Homologous recombination pathway gene variants identified by tumor-only sequencing assays in lung carcinoma patients.

Background: The homologous recombination (HR) repair pathway plays a key role in double-stranded DNA break repair, and germline HR pathway gene variants are associated with increased risk of several cancers, including breast and ovarian cancer. HR deficiency is also a therapeutically targetable phenotype. Methods: Somatic (tumour-only) sequencing was performed on 1,109 cases of lung tumors, and the pathological data were reviewed to filter for lung primary carcinomas. Cases were filtered for variants (disease-associated or of uncertain significance) in 14 HR pathway genes, including BRCA1, BRCA2, and ATM. The clinical, pathological and molecular data were reviewed. Results: Sixty-one HR pathway gene variants in 56 patients with primary lung cancer were identified. Further filtering by variant allele fraction (VAF) of ≥30% identified 17 HR pathway gene variants in 17 patients. ATM gene variants were most the commonly identified (9/17), including two patients with c.7271T>G (p.V2424G), a variant in the germline that is associated with increased familial cancer risk. Four (4/17) patients had a family history of lung cancer, among which three patients had ATM gene variants suspected to be germline in origin. In three other patients with BRCA1/2 or PALB2 gene variants who had undergone germline testing, the variants were confirmed to be germline; lung cancer was the sentinel cancer in two of these patients with a BRCA1 or PALB2 variant. Conclusions: Genomic variants in the HR repair pathway identified in tumor-only sequencing and occurring at higher VAFs (i.e., ≥30%) may suggest a germline origin. Correlating with personal and family history, a subset of these variants is also suggested to be associated with familial cancer risks. Patient age, smoking history and driver mutation status are expected to be a poor screening tool in identifying these patients. Finally, the relative enrichment for ATM variants in our cohort suggests a possible association between ATM mutation and lung cancer risk.

Getting Your Laboratory on Track With Neurotrophic Receptor Tyrosine Kinase.

CONTEXT.­: Neurotrophic receptor tyrosine kinase (NTRK) fusion testing has both diagnostic and therapeutic implications for patient care. With 2 tumor-agnostic US Food and Drug Administration-approved tropomyosin receptor kinase (TRK) inhibitors, testing is increasingly used for therapeutic decision making. However, the testing landscape for NTRK fusions is complex, and optimal testing depends on the clinicopathologic scenario. OBJECTIVE.­: To compare different NTRK testing methods to help pathologists understand test features and performance characteristics and make appropriate selections for NTRK fusion detection for their laboratory and individual patient specimens. DATA SOURCES.­: A literature search for NTRK gene fusions and TRK protein was performed, including papers that discussed treatment, testing methodology, and detection or prevalence of fusion-positive cases. CONCLUSIONS.­: As standard of care in some tumor types, next-generation sequencing (NGS) panel testing is a cost effective and reliable way to detect a broad range of NTRK fusions. The design of the panel and use of DNA or RNA will affect performance characteristics. Pan-TRK immunohistochemistry may be used as a rapid, less expensive screen in cases that will not undergo routine NGS testing, or on specimens unsuitable for NGS testing. Fluorescence in situ hybridization may be appropriate for low-tumor-content specimens that are unsuitable for NGS testing. Quantitative reverse transcription polymerase chain reaction is best suited for monitoring low-level disease of a specific, previously identified target. This information should help laboratories develop a laboratory-specific NTRK testing algorithm that best suits their practice setting and patients' needs.

Proceedings From the ASCO/College of American Pathologists Immune Checkpoint Inhibitor Predictive Biomarker Summit.

PURPOSE: Immune checkpoint inhibition (ICI) therapy represents one of the great advances in the field of oncology, highlighted by the Nobel Prize in 2018. Multiple predictive biomarkers for ICI benefit have been proposed. These include assessment of programmed death ligand-1 expression by immunohistochemistry, and determination of mutational genotype (microsatellite instability or mismatch repair deficiency or tumor mutational burden) as a reflection of neoantigen expression. However, deployment of these assays has been challenging for oncologists and pathologists alike. METHODS: To address these issues, ASCO and the College of American Pathologists convened a virtual Predictive Factor Summit from September 14 to 15, 2021. Representatives from the academic community, US Food and Drug Administration, Centers for Medicare and Medicaid Services, National Institutes of Health, health insurance organizations, pharmaceutical companies, in vitro diagnostics manufacturers, and patient advocate organizations presented state-of-the-art predictive factors for ICI, associated problems, and possible solutions. RESULTS: The Summit provided an overview of the challenges and opportunities for improvement in assay execution, interpretation, and clinical applications of programmed death ligand-1, microsatellite instability-high or mismatch repair deficient, and tumor mutational burden-high for ICI therapies, as well as issues related to regulation, reimbursement, and next-generation ICI biomarker development. CONCLUSION: The Summit concluded with a plan to generate a joint ASCO/College of American Pathologists strategy for consideration of future research in each of these areas to improve tumor biomarker tests for ICI therapy.

CNViz: An R/Shiny Application for Interactive Copy Number Variant Visualization in Cancer.

Copy number variants (CNVs) comprise a class of mutation which includes deletion, duplication, or amplification events that range in size from smaller than a single-gene or exon, to the size of a full chromosome. These changes can affect gene expression levels and are thus implicated in disease, including cancer. Although a variety of tools and methodologies exist to detect CNVs using data from massively parallel sequencing (also referred to as next-generation sequencing), it can be difficult to appreciate the copy number profile in a list format or as a static image. CNViz is a freely accessible R/Bioconductor package that launches an interactive R/Shiny visualization tool to facilitate review of copy number data. As inputs, it requires genomic locations and corresponding copy number ratios for probe, gene, and/or segment-level data. If supplied, loss of heterozygosity (LOH), focal variant data [single nucleotide variants (SNVs) and small insertions and deletions (indels)], and metadata (e.g., specimen purity and ploidy) can also be incorporated into the visualization. The CNViz R/Bioconductor package is an easy-to-use tool built with the intent of encouraging visualization and exploration of copy number variation. CNViz can be used in a clinical setting as well as for research to study patterns in human cancers more broadly. The intuitive interface allows users to visualize the copy number profile of a specimen, dynamically change resolution to explore gene and probe-level copy number changes, and simultaneously integrate LOH, SNV, and indel findings. CNViz is available for download as an R package via Bioconductor. An example of the application is available at rebeccagreenblatt.shinyapps.io/cnviz_example.

TERT gene rearrangement in chordomas and comparison to other TERT-rearranged solid tumors.

Chordomas are rare, slow-growing neoplasms thought to arise from the foetal notochord remnant. A limited number of studies that examined the mutational profiles in chordomas identified potential driver mutations, including duplication in the TBXT gene (encoding brachyury), mutations in the PI3K/AKT signaling pathway, and loss of the CDKN2A gene. Most chordomas remain without clear driver mutations, and no fusion genes have been identified thus far. We discovered a novel TERT in-frame fusion involving RPH3AL (exon 5) and TERT (exon 2) in the index chordoma case. We screened a discovery cohort of 18 additional chordoma cases for TERT gene rearrangement by FISH, in which TERT rearrangement was identified in one additional case. In our independent, validation cohort of 36 chordomas, no TERT rearrangement was observed by FISH. Immunohistochemistry optimized for nuclear TERT expression showed at least focal TERT expression in 40/55 (72.7%) chordomas. Selected cases underwent molecular genetic profiling, which showed low tumor mutational burdens (TMBs) without obvious driver oncogenic mutations. We next examined a cohort of 1,913 solid tumor patients for TERT rearrangements, and TERT fusions involving exon 2 were observed in 7/1,913 (0.4%) cases. The seven tumors comprised five glial tumors, and two poorly differentiated carcinomas. In contrast to chordomas, the other TERT-rearranged tumors were notable for higher TMBs, frequent TP53 mutations (6/7) and presence of other driver oncogenic mutations, including a concurrent fusion (TRIM24-MET). In conclusion, TERT gene rearrangements are seen in a small subset (2/55, 3.6%) of chordomas. In contrast to other TERT-rearranged tumors, where the TERT rearrangements are likely passenger events, the possibility that TERT protein overexpression representing a key event in chordoma tumorigenesis is left open.

A Curriculum for Genomic Education of Molecular Genetic Pathology Fellows: A Report of the Association for Molecular Pathology Training and Education Committee.

Molecular genetic pathology (MGP) is a subspecialty of pathology and medical genetics and genomics. Genomic testing, which is defined as that which generates large data sets and interrogates large segments of the genome in a single assay, is increasingly recognized as essential for optimal patient care through precision medicine. The most common genomic testing technologies in clinical laboratories are next-generation sequencing and microarray. It is essential to train in these methods and to consider the data generated in the context of the diagnosis, medical history, and other clinical findings of individual patients. Accordingly, updating the MGP fellowship curriculum to include genomics is timely, important, and challenging. At the completion of training, an MGP fellow should be capable of independently interpreting and signing out results of a wide range of genomic assays and, given the appropriate context and institutional support, of developing and validating new assays in compliance with applicable regulations. The Genomics Task Force of the MGP Program Directors, a working group of the Association for Molecular Pathology Training and Education Committee, has developed a genomics curriculum framework and recommendations specific to the MGP fellowship. These recommendations are presented for consideration and implementation by MGP fellowship programs with the understanding that MGP programs exist in a diversity of clinical practice environments with a spectrum of available resources.

Bayesian approach to interpreting somatic cancer sequencing data: a case in point.

Cancer lineage/tissue-of-origin assignment in cancers of unknown primary remains a challenge even when aided by massively parallel sequencing. The stakes are high for patients as many contemporary therapeutic strategies are disease-specific, and the biological differences can influence the patients' responses. Herein, we provide an example of how Bayesian analysis can be used to merge data from clinical history, histology, immunohistochemistry (IHC) and cancer DNA sequencing to assist in tissue-of-origin assignment. Iterative Bayesian analysis is performed through a set of simple calculations to calculate the OR between the differential diagnoses. We illustrate a clinical case, where the distinction between a primary lung versus metastatic bladder cancer was aided meaningfully by iterative Bayesian analyses, incorporating IHC and sequencing data.

Long Non-coding RNAs in Pulmonary Neuroendocrine Neoplasms.

Pulmonary neuroendocrine neoplasms (NENs) are classified into low-grade neuroendocrine tumors and high-grade neuroendocrine carcinomas (NECs). There are significant differences in therapeutic strategies of the different NEN subtypes, and therefore, precise classification of pulmonary NENs is critical. However, challenges in pulmonary NEN classification include overlap of diagnostic histological features among the subtypes and reduced or negative expression of neuroendocrine markers in poorly differentiated pulmonary NECs. Recently, transcription factor insulinoma-associated protein 1 (INSM1) was identified as a sensitive marker of neuroendocrine and neuroepithelial differentiation. In this study, INSM1 expression was detected by immunohistochemistry in greater than 94% of pulmonary NENs, indicating that it is a highly sensitive marker of pulmonary NENs and is useful to detect poorly differentiated pulmonary NECs. Although there are well-established morphological and immunohistologic criteria to diagnose pulmonary NENs, there is no universal consensus regarding prognostic markers of pulmonary NENs. Studies have shown that non-small cell lung cancers express long non-coding RNAs (lncRNAs), which regulate gene expression, epithelial-to-mesenchymal transition, and carcinogenesis. We characterized expression and function of lncRNAs, including HOX transcript antisense RNA (HOTAIR), maternally expressed 3 (MEG3), and prostate cancer antigen 3 (PCA3) in pulmonary NENs, including typical carcinoid tumors, atypical carcinoid tumors, small cell lung carcinoma (SCLC/NEC), and large cell neuroendocrine carcinoma (LCNEC/NEC). In situ hybridization and real-time polymerase chain reaction studies showed higher expression (p < 0.01) of all lncRNAs in SCLC/NEC. Small interfering RNA studies indicated a role for MEG3 and PCA3 in tumor proliferation. Therefore, these lncRNAs may serve as prognostic indicators of pulmonary NEN aggressiveness and as possible therapeutic targets.

Development and Validation of Molecular Assays for Limited Tissue Samples.

As the value of molecular testing of cancer specimens increases, the number of tests imposed on tumor specimens also increases, often in tension with the amount of tumor material available. To develop and validate molecular assays for limited specimens, there are specific concerns that must be addressed, including DNA quality, quantity, and abundance; the number of targets/ability to multiplex; and the analytical sensitivity and specificity of the assay itself. Ultimately, weighing these considerations during assay validation in the overall context of clinical utility and laboratory workflow is critical for delivering the highest level of personalized care to patients.

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms.

CONTEXT.­: Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. OBJECTIVE.­: To validate an anchored multiplex polymerase chain reaction-based panel for the detection of fusion transcripts in a university hospital-based clinical molecular diagnostics laboratory. DESIGN.­: We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. RESULTS.­: Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. CONCLUSIONS.­: This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

Impact of KRAS and TP53 Co-Mutations on Outcomes After First-Line Systemic Therapy Among Patients With STK11-Mutated Advanced Non-Small-Cell Lung Cancer.

PURPOSE: The STK11 gene encodes a serine/threonine protein kinase that regulates cell polarity and functions as a tumor suppressor. Patients with non-small-cell lung cancer (NSCLC) and STK11 mutations often have other co-mutations. We evaluated the impact of KRAS and TP53 co-mutations on outcomes after first-line systemic therapy for patients with metastatic or recurrent NSCLC that harbors STK11 mutations. METHODS: We conducted a retrospective review of patients with metastatic NSCLC and STK11 mutations treated at the University of Pennsylvania. STK11 mutations were identified through next-generation sequencing (NGS) in tissue or plasma. Cox proportional hazard models were used to determine the relationship between STK11 co-mutations and survival outcomes. The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). RESULTS: From February 2013 to December 2016, samples from 1,385 patients with NSCLC were analyzed by NGS; of these, 77 patients (6%) harbored an STK11 mutation (n = 56, tissue; n = 21, plasma). Of the 62 patients included, 18 had an STK11 mutation alone, 19 had STK11/KRAS, 18 had STK11/TP53, and seven had STK11/KRAS/TP53. Patients with STK11/KRAS co-mutations had a worse median PFS (2.4 months) compared with STK11 alone (5.1 months; log-rank P = .048), STK11/TP53 (4.3 months; log-rank P = .043), and STK11/KRAS/ TP53 (13 months; log-rank P = .03). Patients with STK11/KRAS co-mutation experienced shorter median OS (7.1 months) compared with STK11 alone (16.1 months; log-rank P < .001), STK11/TP53 (28.3 months; log-rank P < .001), and STK11/KRAS/TP53 (22 months; log-rank P = .025). CONCLUSION: Among patients with advanced NSCLC and STK11 mutations treated with first-line systemic therapy, co-mutation with KRAS was associated with significantly worse PFS and OS. By contrast, co-mutation of STK11 with TP53 conferred a better prognosis.

Cyclin D1 expression and novel mutational findings in Rosai-Dorfman disease.

Rosai-Dorfman disease (RDD) is an enigmatic histiocytic disorder classically diagnosed by a distinctive combination of pathological features: emperipolesis, or migration of intact haematological cells through the voluminous cytoplasm of lesional histiocytes, and expression of S100 by these histiocytes. The pathogenesis has long been elusive until the recent detection of recurrent and mutually exclusive mutations in several oncogenes in the mitogen-activated protein kinase (MAPK) pathway. Based on these findings, we investigated a cohort of 21 RDD patients and found that the lesional histiocytes in 86% (18/21) of patients exhibited strong and diffuse nuclear Cyclin D1 expression, which not only may provide a diagnostic marker for this sometimes pathologically challenging disease, but also probably reflects constitutive MAPK pathway activation because we additionally identified phosphorylated-ERK expression in 90% (19/21) of cases. Further, we performed massively parallel sequencing on a subset (6/18) of the CyclinD1 positive cases, identifying several mutations that have not been previously reported in RDD. Taken together, our findings bolster the concept of RDD as a disease of MAPK activation in a substantial percentage of cases and enhance the current understanding of the pathogenesis of RDD.

Histologic, immunohistochemical, and molecular features of pituicytomas and atypical pituicytomas.

Pituicytoma is a rare, poorly characterized tumor of the sellar region that is thought to be derived from neurohypophyseal pituicytes. Resection of pituicytomas is often associated with significant morbidity including diabetes insipidus and panhypopituitarism. Most of the literature on this tumor exists as small case series or case reports. Here we describe a cohort of fourteen pituicytoma resections from eleven patients. The average follow-up on these cases is 3.7 years with some patients having over 10 years of follow-up data available in the electronic medical record. Pituicytomas were frequently misdiagnosed on pre-operative imaging, and surgical resection was associated with persistent endocrine abnormalities. Histologically, the tumors showed a range of morphologies from epithelioid to spindled. All tumors were positive for TTF-1 with variable immunostaining for other markers including GFAP, EMA, S100, SSTR2A, and synaptophysin. Within this cohort are two patients with atypical pituicytomas which showed increased cellularity, pleomorphism, mitoses and elevated Ki-67 proliferation indexes when compared to non-atypical pituicytomas. Next generation sequencing performed on three tumors revealed alterations in genes involved in the MAPK pathway. Additionally, immunohistochemical staining for phosphorylated-ERK was positive in the majority of tumors. Increased awareness of the neoplastic entity and identification of targetable mutations have the potential to decrease the morbidity associated with resection of pituicytomas.

Thyroid-Like Follicular Carcinoma of the Kidney With Extensive Sarcomatoid Differentiation: A Case Report and Review of the Literature.

Thyroid-like follicular carcinoma of the kidney (TLFCK) is an extremely rare primary renal malignancy that typically has an indolent course and good prognosis. Histologically, this tumor mimics follicular carcinoma of the thyroid; however, typical thyroid markers are negative. There are fewer than 40 cases reported in the literature, and thus, the prognosis and course of disease is not well understood. Sarcomatoid differentiation has never been reported in a case of TLFCK. We present a case of a 48-year-old woman with an aggressive TLFCK with extensive sarcomatoid differentiation and metastatic disease at presentation. We performed targeted next-generation sequencing of both the thyroid-like component and the poorly differentiated sarcomatoid component using our solid tumor panel to evaluate for any disease-associated mutations and to better understand the molecular profile of these tumors.

Expression of Insulinoma-Associated Protein 1 (INSM1) and Orthopedia Homeobox (OTP) in Tumors with Neuroendocrine Differentiation at Rare Sites.

Insulinoma-associated protein 1 (INSM1) and orthopedia homeobox (OTP) are transcription factors that play a critical role in neuroendocrine (NE) and neuroepithelial cell development. INSM1 has been identified in multiple tumors of NE or neuroepithelial origin, whereas OTP expression has been mainly studied in NE tumors of pulmonary origin. Expression of OTP appears to correlate with poorer prognosis in pulmonary carcinoids; however, its expression patterns in other NE/neuroepithelial tumors need further investigation. Here, we assessed the diagnostic utility of INSM1 and OTP in tumors with NE differentiation at relatively uncommon sites including prostate, breast, and tumors of gynecologic origin. Thirty-two formalin-fixed, paraffin-embedded cases were used to construct a tissue microarray. Immunohistochemistry for INSM1 and OTP was performed and scored semi-quantitatively. INSM1 was diffusely expressed in 60% of gynecologic tumors, 71.4% of mammary carcinoma, and 25% of prostate adenocarcinoma with NE differentiation. Diffuse expression of OTP was detected in 50% of prostate adenocarcinoma with NE differentiation and 100% neuroendocrine carcinoma of the ovary. Immunostain for achaete-scute homolog 1, chromogranin, synaptophysin, and CD56 supported the NE and/or neuroepithelial differentiation of the tumors. In summary, INSM1 is expressed in most of the tumors with NE and neuroepithelial differentiation in this study, confirming the diagnostic utility of INSM1 as a novel and sensitive marker of NE/neuroepithelial differentiation. The expression of OTP in some NE tumors outside of lung expands the spectrum of tumors that may express this biomarker and should be considered when working up a NE tumor of unknown primary site.

Validation of a next-generation sequencing oncology panel optimized for low input DNA.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

SETD2 mutations in primary central nervous system tumors.

Mutations in SETD2 are found in many tumors, including central nervous system (CNS) tumors. Previous work has shown these mutations occur specifically in high grade gliomas of the cerebral hemispheres in pediatric and young adult patients. We investigated SETD2 mutations in a cohort of approximately 640 CNS tumors via next generation sequencing; 23 mutations were detected across 19 primary CNS tumors. Mutations were found in a wide variety of tumors and locations at a broad range of allele frequencies. SETD2 mutations were seen in both low and high grade gliomas as well as non-glial tumors, and occurred in patients greater than 55 years of age, in addition to pediatric and young adult patients. High grade gliomas at first occurrence demonstrated either frameshift/truncating mutations or point mutations at high allele frequencies, whereas recurrent high grade gliomas frequently harbored subclones with point mutations in SETD2 at lower allele frequencies in the setting of higher mutational burdens. Comparison with the TCGA dataset demonstrated consistent findings. Finally, immunohistochemistry showed decreased staining for H3K36me3 in our cohort of SETD2 mutant tumors compared to wildtype controls. Our data further describe the spectrum of tumors in which SETD2 mutations are found and provide a context for interpretation of these mutations in the clinical setting.

Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Insulinoma-associated 1: A novel nuclear marker in Merkel cell carcinoma (cutaneous neuroendocrine carcinoma).

Merkel cell carcinoma (MCC) is a rare, clinically aggressive, cutaneous neuroendocrine (NE) neoplasm. As a tumor with small, round, blue cells, the histologic differential diagnosis for MCC can include melanoma, metastatic small cell carcinoma (SCC), nodular hematopoietic tumors, basal cell carcinoma (BCC), atypical variants of squamous carcinoma and the uncommon occurrence of primary cutaneous Ewing sarcoma. In cases with atypical histology or without the classic immunophenotype, the diagnosis can be challenging. Ultimately, immunohistochemistry (IHC) is essential to the definitive diagnosis of MCC and in difficult cases, the diagnosis may hinge entirely on the immunophenotype of the tumor cells. Insulinoma-associated 1 (INSM1) is a transcription factor expressed in tissues undergoing terminal NE differentiation. As a nuclear protein tied to both differentiation and the cell cycle, INSM1 may offer additional utility in comparison to traditional, cytoplasmic markers of NE differentiation.

The Evolving Role of Companion Diagnostics for Breast Cancer in an Era of Next-Generation Omics.

A companion diagnostic is a test for a specific biomarker-approved by the United States Food and Drug Administration-qualifying a patient to receive a specific, associated therapy. As interest has grown in precision medicine over the past decade, the principle of companion diagnostics has gained increasing purchase among laboratory professionals, clinicians, regulators, and even patients. The evolution of the biomarkers used to stratify and treat breast cancer illustrates the history of companion diagnostics and provides a lens through which to examine potential challenges. As new targeted therapies and corresponding biomarkers accumulate, algorithms for diagnosis and treatment necessarily become lengthier and more complex. To accommodate future needs of breast cancer patients, the companion diagnostic model will continue to adapt and evolve.