The Missing LNK: Evolution from Cytosis to Chronic Myelomonocytic Leukemia in a Patient with Multiple Sclerosis and Germline SH2B3 Mutation.

Chronic myelomonocytic leukemia (CMML) is a rare but distinct hematological neoplasm with overlapping features of myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Individuals with CMML have persistent monocytosis and bone marrow dyspoiesis associated with various constitutional symptoms like fevers, unintentional weight loss, or night sweats. It is established that there is a strong association of CMML with preceding or coexisting autoimmune diseases and systemic inflammatory syndromes affecting around 20% of patients. Various molecular abnormalities like TET2, SRSF2, ASXL1, and RAS are reported in the pathogenesis of CMML, but no such mutations have been described to explain the strong association of autoimmune diseases and severe inflammatory phenotype seen in CMML. Germline mutation in SH2B adaptor protein 3 (SH2B3) had been reported before to affect a family with autoimmune disorders and acute lymphoblastic leukemia. In this report, we describe the first case of a female subject with many years of preceding history of multiple sclerosis before the diagnosis of CMML. We outline the evidence supporting the pathogenic role of SH2B3 p.E395K germline mutation, connecting the dots of association between autoimmune diseases and CMML genesis.

A Primer on Chimeric Antigen Receptor T-cell Therapy: What Does It Mean for Pathologists?

CONTEXT.­: Chimeric antigen receptor T-cell (CAR-T) technology has shown great promise in both clinical and preclinical models in mediating potent and specific antitumor activity. With the advent of US Food and Drug Administration-approved CAR-T therapies for B-cell lymphoblastic leukemia and B-cell non-Hodgkin lymphomas, CAR-T therapy is poised to become part of mainstream clinical practice. OBJECTIVE.­: To educate pathologists on CAR-T and chimeric antigen receptor-derived cellular therapy, provide a better understanding of their role in this process, explain important regulatory aspects of CAR-T therapy, and advocate for pathologist involvement in the delivery and monitoring of chimeric antigen receptor-based treatments. Much of the focus of this article addresses US Food and Drug Administration-approved therapies; however, more general issues and future perspectives are considered for therapies in development. DESIGN.­: A CAR-T workgroup, facilitated by the College of American Pathologists Personalized Health Care Committee and consisting of pathologists of various backgrounds, was convened to develop a summary guidance paper for the College of American Pathologists Council on Scientific Affairs. RESULTS.­: The workgroup identified gaps in pathologists' knowledge of CAR-T therapy, including uncertainty in the role of the clinical laboratory in supporting CAR-T therapy. The workgroup considered these issues and summarized the findings to assist pathologists to become stakeholders in CAR-T therapy administration. CONCLUSIONS.­: This manuscript serves to both educate pathologists on CAR-T therapy and serve as a point of initial discussions in areas of CAR-T science, clinical therapy, and regulatory issues as CAR-T therapies continue to be introduced into clinical practice.

Locally Advanced Gastrointestinal Stromal Tumor in a 33-Year-Old Woman Seeking to Conceive.

Gastrointestinal stromal tumors (GISTs) are rare neoplasms of the gastrointestinal tract. They commonly present with nonspecific symptoms and thus are often discovered incidentally. They are best identified by CT scan, and most stain positive for CD117 (C-Kit), CD34, and/or DOG-1. Several risk stratification classification systems have been developed based on tumor size, mitotic rate, location, and perforation. Traditional chemotherapy and radiation therapy have been very ineffective, making surgery the mainstay of treatment. The discovery of mutations associated with these tumors has revolutionized the treatment approach. Imatinib mesylate, a selective tyrosine kinase receptor inhibitor, used as adjuvant or neoadjuvant therapy, has greatly improved the morbidity and mortality associated with GISTs. As the survival of patients has increased with the long-term use of targeted therapies, quality-of-life issues now have become much more relevant and have come to the forefront of care. We present a young woman who was successfully treated for GIST but now faces associated long-term adverse effects of imatinib, including the challenge of preserving fertility and the potential for childbearing.

Investigating the utility of extended mutation analysis in gastrointestinal peritoneal metastasis.

BACKGROUND AND OBJECTIVES: Outcomes for gastrointestinal peritoneal metastases (GI-PM) are worse compared to systemic metastases, with a paucity of data exploring extended mutation profiling. An exploratory mutation analysis in GI-PMs was performed as a "proof of concept" of potential predictive values of profiling in GI-PM and rates of actionable mutations. METHODS: The study included 40 GI-PM patients: 14 low-grade mucinous carcinoma peritonei and 26 HG-PM (12 colons, 10 appendix, 4 small bowels). Demographics, histologies, peritoneal cancer indexes, cytoreduction scores, and survival data were collected. NGS 50-gene mutation profiling was performed on 38 specimens. The association of mutations with survival was evaluated in high-grade PM. RESULTS: KRAS, TP53, and SMAD4 mutations were observed in 61%, 29%, and 8% of cases across all tumor histologies. In 66% cases >1 mutations occurred, associated with decreased survival in HG-PM: 32 vs 73 months, P = .03. TP53 or SMAD4 mutations were associated with decreased survival in HG-PM: 22 vs 48 months, P = .02. Actionable mutations were detected in 70%. CONCLUSION: Actionable mutations were detected at high rates. GI-PMs have similar mutational profiles and TP53, SMAD4, and/or >1 mutation were associate with decreased survival in HG-PM. This data supports the concept of the extended mutation profiling utility in GI-PM warranting further investigation.

Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine.

Biospecimens acquired during routine medical practice are the primary sources of molecular information about patients and their diseases that underlies precision medicine and translational research. In cancer care, molecular analysis of biospecimens is especially common because it often determines treatment choices and may be used to monitor therapy in real time. However, patient specimens are collected, handled, and processed according to routine clinical procedures during which they are subjected to factors that may alter their molecular quality and composition. Such artefactual alteration may skew data from molecular analyses, render analysis data uninterpretable, or even preclude analysis altogether if the integrity of a specimen is severely compromised. As a result, patient care and safety may be affected, and medical research dependent on patient samples may be compromised. Despite these issues, there is currently no requirement to control or record preanalytical variables in clinical practice with the single exception of breast cancer tissue handled according to the guideline jointly developed by the American Society of Clinical Oncology and College of American Pathologists (CAP) and enforced through the CAP Laboratory Accreditation Program. Recognizing the importance of molecular data derived from patient specimens, the CAP Personalized Healthcare Committee established the Preanalytics for Precision Medicine Project Team to develop a basic set of evidence-based recommendations for key preanalytics for tissue and blood specimens. If used for biospecimens from patients, these preanalytical recommendations would ensure the fitness of those specimens for molecular analysis and help to assure the quality and reliability of the analysis data.

A Model Information Management Plan for Molecular Pathology Sequence Data Using Standards: From Sequencer to Electronic Health Record.

Incorporating genetic variant data into the electronic health record (EHR) in discrete computable fashion has vexed the informatics community for years. Genetic sequence test results are typically communicated by the molecular laboratory and stored in the EHR as textual documents. Although text documents are useful for human readability and initial use, they are not conducive for data retrieval and reuse. As a result, clinicians often struggle to find historical gene sequence results on a series of oncology patients within the EHR that might influence the care of the current patient. Second, identification of patients with specific mutation results in the EHR who are now eligible for new and/or changing therapy is not easily accomplished. Third, the molecular laboratory is challenged to monitor its sequencing processes for nonrandom process variation and other quality metrics. A novel approach to address each of these issues is presented and demonstrated. The authors use standard Health Level 7 laboratory result message formats in conjunction with international standards, Systematized Nomenclature of Medicine Clinical Terms and Human Genome Variant Society nomenclature, to represent, communicate, and store discrete gene sequence data within the EHR in a scalable fashion. This information management plan enables the support of the clinician at the point of care, enhances population management, and facilitates audits for maintaining laboratory quality.

Epidermal Growth Factor Receptor Mutational Status and Brain Metastases in Non-Small-Cell Lung Cancer.

INTRODUCTION: Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancers (NSCLC) may be more common in patients with brain metastases. Previous studies, however, did not adjust for effects of confounding variables. METHODS: This retrospective study included 1,522 consecutive patients with NSCLC, whose tumors were diagnosed and tested for EGFR mutations at the University of Nebraska Medical Center (Omaha, NE) and Tata Memorial Hospital (Mumbai, India). Multivariate logistic regression was used to identify any association between EGFR status and clinical factors. RESULTS: EGFR mutations were more common in females than males (38.7% v 24.8%), Asians than whites (31.3% v 13.4%), nonsmokers than smokers (40.2% v 14.6%), alcohol nonconsumers than users (32.4% v 15.8%), adenocarcinoma than other histology types (32.7% v 10.3%), and patients with brain metastases than extracranial or no metastases (39.4% v 29.8% v 15.1%; P < .001 for all comparisons). There was a higher likelihood of an EGFR mutation among patients with brain metastases (odds ratio, 1.8; P < .001). The median overall survival (OS) was 19.8 months. Patients with brain metastases had a shorter median OS (15 v 20.6 months; P = .02). However, in the cohort of EGFR mutation-positive patients, there was no difference in median OS between patients with and without brain metastases (20.8 v 25.1 months; P = .11). CONCLUSION: There is a nearly two-fold higher incidence of EGFR mutations in NSCLC among patients with brain metastases at diagnosis. EGFR mutations did not predict for outcomes from brain metastases.

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology.

OBJECTIVES: - To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. METHODS: - The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. RESULTS: - Twenty-one guideline statements were established. CONCLUSIONS: - Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and the American Society of Clinical Oncology.

Purpose Molecular testing of colorectal cancers (CRCs) to improve patient care and outcomes of targeted and conventional therapies has been the center of many recent studies, including clinical trials. Evidence-based recommendations for the molecular testing of CRC tissues to guide epidermal growth factor receptor (EGFR) -targeted therapies and conventional chemotherapy regimens are warranted in clinical practice. The purpose of this guideline is to develop evidence-based recommendations to help establish standard molecular biomarker testing for CRC through a systematic review of the literature. Methods The American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and the American Society of Clinical Oncology (ASCO) convened an Expert Panel to develop an evidence-based guideline to help establish standard molecular biomarker testing, guide targeted therapies, and advance personalized care for patients with CRC. A comprehensive literature search that included over 4,000 articles was conducted to gather data to inform this guideline. Results Twenty-one guideline statements (eight recommendations, 10 expert consensus opinions and three no recommendations) were established. Recommendations Evidence supports mutational testing for genes in the EGFR signaling pathway, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize molecular testing for predictive and prognostic molecular biomarkers involve selection of assays, type of specimens to be tested, timing of ordering of tests and turnaround time for testing results. Additional information is available at: www.asco.org/CRC-markers-guideline and www.asco.org/guidelineswiki.

Molecular Biomarkers for the Evaluation of Colorectal Cancer.

Objectives: To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. Methods: The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. Results: Twenty-one guideline statements were established. Conclusions: Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented.

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology.

OBJECTIVES: To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. METHODS: The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. RESULTS: Twenty-one guideline statements were established. CONCLUSIONS: Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented. Key Words: Molecular diagnostics; Gastrointestinal; Histology; Genetics; Oncology.

QIAGEN Therascreen KRAS RGQ Assay, QIAGEN KRAS Pyro Assay, and Dideoxy Sequencing for Clinical Laboratory Analysis of KRAS Mutations in Tumor Specimens.

OBJECTIVE: To compare the performance of assays used to assess KRAS mutations in tumor specimens. METHODS: We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method. RESULTS: We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86% for the RGQ assay compared with 100% for the Pyro but 100% for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ. CONCLUSION: The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.

Clinical utility of KRAS and BRAF mutations in a cohort of patients with colorectal neoplasms submitted for microsatellite instability testing.

BACKGROUND: Molecular analysis has become important in colorectal carcinoma (CRC) evaluation. Alterations in KRAS, BRAF, or mismatch repair (MMR) genes may determine therapeutic response or define a hereditary cancer syndrome. Correlation of DNA studies with clinical findings will further clarify the clinical utility of these markers. PATIENTS AND METHODS: A retrospective study was performed on 111 paraffin-embedded tumor specimens submitted for microsatellite instability (MSI) testing based on clinical history or histologic examination, or both. DNA samples were screened for 7 KRAS mutations and the BRAF p.V600E mutation using fluorescent allele-specific polymerase-chain reaction (PCR) and capillary electrophoresis. Clinical data were collected through chart review. RESULTS: Fifty-eight male and 53 female patients were studied. The incidence of KRAS and BRAF mutations was 49.5% and 7.2%, respectively. Dideoxy sequencing verified KRAS mutation status in 46 of 49 specimens tested. There was a trend toward significance of individual KRAS mutations on survival (P = .003). Dually positive KRAS and MSI tumors exclusively demonstrated p.G12D and p.G13D mutations (G>A transitions). BRAF-mutated tumors were predominantly right-sided and associated with a borderline worse prognosis. Forty-eight percent of tumors with MSI were present in the left colon or rectum. CONCLUSION: Allele-specific PCR is an accurate and convenient method to assess KRAS and BRAF mutations and may detect mutations not identified by dideoxy sequencing. KRAS mutation status, in conjunction with morphologic or clinical parameters, may be useful in determining whether a tumor should be tested for MSI. MSI testing should not be considered exclusively in right-sided lesions. BRAF analysis may not be useful in rectal adenocarcinomas and should be evaluated in larger studies.

Comparison study of the performance of the QIAGEN EGFR RGQ and EGFR pyro assays for mutation analysis in non-small cell lung cancer.

OBJECTIVES: To compare 2 laboratory assays commonly used in the evaluation of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). METHODS: Fifty-three formalin-fixed, paraffin-embedded NSCLC specimens were selected. Extracted DNA was analyzed using the EGFR RGQ Amplification Refractory Mutation System Scorpions probe-based real-time polymerase chain reaction (PCR) assay and the EGFR Pyro pyrosequencing assay. RESULTS: Fourteen EGFR mutations were identified in 13 specimens using at least 1 of the assays, with a mutation concordance rate of 92.9%. Using dideoxy sequencing as the gold standard, clinical sensitivity was 73.7% and 68.4% by the RGQ and Pyro assays, respectively, but 100% by both for common drug sensitivity mutations. Performance observations included the following: the RGQ system requires higher DNA input, the RGQ system is a single-step procedure, the EGFR Pyro assay is a 2-step procedure, only the RGQ system can identify exon 20 insertions, the RGQ system is more sensitive, and the Pyro system can specify exact mutations for all interrogated sites. CONCLUSIONS: Both the RGQ real-time PCR and Pyro assays adequately detect common EGFR mutations; however, the RGQ system is more clinically and analytically sensitive. Performance characteristics should be considered when evaluating these EGFR mutation assays for clinical adoption.

Molecular diagnostics of soft tissue tumors.

CONTEXT: Soft tissue pathology encompasses a remarkably diverse assortment of benign and malignant soft tissue tumors. Rendering a definitive diagnosis is complicated not only by the large volume of existing histologic subtypes (>100) but also frequently by the presence of overlapping clinical, histologic, immunohistochemical, and/or radiographic features. During the past 3 decades, mesenchymal tumor-specific, cytogenetic and molecular genetic abnormalities have demonstrated an increasingly important, ancillary role in mesenchymal tumor diagnostics. OBJECTIVES: To review molecular diagnostic tools available to the pathologist to further classify specific soft tissue tumor types and recurrent aberrations frequently examined. Advantages and limitations of individual approaches will also be highlighted. DATA SOURCES: Previously published review articles, peer-reviewed research publications, and the extensive cytogenetic and molecular diagnostic experience of the authors to include case files of The University of Nebraska Medical Center. CONCLUSIONS: Cytogenetic and molecular genetic assays are used routinely for diagnostic purposes in soft tissue pathology and represent a powerful adjunct to complement conventional microscopy and clinicoradiographic evaluation in the formulation of an accurate diagnosis. Care should be taken, however, to recognize the limitations of these approaches. Ideally, more than one technical approach should be available to a diagnostic laboratory to compensate for the shortcomings of each approach in the assessment of individual specimens.

PU.1 regulates the expression of the vav proto-oncogene.

Vav is a guanine nucleotide exchange factor for the rho/rac GTPases that is upregulated in the embryo during the transition from primitive to definitive hematopoiesis. It is one of several genetic markers that correlates with the differentiation of the intraembryonic definitive hematopoietic stem cell. Subsequently, in the adult, vav is expressed predominantly in cells of the hematopoietic system. A heat-resistant protein complex that bound to a 23-bp segment, which is essential for vav promoter activity, was found to be present in myeloid cells but not T-cells. The complex was absent in non-hematopoietic cells which normally do not express vav. Using a saturation mutagenesis method, Mutex, a "footprint" of the protein binding site (AGAGGAAGT) was obtained that was consistent with the consensus binding site for PU.1. A specific antibody to PU.1 supershifted the complex and identified the presence of PU.1 within the complex. A GST fusion protein of the human PU.1 bound to the same consensus sequence as the heat-resistant complex from myeloid lineages. Specific mutation of the GGAA PU.1 core binding site silenced vav promoter activity and a dominant negative PU.1 inhibited the transactivation of PU.1 at the vav promoter as measured by the expression of the EGFP reporter gene. In addition, PCR analysis of immunoprecipitated chromatin using specific antibodies for PU.1 detected the co-immunoprecipitation of DNA containing the vav promoter. These results suggest that PU.1 is essential for transcriptional activity of the vav promoter in myeloid cells.