromosome arm 8q was most prevalent among tumors with microsatellite instability, including a case of translocation involving 8q. Subclonal analysis identified co-occurring driver mutations previously known to be exclusive.
Subject(s)
Chromosomal Instability , Chromosomes, Human, Pair 8 , Colorectal Neoplasms/genetics , DNA Mismatch Repair , Genotype , Humans , Microsatellite Repeats , MutS Homolog 3 Protein/genetics , MutS Homolog 3 Protein/metabolism , Neoplasm Proteins/genetics , Whole Genome SequencingABSTRACT
BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far. METHODS AND RESULTS: Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.
Subject(s)
DNA Mutational Analysis/methods , Microscopy, Fluorescence/methods , Neoplasms/genetics , Oligonucleotide Probes/genetics , Base Sequence , Cell Line, Tumor , Enzymes , Fluorescent Dyes/chemistry , Humans , Molecular Sequence Data , Oligonucleotides , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins B-raf/geneticsABSTRACT
BACKGROUND: Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis. RESULTS: Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity. CONCLUSIONS: We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.
Subject(s)
Evolution, Molecular , Krukenberg Tumor/genetics , Ovarian Neoplasms/genetics , Protein Serine-Threonine Kinases/genetics , Receptors, Transforming Growth Factor beta/genetics , Stomach Neoplasms/genetics , Adult , Animals , Antigens, CD , Cadherins/genetics , Female , Genetic Variation , Humans , Krukenberg Tumor/pathology , Krukenberg Tumor/secondary , Mice , Mice, Transgenic , Molecular Sequence Data , Neoplasms, Experimental/genetics , Ovarian Neoplasms/pathology , Ovarian Neoplasms/secondary , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Transforming Growth Factor-beta Type II , Stomach Neoplasms/pathology , Tumor Cells, Cultured , Tumor Suppressor Protein p53/geneticsABSTRACT
For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffin embedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas.