Cryptic and atypical KMT2A-USP2 and KMT2A-USP8 rearrangements identified by mate pair sequencing in infant and childhood leukemia.

Infant leukemias are a rare group of neoplasms that are clinically and biologically distinct from their pediatric and adult counterparts. Unlike leukemia in older children where survival rates are generally favorable, infants with leukemia have a 5-year event-free survival rate of <50%. The majority of infant leukemias are characterized by KMT2A (MLL) rearrangements (~70 to 80% in acute lymphoblastic leukemia), which appear to be drivers of early leukemogenesis. In this report, we describe three cases: a 9-month-old female infant with B-acute lymphoblastic leukemia (B-ALL), an 8-month-old female presenting with B/myeloid mixed phenotype acute leukemia (MPAL), and a 16-month-old male with B-ALL. The first case had a normal karyotype and B-ALL FISH results consistent with an atypical KMT2A rearrangement. The second case had trisomy 10 as the sole chromosomal abnormality and a normal KMT2A FISH result. Case 3 had trisomy 8 and a t(11;15)(q23;q21), an atypical KMT2A rearrangement by FISH studies, and a focal deletion of 15q with a breakpoint within the USP8 gene by chromosomal microarray. Mate pair sequencing was performed on all three cases and identified a KMT2A-USP2 rearrangement (cases 1 and 2) or a KMT2A-USP8 rearrangement (case 3). These recently characterized KMT2A fusions have been described exclusively in infant and pediatric leukemia cases where the incidence varies vary according to leukemia subtype, are considered high-risk, with a high incidence of central nervous system involvement, poor response to initial prednisone treatment, and poor event free survival. Additionally, approximately half of cases are unable to be resolved using standard cytogenetic approaches and are likely under recognized. Therefore, targeted molecular approaches are suggested in genetically unresolved infant leukemia cases to characterize these prognostically relevant clones.

First Report of Prenatal Ascertainment of a Fetus With Homozygous Loss of the SOX10 Gene and Phenotypic Correlation by Autopsy Examination.

The SOX10 gene plays a vital role in neural crest cell development and migration. Abnormalities in SOX10 are associated with Waardenburg syndrome Types II and IV, and these patients have recognizable clinical features. This case report highlights the first ever reported homozygous loss of function of the SOX10 gene in a human. This deletion is correlated using family history, prenatal ultrasound, microarray analysis of amniotic fluid, and ultimately, a medical autopsy examination to further elucidate phenotypic effects of this genetic variation. Incorporating the use of molecular pathology into the autopsy examination of fetuses with suspected congenital anomalies is vital for appropriate family counseling, and with the ability to use formalin-fixed and paraffin-embedded tissues, has become a practical approach in autopsy pathology.

Next-Generation Sequencing Informatic Architecture Considerations.

Clinically relevant sequencing methodologies continue to expand in number, diversity, complexity, and scale. This evolving and varied landscape requires unique implementations in all aspects of the assay, including the wet bench, bioinformatics, and reporting. Following implementation, the informatics of many of these tests continue to change over time, from software and annotation source updates, guidelines, and knowledgebase changes to changes in underlying information technology (IT) infrastructure. Key principles can be applied when implementing the informatics of a new clinical test which can greatly improve the lab's ability to deal with these updates rapidly and reliably. In this chapter, we discuss a variety of informatics issues which span all NGS applications. In particular, there is the need for implementing a reliable, repeatable, redundant, and version-controlled bioinformatics pipeline and architecture and a discussion of common methodologies to address these needs.

Liquid Biopsy for Advanced Cancer: An Amplicon-Based Massively Parallel Sequencing Panel Approach to Precision Oncology.

Although discovered in the 1940s (Mandel and Metais, C R Seances Soc Biol Fil 142:241-243, 1948), cell-free DNA has only recently become a tool practical for use in clinical settings. The challenges associated with detection of circulating tumor DNA (ctDNA) in patient plasma are many and exist in the pre-analytical, analytical, and post-analytical periods. Initiation of a ctDNA program in a small academic clinical laboratory setting can be challenging. Thus, cost-effective, fast methods should be leveraged to promote a self-supporting system. Any assay should be based on clinical utility and have the potential to adapt in order to maintain relevance in a rapidly developing genomic landscape. Herein is described one of many approaches to ctDNA mutation testing - a massively parallel sequencing (MPS) method that is widely applicable and relatively easy to perform. Sensitivity and specificity are enhanced by unique molecular identification tagging and deep sequencing.

Managing a PCR Contamination Event in a Molecular Pathology Laboratory.

Contamination in a molecular laboratory may lead to erroneous results with potential to cause patient harm if not promptly identified and corrected. A general overview of the practices used in molecular laboratories to identify and address contamination once an event has occurred is discussed. The process used to assess the risk associated with the identified contamination event, determine the appropriate course of immediate action, perform a root cause analysis to determine the source of contamination, and assess and document the results of the decontamination process will be reviewed. Finally, the chapter will discuss a return to normal with consideration of appropriate corrective actions to mitigate future contamination events.

Novel high-risk acute myeloid leukemia subgroup with ERG amplification and Biallelic loss of TP53.

ETS-related gene (ERG) amplification, observed in 4-6% of acute myeloid leukemia (AML), is associated with unfavorable prognosis. To determine coincident effects of additional genomic abnormalities in AML with ERG amplification (ERGamp), we examined 11 ERGamp cases of 205 newly diagnosed AML using chromosomal microarray analysis and next generation sequencing. ERGamp cases demonstrated a distinct pattern of high genetic complexity: loss of 5q, chromothripsis and TP53 loss of function variants. Remarkably, allelic TP53 loss or loss of heterozygosity (LOH) co-occurring with TP53 inactivating mutation dramatically effected ERGamp tumor patient outcome. In the presence of homozygous TP53 loss of function, ERGamp patients demonstrated no response to induction chemotherapy with median overall survival (OS) of 3.8 months (N = 9). Two patients with heterozygous loss of TP53 function underwent alloSCT without evidence of relapse at one year. Similarly, a validation TCGA cohort, 6 of the 8 ERGamp cases with TP53 loss of function demonstrated median OS of 2.5 months. This suggests that with TP53 mutant ERGamp AML, successive loss of the second TP53 allele, typically by 17p deletion or LOH identifies a specific high-risk subtype of AML patients who are resistant to standard induction chemotherapy and need novel approaches to avert the very poor prognosis.

Clinical utility of chromosomal microarray in establishing clonality and high risk features in patients with Richter transformation.

Richter transformation (RT) refers to the development of an aggressive lymphoma in patients with pre-existing chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). It carries a poor prognosis secondary to poor response to therapy or rapid disease relapse. Currently there are no randomized trials to guide treatment. Therapeutic decisions are often influenced by the presence or absence of a clonal relationship between the underlying CLL/SLL and the new lymphoma given the poor prognosis of patients with clonally related RT. Chromosomal microarray analysis (CMA) can help to establish clonality while also detecting genomic complexity and clinically relevant genetic variants such as loss of CDKN2A and/or TP53. As a result, CMA has potential prognostic and therapeutic implications. For this study, CMA results from patients with Richter transformation were evaluated in paired CLL/SLL and transformed lymphoma samples. CMA revealed that 86% of patients had common aberrations in the two samples indicating evidence of common clonality. CMA was also useful in detecting aberrations associated with a poor prognosis in 71% of patients with RT. This study highlights the potential clinical utility of CMA to investigate the clonal relationship between CLL/SLL and RT, provide prognostic information, and possibly guide therapeutic decision making for patients with Richter transformation.

Molecular profiling of vitreous fluid by massively parallel sequencing: a case series.

INTRODUCTION: In cases of suspected intraocular malignancy, vitreous may be the preferred pathologic sample; however, cellularity may be insufficient for definitive cytopathological diagnosis. Ancillary methodology to study vitreous fluid aspiration for mutational analysis may assist in treatment decisions. MATERIALS AND METHODS: Three individual patient vitreous humor samples were received in the laboratory for mutation testing. The samples were collected during standard of care and analyzed for routine cytopathology. In each case, cytopathology was inconclusive and mutational analyses to support diagnostic suspicions were clinically requested. Based on the clinically and pathologically suspected diagnoses, an appropriate massively parallel sequencing assay previously validated for clinical use was performed using DNA extracted from vitreous samples that had previously undergone various processing. Nucleic acid yield was assessed by fluorometric or spectrophotometric methods, with yield ranging from 2.7 to 86.5 ng. Library preparations were performed using standard laboratory protocols. RESULTS: Two of the cases were suspicious for melanoma and a 50-gene solid tumor panel was performed. The third case was worrisome for vitreoretinal lymphoma and a 49-gene myeloid panel was performed. CONCLUSIONS: In all cases, the molecular profiling assisted with the clinical assessment and/or management of each patient.

Integrated genomic analysis using chromosomal microarray, fluorescence in situ hybridization and mate pair analyses: Characterization of a cryptic t(9;22)(p24.1;q11.2)/BCR-JAK2 in myeloid/lymphoid neoplasm with eosinophilia.

The 2016 World Health Organization entity 'Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2' encompasses a group of rare neoplasms that result from the formation of a fusion gene that leads to expression of an aberrant tyrosine kinase. This entity also contains variant JAK2 fusion partners, and detection of this defining event can be facilitated by various cytogenetic and molecular methods. Cryptic rearrangements of 9p24/JAK2 can be particularly challenging to identify. We describe the use of chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) with a probe for JAK2, and genomic mate pair analysis to describe a complex karyotype with a t(9;22) that produced a functional BCR-JAK2 fusion, leading to the appropriate diagnosis for the patient. This case highlights the importance of using an integrated genomic approach to fully define complex aberrations to assign proper diagnoses.

Benign Fibromyxoid Lesion of the Breast: A Distinct Entity From Benign Spindle Cell Tumors of the Mammary Stroma?

Myxoid lesions of the breast can be diagnostically challenging entities. We report 4 cases of CD34+ fibromyxoid lesion that have been previously diagnosed as "benign myxoid lesion," "nodular mucinosis," or "mammary myofibroblastoma, myxoid type" on the basis of CD34-positivity. The lesions were microscopically well circumscribed and composed of a paucicellular spindle cell proliferation in a background of myxoid stroma. No epithelial component was identified. The spindle cells showed immunohistochemical reactivity for CD34 and smooth muscle actin. Based on morphologic and immunohistochemical similarities between these cases and myxoid myofibroblastoma, we compared 4 myxoid lesions with cases of typical myofibroblastoma, utilizing retinoblastoma (Rb) antibody and fluorescent in situ hybridization for 13q14 gene rearrangement (encoding the Rb gene). The myxoid lesions showed retention of Rb protein by immunohistochemistry, whereas Rb expression was lost in cases of myofibroblastoma. We identified loss of 13q14 in 3 of 4 cases of myofibroblastoma. Notably, 13q14 gene rearrangement was not observed in any of the myxoid lesions. Our data show that there is at least a subset of CD34+ fibromyxoid lesions that, despite overlapping morphologic and immunohistochemical phenotype and proposed common histogenesis with myofibroblastomas, is genetically distinct from the latter based on Rb analysis.

IgM Myeloma with Plasma Cell Leukemia: Case Report and Literature Review.

IgM multiple myeloma (MM) is a rare entity representing approximately 0.5% of all MM. It should be distinguished from malignant neoplasms of B cells with plasmacytic differentiation such as Waldenstrom macroglobulinemia (WM) and marginal zone lymphoma with plasmacytic differentiation. Plasma cell leukemia (PCL) is a rare and aggressive variant of MM characterized by the presence of circulating plasma cells. We present a case report of a patient who presented with IgM MM in primary PCL phase with high-risk cytogenetics. To our knowledge, this is the first reported case of IgM MM with primarily leukemic presentation in the era of novel drugs. We demonstrate that it is important to distinguish IgM MM from WM and review the data from clinical trials that was used to devise a treatment strategy for this high-risk patient. This case adds to the understanding of the diagnosis and management of IgM MM in leukemic phase.

Amyloidosis and Unexpected Death: A Review of Seven Cases.

Amyloidosis, deposition of improperly folded insoluble proteins, may affect one organ or may be systemic. Although plasma cell dyscrasia is frequently implicated in etiology and is due to immunoglobulin light chain production (AL amyloidosis), age-related amyloidosis is believed to be secondary to transthyretin production (ATTR), chronic inflammation-related amyloidosis is thought due to acute phase reactants (AA amyloidosis), and dozens of others are also described. Clinical presentation is dependent upon the organ(s) involved and those associated with unexpected death are expected to involve the cardiovascular system. All cases received for forensic postmortem examination at the Medical University of South Carolina from 2008 to May 15, 2015 were searched to identify any in which amyloidosis was listed as a final diagnosis. Seven cases were identified that met the criteria and were reviewed for demographic information, presentation, cause and manner of death, and assessment of pertinence of the diagnosis of amyloidosis to the cause and manner of death. Interestingly, gross examination of the heart was suggestive of amyloidosis or other infiltrative process in only two of the cases reviewed and a history of myeloma was only noted in one individual. Common gross and microscopic findings are described and relevant medical history and toxicology findings are compared.

A 27-Year Retrospective Study of Pediatric Suicide Cases Referred to the Medical University of South Carolina (1988-2014).

Suicide by individuals under 18 years of age is a tragic reality of society. To assess local trends and demographics, we retrospectively reviewed all pediatric cases referred to our institution from 1988-2014 (27 years). Pediatric cases were defined as individuals younger than 18 years of age. The incidence of reported suicides declined from 3.8 cases per year to 2.4 cases per year as compared to observations at our institution over a previous ten-year period (1988-1998). In concert with the overall decrease in cases were increases in the proportion of adolescents younger than 15 and the female demographic. Furthermore, a shift in suicidal methodology was noted, with an increase in suicide by hanging. Indeed, for females, hanging became the most common suicide modality, replacing firearms. Our findings are congruent with national trends and underline the need for global suicide preventive interventions targeted toward increasingly younger adolescents and females.

Urothelial carcinoma of donor origin in a kidney transplant patient.

BACKGROUND: Malignancy after transplantation is an uncommon multifactorial occurrence. Immunosuppression to prevent graft rejection is described as a major risk factor in malignancy development in the post-transplant state. Donor-derived malignancy is a rare reported complication. Herein, we review our patient history and discuss diagnostic strategies and the implications of immunosuppression for donor-derived malignancy. CASE PRESENTATION: This is a 69-year-old man with post-renal-transplant urothelial carcinoma determined to be of donor origin. His course was complicated by BK virus at six years post-transplant; urothelial carcinoma was identified nine years post-transplant. Cystectomy was performed, but because of immunosuppression and underlying chronic kidney disease, the patient was considered ineligible for adjuvant chemotherapy. Two years after resection, screening MRI demonstrated retroperitoneal lymphadenopathy and a right upper pole mass in the transplanted kidney. Urine cytology confirmed the presence of malignant cells; FISH showed 2-8 copies of the X chromosome and no Y chromosome consistent with female origin of the malignant cells. CT-guided renal mass and paraaortic lymph node biopsies demonstrated that about 50 % of cells had an XY complement, while the remainder showed a XX genotype by chromosomal SNP microarray analysis. Immunosuppression was discontinued and the donor kidney removed. X/Y FISH of the urothelial carcinoma identified in the explanted kidney confirmed that the malignant cells were of female donor origin. Follow-up at 3, 6 and 12 months after discontinuation of immunosuppression and surgery demonstrated normalization of the lymphadenopathy and absence of new lesions. CONCLUSIONS: Immunosuppression is a major risk factor for development of malignancy in transplant recipients. Donor-derived malignancy can arise and current molecular studies allow an accurate diagnosis. Withdrawal of immunosuppression and surgical resection of the transplant kidney proved an effective treatment in our case.

A case example of asphyxia due to occupational exposure to airborne chemicals and review of workplace fatalities.

Although worker injury and fatalities have decreased since adoption of the Occupational Safety and Health Act in 1970, it remains an important safety issue. This article describes a 27-year-old white male who died from occupational exposure to airborne chemicals. Several trends in the last several decades, both in the types of injuries and the occupations associated with fatalities, are noted. Additionally, individual risk factors such as age, gender, chronic disease, smoking, and alcohol and drug use are implicated in worker health and safety. The role of the forensic pathologist in the investigation of workplace deaths is highlighted, in addition to the future of occupational safety and current improvements brought about by such incidents.

Sudden Cardiac Death in a Young Man with Migraine-associated Arrhythmia.

A 31-year-old man with migraine-induced syncope and bradycardia with subsequent pacemaker implantation died unexpectedly. Clinically unsuspected cardiac anomalies were found at autopsy including myocardial bridging of the left anterior descending artery and shelf-like coronary artery ostia. Nortriptyline was identified by toxicologic analysis. A review of the autopsy findings, the historical information, and the effects of the possible arrhythmogenic circumstances is undertaken and the potential contributions to the death are discussed. Cardiac arrhythmias have been documented during migraines. Coronary artery bridging has been known to lead to ischemia and infarction, ventricular tachycardia, and sudden death; however, these are very rare sequelae. Congenital coronary artery anomalies have been linked to sudden cardiac death, but only rarely cause death in people younger than 31 years. Migraines and the autopsy findings described have been associated with cardiac arrhythmia and sudden death. Altogether, they led to the unexpected death of this young man.

The importance of microscopic examination of the lungs in decedents with sustained central intravascular catheters: a nine-case series.

Indwelling intravascular catheters provide convenient access to healthcare personnel and also recreational intravenous drug users who inject suspensions of oral medications. A nine-case series of autopsies of clinically stable decedents with indwelling catheters and sudden death is herein presented. Pulmonary histologic findings were consistent with intravenous administration of oral medications in all cases. In eight, the mechanism of death was directly attributed to occlusive vascular embolization of foreign material, with or without contribution of acute drug toxicity. In one, the mechanism of death was solely attributed to acute drug toxicity. Acute, massive embolization of foreign material may explain sudden death by vascular obstruction, whereas chronic repeated injections lead to obliteration of the pulmonary vasculature, increased pulmonary vascular resistance, and cardiac failure. Therefore, a complete autopsy with histologic examination of the lungs and toxicology testing is recommended in patients with indwelling catheters to determine the cause and mechanism of death.

Fat embolism in pediatric patients: an autopsy evaluation of incidence and etiology.

INTRODUCTION: Little is known about the incidence and etiology of fat embolism in pediatric patients. We sought to determine the incidence, time course, and associated factors of pulmonary fat embolism (PFE), cerebral fat embolism (CFE), and kidney fat embolism (KFE) in trauma and nontrauma pediatric patients at the time of autopsy. METHODS: Retrospectively, a convenience sample of consecutive pediatric patients (age, ≤10 years) who had undergone autopsy between 2008 and 2012 were evaluated for fat embolism. Patients who had no documented cause of death or who were hospital births and died during the same hospitalization were excluded. Formalin-fixed paraffin sections were reviewed by a forensic pathologist for evidence of fat embolism and nuclear elements. Autopsy reports were used to determine cause of death, injuries, resuscitative efforts taken, sex, height, weight, and age. RESULTS: Sixty-seven decedents were evaluated. The median age was 2.0 years (interquartile range, 0.75-4), median body mass index (BMI) was 18.0 kg/m(2) (interquartile range, 15.7-19.0 kg/m(2)), and 55% of the patients were male. Pulmonary fat embolism, CFE, and KFE were present in 30%, 15%, and 3% of all patients, respectively. The incidence of PFE was not significantly different by cause of death (trauma 33%, drowning 36%, burn 14%, medical 28%). Patients with PFE but not CFE had significantly higher age, height, weight, and BMI. Half of the PFE and 57% of the CFE occurred in patients who lived less than 1 hour after beginning of resuscitation. Seventy-one percent of patients with CFE did not have a patent foramen ovale. Multivariate regression revealed an increased odds ratio of PFE based on BMI (1.244 [95% confidence interval, 1.043-1.484], P = .015). None of the samples evaluated demonstrated nuclear elements. CONCLUSIONS: Pulmonary fat embolism, CFE, and KFE are common in pediatric trauma and medical deaths. Body mass index is independently associated with the development of PFE. Absence of nuclear elements suggests that fat embolism did not originate from intramedullary fat.

The significance of adrenal hemorrhage: undiagnosed Waterhouse-Friderichsen syndrome, a case series.

A retrospective series of five cases of nontraumatic gross adrenal hemorrhage were identified in 800 consecutive forensic autopsies. All patients were males, of different ethnicities and with ages ranging from 2 to 48 years. All patients had a clinical history and autopsy findings suggestive of sepsis. Pre- or postmortem microbiological cultures were variably positive for Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. The fifth case was positive for yeast and a coagulase negative staphylococcus; contamination of this culture medium cannot be excluded. No cases had a culture positive for Neisseria meningitidis. We find that the reviewed patients with grossly or microscopically identifiable adrenal hemorrhage were otherwise healthy individuals who died suddenly as a consequence of bacterial infection. In each case, signs and symptoms compatible with premortem adrenal insufficiency were reported; in no instance was the adrenal hemorrhage clinically identified.