Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Analytic validation of NeXT Dx™, a comprehensive genomic profiling assay.

We describe the analytic validation of NeXT Dx, a comprehensive genomic profiling assay to aid therapy and clinical trial selection for patients diagnosed with solid tumor cancers. Proprietary methods were utilized to perform whole exome and whole transcriptome sequencing for detection of single nucleotide variants (SNVs), insertions/deletions (indels), copy number alterations (CNAs), and gene fusions, and determination of tumor mutation burden and microsatellite instability. Variant calling is enhanced by sequencing a patient-specific normal sample from, for example, a blood specimen. This provides highly accurate somatic variant calls as well as the incidental reporting of pathogenic and likely pathogenic germline alterations. Fusion detection via RNA sequencing provides more extensive and accurate fusion calling compared to DNA-based tests. NeXT Dx features the proprietary Accuracy and Content Enhanced technology, developed to optimize sequencing and provide more uniform coverage across the exome. The exome was validated at a median sequencing depth of >500x. While variants from 401 cancer-associated genes are currently reported from the assay, the exome/transcriptome assay is broadly validated to enable reporting of additional variants as they become clinically relevant. NeXT Dx demonstrated analytic sensitivities as follows: SNVs (99.4%), indels (98.2%), CNAs (98.0%), and fusions (95.8%). The overall analytic specificity was >99.0%.

Prostatic adenocarcinoma with novel NTRK3 gene fusion: a case report.

TMPRSS2-ERG gene fusion occurs in approximately 50% of prostatic adenocarcinoma and their expression is associated with aggressive phenotype, higher tumor stage, and tumor metastasis. A case of prostatic adenocarcinoma with IRF2BP2-NTRK1 translocation was previously reported. We report a prostatic adenocarcinoma with novel NTRK3 gene fusion that occurs in a 71-year-old male patient with aggressive histologic phenotype and multiple bony metastases. Prostatic biopsy revealed that there is a prostatic adenocarcinoma with a Gleason score of 9 (4+5), grade group 5, and multiple sites of perineural and ganglional invasion. Fluorescence in-situ hybridization (FISH) and next-generation sequencing were performed. FISH studies showed a breakage within the NTRK3 gene in prostatic adenocarcinoma cells. Next-generation sequencing confirmed that there is a PRPSAP1-NTRK3 translocation in the prostatic adenocarcinoma. In addition, ASXL1, KIF5B, MED12, PIK3CA mutations were found. NTRK alterations or dysregulation of PI3K signaling pathway were found in many types of cancers. TRK inhibitors including larotrectinib and entrectinib were approved by the US Food and Drug Administration for treating TRK fusion-positive malignant tumors and PI3K/AKT/mTOR pathway inhibitors were under clinical studies on various cancers including prostate cancer. In our current case, both NTRK3 and PIK3CA may serve as biomarkers for precision targeted therapy.

Incidental Detection of Maternal Neoplasia in Noninvasive Prenatal Testing.

BACKGROUND: Noninvasive prenatal testing (NIPT) uses cell-free DNA (cfDNA) as an analyte to detect copy-number alterations in the fetal genome. Because maternal and fetal cfDNA contributions are comingled, changes in the maternal genome can manifest as abnormal NIPT results. Circulating tumor DNA (ctDNA) present in cases of maternal neoplasia has the potential to distort the NIPT readout to a degree that prevents interpretation, resulting in a nonreportable test result for fetal aneuploidy. METHODS: NIPT cases that showed a distortion from normal euploid genomic representation were communicated to the caregiving physician as nonreportable for fetal aneuploidy. Follow-up information was subsequently collected for these cases. More than 450000 pregnant patients who submitted samples for clinical laboratory testing >3 years are summarized. Additionally, in-depth analysis was performed for >79000 research-consented samples. RESULTS: In total, 55 nonreportable NIPT cases with altered genomic profiles were cataloged. Of these, 43 had additional information available to enable follow-up. A maternal neoplasm was confirmed in 40 of these cases: 18 malignant, 20 benign uterine fibroids, and 2 with radiological confirmation but without pathological classification. CONCLUSIONS: In a population of pregnant women who submitted a blood sample for cfDNA testing, an abnormal genomic profile not consistent with fetal abnormalities was detected in about 10 out of 100000 cases. A subset of these observations (18 of 43; 41.9%) was attributed to maternal malignant neoplasms. These observational results suggest the need for a controlled trial to evaluate the potential of using cfDNA as an early biomarker of cancer.

Initial assessment of the benefits of implementing pharmacogenetics into the medical management of patients in a long-term care facility.

The health care costs associated with prescription drugs are enormous, particularly in patients with polypharmacy (taking more than five prescription medications), and they continue to grow annually. The evolution of pharmacogenetics has provided clinicians with a valuable tool that allows for a smarter, more fine-tuned approach to treating patients for a number of clinical conditions. Applying a pharmacogenetics approach to the medical management of patients can provide a significant improvement to their care, result in cost savings by reducing the use of ineffective drugs, and decrease overall health care utilization. AltheaDx has begun a study to look at the benefits associated with incorporating pharmacogenetics into the medical management of patients who are on five or more medications. Applying pharmacogenetic guided PharmD recommendations across this patient population resulted in the elimination and/or replacement of one to three drugs, for 50% of the polypharmacy patient population tested, and an estimated US$621 in annual savings per patient. The initial assessment of this study shows that there is a clear opportunity for concrete health care savings solely from prescription drug management when incorporating pharmacogenetic testing.

EGFR-mutated lung cancer with T790M-acquired resistance in the brain and histologic transformation in the lung.

This case report describes the rare occurrence of a T790M resistance mutation found in a central nervous system (CNS) parenchymal metastasis. A concomitant squamous histology transformation in a lung non-T790M-resistant metastasis is also described. The authors hypothesize that this CNS resistance and histology transformation may have resulted from intermittent use of erlotinib treatment. This case report emphasizes the complexities of using erlotinib in the induction setting.

Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.

OBJECTIVE: To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. PARTICIPANTS: Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. EVIDENCE: Three unbiased literature searches of electronic databases were performed to capture published articles from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. EVIDENCE was formally graded for each recommendation. CONSENSUS PROCESS: Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). CONCLUSIONS: The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.

OBJECTIVE: To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. PARTICIPANTS: Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. EVIDENCE: Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. CONSENSUS PROCESS: Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). CONCLUSIONS: The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.

OBJECTIVE: To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. PARTICIPANTS: Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. EVIDENCE: Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. CONSENSUS PROCESS: Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). CONCLUSIONS: The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

p53 Testing for Li-Fraumeni and Li-Fraumeni-like syndromes.

Li-Fraumeni Syndrome (LFS; OMIM #151623) is an autosomal dominant cancer predisposition syndrome characterized by early onset tumors including sarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Li-Fraumeni syndrome is primarily attributed to germline mutations in the p53 tumor suppressor gene, which encodes tumor protein 53. In addition to germline p53 mutations, the p53 gene is the most commonly mutated gene in human cancers, with as much as 50% of tumors containing somatic p53 mutations. This unit provides a protocol to perform germline mutation analysis of the p53 gene. The protocol includes steps for amplification and sequencing of the entire coding region of the p53 gene (exons 2 to 11). The protocol was designed for detecting germline alterations from DNA extracted from blood; however, with some additional optimization, it could also be used to detect somatic mutations in DNA extracted from tumors.

Database of somatic mutations in EGFR with analyses revealing indel hotspots but no smoking-associated signature.

We created an Epidermal Growth Factor Receptor (EGFR) Mutation Database (http://www.cityofhope.org/cmdl/egfr_db) that curates a convenient compilation of somatic EGFR mutations in non-small-cell lung cancer (NSCLC) and associated epidemiological and methodological data, including response to the tyrosine kinase inhibitors Gefitinib and Erlotinib. Herein, we analyze 809 mutations collected from 26 publications. Four super hotspots account for 70% of reported mutations while two-thirds of 131 unique mutations have been reported only once and account for only 11% of reported mutations. Consistent with strong biological selection for gain of function, the reported mutations are virtually all missense substitutions or in-frame microdeletions, microinsertions, or microindels (colocalized insertion and deletion with a net gain or loss of 1-50 nucleotides). Microdeletions and microindels are common in a region of exon 19. Microindels, which account for 8% of mutations, have smaller inserted sequences (95% are 1 to 5 bp) and are elevated 16-fold relative to mouse somatic microindels and to human germline microindels. Microdeletions/microindels are significantly more frequent in responders to Gefitinib or Erlotinib (P = 0.003). In addition, EGFR mutations in smokers do not carry signatures of mutagens in cigarette smoke. Otherwise, the mutation pattern does not differ significantly with respect to gender, age, or tumor histology. The EGFR Mutation Database is a central resource of EGFR sequence variant data for clinicians, geneticists, and other researchers. Authors are encouraged to submit new publications with EGFR sequence variants to be included in the database or to provide direct submissions via The WayStation submission and publication process (http://www.centralmutations.org).

EGF receptor testing for non-small cell lung carcinomas.

Non-small cell lung cancer (NSCLC) is one of the most common cancers worldwide. An estimated 170,000 new diagnoses are expected this year. Recently, small molecule inhibitors directed at the EGFR kinase domain were approved for the treatment of advanced stages of NSCLC. Genotyping of the EGFR kinase domain has proven to be a useful marker for predicting who will respond to these novel medications. This unit provides a protocol to perform mutation analysis on the EGFR kinase domain where mutations have been associated with significant responsiveness to these EGFR inhibitors. The protocol includes microdissection of tumor tissue from slides, DNA digestion of these cells, amplifying and sequencing pertinent segments of the EGFR gene, and interpretation of the data. The protocol is designed with appropriate redundancy to eliminate allele dropout and to maximize detection of somatic mutations within the tumor.