Importance Of Next-generation Sequencing Research Articles

Exceptional Clinical Response to Alpelisib in a Patient with Metastatic Breast Cancer with Hyperbilirubinemia and a PIK3CA Mutation

In the era of personalized and precision medicine, the importance of next-generation sequencing in metastatic breast cancer has been increasingly recognized. This case highlights one such example in which a patient with heavily pretreated, hormone receptor-positive metastatic breast cancer was discovered to have a PIK3CA mutation and started on a PIK3 inhibitor with an exceptional response. This patient had been facing a visceral crisis with diffuse liver metastasis and hyperbilirubinemia. Alpelisib was able to achieve rapid disease control within a matter of weeks with clinical and objective laboratory improvement. To date, there has been no published data regarding the safety of alpelisib in patients with elevated bilirubin and this case report lends support to safety and efficacy in this situation.

Implementing laboratory automation for next-generation sequencing: benefits and challenges for library preparation.

In the wake of COVID-19, the importance of next-generation sequencing (NGS) for diagnostic testing and surveillance-based screening has never been more evident. Considering this, continued investment is critical to ensure more public health laboratories can adopt these advanced molecular technologies. However, many facilities may face potential barriers such as limited staff available to routinely prepare, test, and analyze samples, lack of expertise or experience in sequencing, difficulties in assay standardization, and an inability to handle throughput within expected turnaround times. Workflow automation provides an opportunity to overcome many of these challenges. By identifying these types of sustainable solutions, laboratories can begin to utilize more advanced molecular-based approaches for routine testing. Nevertheless, the introduction of automation, while valuable, does not come without its own challenges. This perspective article aims to highlight the benefits and difficulties of implementing laboratory automation used for sequencing. We discuss strategies for implementation, including things to consider when selecting instrumentation, how to approach validations, staff training, and troubleshooting.

High diagnostic yield with algorithmic molecular approach on hereditary neuropathies.

Charcot-Marie-Tooth disease covers a group of inherited peripheral neuropathies. The aim of this study was to investigate the effect of targeted next-generation sequencing panels on the molecular diagnosis of Charcot-Marie-Tooth disease and its subtypes in routine clinical practice, and also to show the limitations and importance of next-generation sequencing in the diagnosis of Charcot-Marie-Tooth diseases. This is a retrospective study. Three different molecular methods (multiplex ligation probe amplification, next-generation sequencing, and whole-exome sequencing) were used to detect the mutations related to Charcot-Marie-Tooth disease. In total, 64 patients (33 males and 31 females) with suspected Charcot-Marie-Tooth disease were analyzed for molecular etiology. In all, 25 (39%) patients were diagnosed by multiplex ligation probe amplification. With an extra 11 patients with normal PMP22 multiplex ligation probe amplification results that were consulted to our laboratory for further genetic analysis, a total of 50 patients underwent next-generation sequencing for targeted gene panels associated with Charcot-Marie-Tooth disease. Notably, 18 (36%) patients had pathogenic/likely pathogenic variants. Whole-exome sequencing was performed on five patients with normal next-generation sequencing results; the diagnostic yield by whole-exome sequencing was 80% and it was higher in the childhood group. The molecular etiology in Charcot-Marie-Tooth disease patients can be determined according to pre-test evaluation, deciding the inheritance type with pedigree analysis, the clinical phenotype, and an algorithm for the genetic analysis. The presence of patients without a molecular diagnosis in all the literature suggests that there are new genes or mechanisms waiting to be discovered in the etiology of Charcot-Marie-Tooth disease.

P1531: HETEROGENEITY OF DISEASE-CAUSING GENOTYPES BETWEEN FAMILIES WITH HEREDITARY SPHEROCYTOSIS: A SINGLE-CENTER COHORT STUDY

Background: Hereditary spherocytosis (HS) is a phenotypically and genetically heterogeneous disease, in which erythrocytes are unable to maintain their normal biconcave shape due to genetic mutations in membrane/cytoskeletal proteins that play a role in structural morphologic stability. Recent progress in genomic analyses technologies has helped to shed light on the complexity of genotype/phenotype correlations within this orphan hemolytic red blood cell disorder. Aims: In this retrospective investigation, we aim to investigate the genotype-phenotype correlation based on the clinical severity of the disease and the gene-wise predicted pathogenicity comparison. Furthermore, we want to highlight the importance of next generation sequencing (NGS) technologies in the diagnosis of HS. Methods: We provide the results of a single-center study in Austria on a total of 69 patients (core group, N=35; affected family members, N=34) with HS, focusing on NGS results (both exome and panel sequencing), ancestry, laboratory findings, clinics, as well as genotype-phenotype correlations. Results: A pathogenic (N=59/69; core group, N=29/35) or likely pathogenic variant (N=6/69; core group N=3/35) was identified in 94% (core group 91%) of the patients, including 18 novel variants. Variants in ANK1 (46%; European ancestry 37%, non-European 9%) were most encountered, followed by SPTB (31%, all European) and SLC4A1 (14%; European 11%, non-European 3%). In three patients (9%, all European) we were unable to identify the genetic cause unequivocally. Except for a heterozygous intronic ANK1 c.1504-9G>A variant, which was identified in two unrelated European families, there was no overlap on causative variants. The majority of mutations (N=25; 81%) were of high impact and include frameshifts, N=14 (45%); nonsense, N=8 (26%); CNVs, N=2 (6%) and splice-site variants, N=1 (3%). Sufficient data for phenotype-genotype correlation testing were available for 56 patients (core group, N=35), and phenotypes were milder in the patients with SLC4A1 variants. Patients with ANK1 and SPTB variants had comparable phenotypes. Summary/Conclusion: To our knowledge, this is the first study describing genomics (N=69) and genotype-phenotype correlation (N=56) in patients with HS in Central-Europe. We were able to identify causative variants in 94% of the 69 patients, including atypical disease-causing genetic findings such as intronic and CNV mutations. This evidenced the compelling need of a combination of standardized NGS analysis and clinical deep investigation in order to accurately characterize and treat HS patients.

Unmet needs in the treatment of intrahepatic cholangiocarcinoma harboring FGFR2 gene rearrangements.

Intrahepatic cholangiocarcinoma, a malignancy of the intrahepatic bile ducts, is the second most common primary liver malignancy and has been rising in incidence over the past several decades. Given its poor prognosis and diagnosis at a late stage, novel therapies are urgently needed to improve outcomes. Intrahepatic cholangiocarcinoma harbors a high rate of targetable mutations, spurring an increased interest in drug development in this disease. FGFR2gene rearrangements occur in approximately 10-16% of these tumors and this underscores the importance of next generation sequencing in this population. There are now several FGFR inhibitors in development, and these agents may help improve outcomes for these patients. However, both primary and secondary resistance remain a challenge.

Genetic Testing Contributes to Diagnosis in Cerebral Palsy: Aicardi-Goutières Syndrome as an Example.

Cerebral palsy (CP) is a non-progressive neurodevelopmental disorder characterized by motor impairments, often accompanied by co-morbidities such as intellectual disability, epilepsy, visual and hearing impairment and speech and language deficits. Despite the established role of hypoxic–ischemic injury in some CP cases, several studies suggest that birth asphyxia is actually an uncommon cause, accounting for <10% of CP cases. For children with CP in the absence of traditional risk factors, a genetic basis to their condition is increasingly suspected. Several recent studies indeed confirm copy number variants and single gene mutations with large genetic heterogeneity as an etiology in children with CP. Here, we report three patients with spastic cerebral palsy and a genetically confirmed diagnosis of Aicardi-Goutières syndrome (AGS), with highly variable phenotypes ranging from clinically suggestive to non-specific symptomatology. Our findings suggest that AGS may be a rather common cause of CP, that frequently remains undiagnosed without additional genetic testing, as in only one case a clinical suspicion of AGS was raised. Our data show that a diagnosis of AGS must be considered in cases with spastic CP, even in the absence of characteristic brain abnormalities. Importantly, a genetic diagnosis of AGS may have significant therapeutic consequences, as targeted therapies are being developed for type 1 interferonopathies, the group of diseases to which AGS belongs. Our findings demonstrate the importance of next generation sequencing in CP patients without an identifiable cause, since targeted diagnostic testing is hampered by the often non-specific presentation.

Detailed evaluation of cancer sequencing pipelines in different microenvironments and heterogeneity levels.

The importance of next generation sequencing (NGS) rises in cancer research as accessing this key technology becomes easier for researchers. The sequence data created by NGS technologies must be processed by various bioinformatics algorithms within a pipeline in order to convert raw data to meaningful information. Mapping and variant calling are the two main steps of these analysis pipelines, and many algorithms are available for these steps. Therefore, detailed benchmarking of these algorithms in different scenarios is crucial for the efficient utilization of sequencing technologies. In this study, we compared the performance of twelve pipelines (three mapping and four variant discovery algorithms) with recommended settings to capture single nucleotide variants. We observed significant discrepancy in variant calls among tested pipelines for different heterogeneity levels in real and simulated samples with overall high specificity and low sensitivity. Additional to the individual evaluation of pipelines, we also constructed and tested the performance of pipeline combinations. In these analyses, we observed that certain pipelines complement each other much better than others and display superior performance than individual pipelines. This suggests that adhering to a single pipeline is not optimal for cancer sequencing analysis and sample heterogeneity should be considered in algorithm optimization.

Usefulness of NGS for Diagnosis of Dominant Beta-Thalassemia and Unstable Hemoglobinopathies in Five Clinical Cases.

Unstable hemoglobinopathies (UHs) are rare anemia disorders (RADs) characterized by abnormal hemoglobin (Hb) variants with decreased stability. UHs are therefore easily precipitating, causing hemolysis and, in some cases, leading to dominant beta-thalassemia (dBTHAL). The clinical picture of UHs is highly heterogeneous, inheritance pattern is dominant, instead of recessive as in more prevalent major Hb syndromes, and may occur de novo. Most cases of UHs are not detected by conventional testing, therefore diagnosis requires a high index of suspicion of the treating physician. Here, we highlight the importance of next generation sequencing (NGS) methodologies for the diagnosis of patients with dBTHAL and other less severe UH variants. We present five unrelated clinical cases referred with chronic hemolytic anemia, three of them with severe blood transfusion dependent anemia. Targeted NGS analysis was performed in three cases while whole exome sequencing (WES) analysis was performed in two cases. Five different UH variants were identified correlating with patients’ clinical manifestations. Four variants were related to the beta-globin gene (Hb Bristol—Alesha, Hb Debrousse, Hb Zunyi, and the novel Hb Mokum) meanwhile one case was caused by a mutation in the alpha-globin gene leading to Hb Evans. Inclusion of alpha and beta-globin genes in routine NGS approaches for RADs has to be considered to improve diagnosis’ efficiency of RAD due to UHs. Reducing misdiagnoses and underdiagnoses of UH variants, especially of the severe forms leading to dBTHAL would also facilitate the early start of intensive or curative treatments for these patients.

Jeffrey\u2019s insights: Jeffrey Modell Foundation\u2019s global genetic sequencing pilot program to identify specific primary immunodeficiency defects to optimize disease management and treatment

Primary immunodeficiencies (PI) are genetic defects of the immune system that result in chronic and often life-threatening infections and/or life-threatening autoimmunity if not diagnosed and treated. Patients with a suspected PI, but without a genetic diagnosis, commonly undergo a diagnostic odyssey that is costly, time-consuming, and arduous. This delay in diagnosis prevents appropriate disease management and treatment, contributing to prolonged suffering and decreased quality of life. Although next generation sequencing (NGS) can provide these patients with relief from such a diagnostic odyssey, it is often unavailable, mainly due to cost and inaccessibility. In January 2019, the Jeffrey Modell Foundation (JMF) launched a free genetic sequencing pilot program for Jeffrey Modell Centers Network (JMCN) patients clinically diagnosed with an underlying PI. A total of 21 sites within the JMCN were invited to participate. JMF collaborated with Invitae, and testing was comprised of Invitae’s Primary Immunodeficiency Panel, which currently includes 207 genes. A questionnaire was disseminated to each participating physician to evaluate barriers to access to genetic sequencing and changes in disease management and treatment after testing. One hundred fifty-eight patients and 29 family members were tested in this pilot study. Twenty-one percent of patients with a suspected monogenic disorder received a molecular diagnosis, and others received potentially useful diagnostic leads. Based on the results of genetic sequencing, clinical diagnosis was altered in 45% of patients, disease management was altered in 40%, treatment was altered in 36%, and genetic counseling was altered in 62%. The results of this pilot program demonstrate the utility, cost-efficiency, and critical importance of NGS for PI and make the case for broad scale sequence–based diagnostics for PI patients when requested by expert immunologists.

Clinical results and importance of next-generation sequencing (NGS) in detecting targeted mutations in the treatment of metastatic Lung Cancer: Single center initial results

Objective: In Lung cancer (LC), which is one of the most deadly cancers, longer survival has been achieved with targeted agents. For this reason, it is important to find the patients who are suitable for targeted therapies. Next-generation sequencing (NGS) is a method that allows multiple genetic variants to be detected simultaneously by performing massive parallel DNA sequencing at the same time. We wanted to reveal the clinical effects and benefits of genetic variant analysis with NGS for our patients.&#x0D; Material and Methods: Patients with stage 4 non-squamous and not otherwise specified (NOS) Non-small cell LC who underwent genetic variant analysis with NGS were included in the study, retrospectively.&#x0D; Results: Total of the 51 patients, 41 (80.4%) were male and the median age was 64 (35-85) years. According to TNM, 21 (41.2%) patients were stage 4A, 30 (58.8%) patients were stage 4B and 39 (76.5%) patients had adenocarcinoma and 12 (23.5%) had NOS histology. NGS analyzes were performed in median 14 days (8-43) and determined 24 pathogenic variants in 17 (%25) patients: 9EGFR (%17,6), 6PIKC3A (%11,7), 5KRAS (%9,8), 2PTEN (%3,9), 1BRAF (%1,9), 1MET (%1,6) (7 of them concomitantly). Cytotoxic chemotherapy was recommended in 41, anti-EGFR agents in 8 (afatinib in 4, erlotinib in 4 patients) patients and anti-BRAF+MEK inhibitor agent (dabrafenib+trametinib) in 1 patient.&#x0D; Conclusion: With the NGS, in just two weeks, both target and resistance genetic variants of our patients were detected at the same time and individualized treatments were applied. In this way, both time and cost were saved.

New Era of Endoscopic Ultrasound-Guided Tissue Acquisition: Next-Generation Sequencing by Endoscopic Ultrasound-Guided Sampling for Pancreatic Cancer.

Pancreatic cancer is a lethal cancer with an increasing incidence. Despite improvements in chemotherapy, patients with pancreatic cancer continue to face poor prognoses. Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is the primary method for obtaining tissue samples of pancreatic cancer. Due to advancements in next-generation sequencing (NGS) technologies, multiple parallel sequencing can be applied to EUS-TA samples. Genomic biomarkers for therapeutic stratification in pancreatic cancer are still lacking, however, NGS can unveil potential predictive genomic biomarkers of treatment response. Thus, the importance of NGS using EUS-TA samples is becoming recognized. In this review, we discuss the recent advances in EUS-TA application for NGS of pancreatic cancer.

The utility of next generation sequencing in the correct diagnosis of congenital hypochloremic hypokalemic metabolic alkalosis

Persistent hypokalemic hypochloremic metabolic alkalosis represents a heterogeneous group of genetic disorders of which the most common is Bartter syndrome (BS). BS is an inherited renal tubulopathy caused by defective salt reabsorption in the thick ascending loop of Henle, which results in persistent hypokalemic hypochloremic metabolic alkalosis. Here we report a 10-year-old girl of a consanguineous family. She presented prenatally with severe polyhydramnios and distended bowel loops. Thereafter, she displayed failure to thrive and had recurrent admissions due to dehydration episodes associated with diarrhea, and characterized by hypokalemia, hypochloremia and metabolic alkalosis. BS was considered her working diagnosis for several years despite negative genetic analysis of the known genes associated with BS. Whole exome sequencing identified a novel homozygous c.1652delT deleterious frameshift mutation in the SLC26A3 gene, which confirmed the diagnosis of congenital chloride diarrhea (CCD), a rare autosomal recessive disease that mimics biochemically BS. A review of twelve additional reported cases of CCD that were initially misdiagnosed as BS, emphasizes CCD in the differential diagnosis of BS, and highlights the clinical discrepancies between these two entities. Taken together, our report further emphasizes the typical clinical features of CCD, and the importance of next generation sequencing in the diagnosis of syndromes with genetic heterogeneity. We suggest including SLC26A3 in the extended BS targeted gene panels.

Abstract P6-03-18: Outcome of FoundationOne testing in metastatic breast cancer therapy: A single center experience

Abstract Background: Next generation sequencing (NGS) has made genomic, mutation-driven, cancer medicine feasible. However, few studies have reported the importance of NGS in identifying mutation-driven treatment or whether such therapies are effective in women with metastatic breast cancer (MBC). We reviewed the impact of NGS in women with MBC at our institution. Methods: Among the medical oncologists who treat metastatic breast cancers at the City of Hope Comprehensive Cancer Center, we identified 21 patients with HER2 negative cancers, whose tumors were submitted for FoundationOne genomic profiling. We report the individual mutation profile, potential therapeutic options, and treatment outcome. Result: Of the 21 patients (pts), 13 had triple negative breast cancer (TNBC) and 12 of 13 (92%) had actionable mutation(s). Mutation profiles have been heterogeneous. Loss or mutations of TP53 were the most common genomic alteration in TNBC (12 of 13). Four pts received targeted therapies (everolimus in 3, pazopanib in 1) and all experienced at least partial clinical response. An additional 3 pts were treated with directed therapy but are too early to evaluate, 4 pts were not treated because of disease progression or clinical deterioration, and 1 pt was lost to follow-up. All 8 pts with ER+ disease had actionable mutations identified and 7 (88%) received targeted therapy (pazopanib in1, everolimus in 3, palbociclib in 3). All experienced at least partial clinical response; 1 was too ill for treatment. The most common genomic alterations are: PIK3CA mutation, PTEN loss, P53 mutation, FGFR mutation, and ZNF mutation. Conclusion: Targeted genomic sequencing through FoundationOne can identify mutations that are amenable to treatment with agents that are not generally approved for use in breast cancer. In order to test the benefit of target-directed therapy, tumor should be submitted for NGS while patients have a good performance status. Citation Format: Yuan Y, Mortimer JE. Outcome of FoundationOne testing in metastatic breast cancer therapy: A single center experience. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-03-18.