Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Craven, Kelly E.; Fischer, Catherine G.; Jiang, Liqun; Pallavajjala, Aparna; Lin, Ming-Tseh; Eshleman, James R.

doi:10.1016/j.jmoldx.2022.08.006

Cited by 12 publications

(4 citation statements)

References 56 publications

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“…DNA was processed and library-prepped using the KAPA Hyper Prep chemistry (KAPA Biosystems) workflow as previously described, but with an updated JHOP IDT bait set, v5.2 8 . Next-generation sequencing was performed on a NovaSeq.…”

Section: Methodsmentioning

confidence: 99%

“…DNA was processed and library-prepped using the KAPA Hyper Prep chemistry (KAPA Biosystems) workflow as previously described, but with an updated JHOP IDT bait set, v5.2. 8 Next-generation sequencing was performed on a NovaSeq. 6000 (Illumina Biotechnology) using 2×100 paired-end chemistry with a clinically validated laboratory-developed test at the Clinical Laboratory Improvement Amendments-certified Molecular Diagnostics Laboratory at Johns Hopkins, as previously described.…”

Section: Sample Preparation Sequencing and Analysis Workflowmentioning

confidence: 99%

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Positive GPNMB Immunostaining Differentiates Renal Cell Carcinoma With Fibromyomatous Stroma Associated With TSC1/2/MTOR Alterations From Others

Li,

Argani,

Halper-Stromberg

et al. 2023

American Journal of Surgical Pathology

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Renal cell carcinoma with fibromyomatous stroma (RCCFMS) include ELOC/TCEB1-mutated renal cell carcinoma (RCC) and those with TSC1/2/MTOR alterations. Besides morphologic similarity, most of these tumors is known to be diffusely positive for carbonic anhydrase IX and cytokeratin 7 by immunohistochemistry. We previously showed strong and diffuse expression of GPNMB (glycoprotein nonmetastatic B) in translocation RCC and eosinophilic renal neoplasms with known TSC1/2/MTOR alterations. We retrospectively identified molecularly confirmed cases of TCEB1/ELOC-mutated RCC (7 tumors from 7 patients), and RCCFMS with alterations in TSC1/2/MTOR (6 tumors from 5 patients, 1 patient with tuberous sclerosis syndrome). In addition, we included 7 clear cell papillary renal cell tumors (CCPRCTs) and 8 clear cell RCC, as they can also present morphologic overlap with RCCFMS. Morphologically, RCCs with TSC1/2/MTOR alterations and those with TCEB1/ELOC mutations were indistinguishable and characterized by papillary, nested, or tubular architecture, with tumor cells with clear cytoplasm and low nuclear grade. By immunohistochemistry, cytokeratin 7 was positive in 5/7 (71%) of TCEB1/ELOC-mutated RCCs, 6/6 (100%) of RCCs with TSC1/2/mTOR alterations, and 7/7 (100%) of CCPRCTs (P=not significant). Carbonic anhydrase IX was positive in 7/7 TCEB1/ELOC-mutated RCCs, 6/6 (100%) of RCCs with TSC1/2/MTOR alterations, and 7/7 (100%) of CCPRCTs (P=NS). GPNMB was strongly and diffusely positive in all tumors with TSC1/2/MTOR alterations (6/6), while negative in all TCEB1/ELOC-mutated RCCs (0/6), or CCPRCTs (0/7) (P=0.002). Two of 8 clear cell RCC showed focal weak staining, while 6/8 were negative. In conclusion, the results support the use of GPNMB to distinguish RCCFMS with TSC1/2/MTOR alterations from others with similar morphology.

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Section: Methodsmentioning

confidence: 99%

Section: Sample Preparation Sequencing and Analysis Workflowmentioning

confidence: 99%

Positive GPNMB Immunostaining Differentiates Renal Cell Carcinoma With Fibromyomatous Stroma Associated With TSC1/2/MTOR Alterations From Others

Li,

Argani,

Halper-Stromberg

et al. 2023

American Journal of Surgical Pathology

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“…The MGMT and beta-Actin copy numbers were used to calculate the ratio of MGMT/beta-Actin × 1000 (Unmethylated < 2.00, Methylated ≥ 2.00). NGS was performed on resected tumor tissue at the Johns Hopkins Medical Laboratories [22]. The incorporation of routine NGS of clinical samples into the diagnostic workup occurred around 2017.…”

Section: Methodsmentioning

confidence: 99%

Management and Molecular Characterization of Intraventricular Glioblastoma: A Single-Institution Case Series

Parker,

Kalluri,

Materi

et al. 2023

IJMS

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While the central nervous system (CNS) tumor classification has increasingly incorporated molecular parameters, there is a paucity of literature reporting molecular alterations found in intraventricular glioblastoma (IVGBM), which are rare. We present a case series of nine IVGBMs, including molecular alterations found in standardized next-generation sequencing (NGS). We queried the clinical charts, operative notes, pathology reports, and radiographic images of nine patients with histologically confirmed IVGBM treated at our institution (1995–2021). Routine NGS was performed on resected tumor tissue of two patients. In this retrospective case series of nine patients (22% female, median (range) age: 64.3 (36–85) years), the most common tumor locations were the atrium of the right lateral ventricle (33%) and the septum pellucidum (33%). Five patients had preoperative hydrocephalus, which was managed with intraoperative external ventricular drains in three patients and ventriculoperitoneal shunts in one patient. Hydrocephalus was managed with subtotal resection of a fourth ventricular IVGBM in one patient. The most common surgical approach was transcortical intraventricular (56%). Gross total resection was achieved in two patients, subtotal resection was achieved in six patients, and one patient received a biopsy only. Immunohistochemistry for IDH1 R132H mutant protein was performed in four cases and was negative in all four. Genetic alterations common in glioblastoma, IDH-wildtype, were seen in two cases with available NGS data, including EGFR gene amplification, TERT promoter mutation, PTEN mutation, trisomy of chromosome 7, and monosomy of chromosome 10. Following surgical resection, four patients received adjuvant chemoradiation. Median survival among our cohort was 4.7 months (IQR: 0.9–5.8 months). Management of IVGBM is particularly challenging due to their anatomical location, presentation with obstructive hydrocephalus, and fast growth, necessitating prompt intervention. Additional studies are needed to better understand the genetic landscape of IVGBM compared to parenchymal glioblastoma and may further elucidate the unique pathophysiology of these rare tumors.

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“…Given the existence of unusual JAK2 mutations and the high prevalence of additional mutations beyond driver mutations in MF, routine incorporation of NGS-based mutation profiling at the time of diagnosis is imperative. Bioinformatics pipelines for NGS data analysis are known to exhibit variable accuracies in detecting and naming large indels [103]. Modifying the bioinformatics pipeline for large indel detection is essential when devising NGS-based mutation profiling tests for myeloid neoplasms.…”

Section: Laboratory Test Considerationsmentioning

confidence: 99%

Molecular Genetic Profile of Myelofibrosis: Implications in the Diagnosis, Prognosis, and Treatment Advancements

Verma,

Papadantonakis,

Peker Barclift

et al. 2024

Cancers

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Myelofibrosis (MF) is an essential element of primary myelofibrosis, whereas secondary MF may develop in the advanced stages of other myeloid neoplasms, especially polycythemia vera and essential thrombocythemia. Over the last two decades, advances in molecular diagnostic techniques, particularly the integration of next-generation sequencing in clinical laboratories, have revolutionized the diagnosis, classification, and clinical decision making of myelofibrosis. Driver mutations involving JAK2, CALR, and MPL induce hyperactivity in the JAK-STAT signaling pathway, which plays a central role in cell survival and proliferation. Approximately 80% of myelofibrosis cases harbor additional mutations, frequently in the genes responsible for epigenetic regulation and RNA splicing. Detecting these mutations is crucial for diagnosing myeloproliferative neoplasms (MPNs), especially in cases where no mutations are present in the three driver genes (triple-negative MPNs). While fibrosis in the bone marrow results from the disturbance of inflammatory cytokines, it is fundamentally associated with mutation-driven hematopoiesis. The mutation profile and order of acquiring diverse mutations influence the MPN phenotype. Mutation profiling reveals clonal diversity in MF, offering insights into the clonal evolution of neoplastic progression. Prognostic prediction plays a pivotal role in guiding the treatment of myelofibrosis. Mutation profiles and cytogenetic abnormalities have been integrated into advanced prognostic scoring systems and personalized risk stratification for MF. Presently, JAK inhibitors are part of the standard of care for MF, with newer generations developed for enhanced efficacy and reduced adverse effects. However, only a minority of patients have achieved a significant molecular-level response. Clinical trials exploring innovative approaches, such as combining hypomethylation agents that target epigenetic regulators, drugs proven effective in myelodysplastic syndrome, or immune and inflammatory modulators with JAK inhibitors, have demonstrated promising results. These combinations may be more effective in patients with high-risk mutations and complex mutation profiles. Expanding mutation profiling studies with more sensitive and specific molecular methods, as well as sequencing a broader spectrum of genes in clinical patients, may reveal molecular mechanisms in cases currently lacking detectable driver mutations, provide a better understanding of the association between genetic alterations and clinical phenotypes, and offer valuable information to advance personalized treatment protocols to improve long-term survival and eradicate mutant clones with the hope of curing MF.

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Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory

Cited by 12 publications

References 56 publications

Positive GPNMB Immunostaining Differentiates Renal Cell Carcinoma With Fibromyomatous Stroma Associated With TSC1/2/MTOR Alterations From Others

Positive GPNMB Immunostaining Differentiates Renal Cell Carcinoma With Fibromyomatous Stroma Associated With TSC1/2/MTOR Alterations From Others

Management and Molecular Characterization of Intraventricular Glioblastoma: A Single-Institution Case Series

Molecular Genetic Profile of Myelofibrosis: Implications in the Diagnosis, Prognosis, and Treatment Advancements

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