Advancing Global Health Surveillance of Mycotoxin Exposures using Minimally Invasive Sampling Techniques: A State-of-the-Science Review.

Mycotoxins are a heterogeneous group of toxins produced by fungi that can grow in staple crops (e.g., maize, cereals), resulting in health risks due to widespread exposure from human consumption and inhalation. Dried blood spot (DBS), dried serum spot (DSS), and volumetric tip microsampling (VTS) assays were developed and validated for several important mycotoxins. This review summarizes studies that have developed these assays to monitor mycotoxin exposures in human biological samples and highlights future directions to facilitate minimally invasive sampling techniques as global public health tools. A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted. Key assay performance metrics were extracted to provide a critical review of the available methods. This search identified 11 published reports related to measuring mycotoxins (ochratoxins, aflatoxins, and fumonisins) using DBS/DSS and VTS assays. Multimycotoxin assays adapted for DBS/DSS and VTS have undergone sufficient laboratory validation for applications in large-scale population health and human biomonitoring studies. Future work should expand the number of mycotoxins that can be measured in multimycotoxin assays, continue to improve multimycotoxin assay sensitivities of several biomarkers with low detection rates, and validate multimycotoxin assays across diverse populations with varying exposure levels. Validated low-cost and ultrasensitive minimally invasive sampling methods should be deployed in human biomonitoring and public health surveillance studies to guide policy interventions to reduce inequities in global mycotoxin exposures.

Genomic characteristics and prognostic significance of co-mutated ASXL1/SRSF2 acute myeloid leukemia.

The ASXL1 and SRSF2 mutations in AML are frequently found in patients with preexisting myeloid malignancies and are individually associated with poor outcomes. In this multi-institutional retrospective analysis, we assessed the genetic features and clinical outcomes of 43 patients with ASXL1mut SRSF2mut AML and compared outcomes to patients with either ASXL1 (n = 57) or SRSF2 (n = 70) mutations. Twenty-six (60%) had secondary-AML (s-AML). Variant allele fractions suggested that SRSF2 mutations preceded ASXL1 mutational events. Median overall survival (OS) was 7.0 months (95% CI:3.8,15.3) and was significantly longer in patients with de novo vs s-AML (15.3 vs 6.4 months, respectively; P = .04 on adjusted analysis). Compared to ASXL1mut SRSF2wt and ASXL1wt SRSF2mut , co-mutated patients had a 1.4 and 1.6 times increase in the probability of death, respectively (P = .049), with a trend towards inferior OS (median OS = 7.0 vs 11.5 vs 10.9 months, respectively; P = .10). Multivariable analysis suggests this difference in OS is attributable to the high proportion of s-AML patients in the co-mutated cohort (60% vs 32% and 23%, respectively). Although this study is limited by the retrospective data collection and the relatively small sample size, these data suggest that ASXL1mut SRSF2mut AML is a distinct subgroup of AML frequently associated with s-AML and differs from ASXL1mut SRSF2wt /ASXL1wt SRSF2mut with respect to etiology and leukemogenesis.

Rosai-Dorfman Disease and Exocrine Pancreatic Insufficiency in a Patient With a Germline SLC29A3 Mutation.

Rosai-Dorfman disease (RDD) typically presents as bulky lymphadenopathy. Somatic mutations in RAS/MAP kinase pathway genes are common but germline mutations are rare. A patient with RDD and exocrine pancreatic insufficiency was found to have a homozygous germline mutation in SLC29A3, which has been associated with the Histiocytosis/Lymphadenopathy Plus Syndrome. His RDD also was positive for a somatic mutation in lymphoid enhancer binding factor 1 (LEF1). The concurrence of RDD and pancreatic insufficiency should raise consideration of SLC29A3 mutations. Other cases will be needed to confirm this observation and a possible contribution of LEF1 to the development of RDD.

Identifying transcriptional profiles and evaluating prognostic biomarkers of HIV-associated diffuse large B-cell lymphoma from Malawi.

Lymphoma incidence in sub-Saharan Africa (SSA) is increasing due to HIV and population aging. Diffuse Large B-cell lymphoma (DLBCL), the most common lymphoma in SSA and worldwide, is highly associated with HIV, but molecular studies of HIV-associated DLBCL are scarce globally. We describe profiling of DLBCL from Malawi, aiming to elucidate tumor biology and identify clinically meaningful biomarkers specifically for SSA. Between June 1, 2013 and June 1, 2016, 59 cases of DLBCL (32 HIV+/27 HIV-) enrolled in the Kamuzu Central Hospital Lymphoma Study were characterized, of which 54 (92%) were negative for Epstein-Barr virus. Gene expression profiling (GEP) by whole transcriptome sequencing was performed on the first 36 cases (22 HIV+/14 HIV-). Immunohistochemistry (IHC) and GEP results were compared with published data and correlated to clinical outcome and pathologic features. Unsupervised clustering strongly segregated DLBCL by HIV status (p = 0.0003, Chi-squared test), indicating a marked contribution of HIV to expression phenotype. Pathway analysis identified that HIV-associated tumors were enriched in hypoxia, oxidative stress, and metabolism related gene expression patterns. Cell-of-origin subtype, determined by sequencing and IHC, did not associate with differences in overall survival (OS), while Ki-67 proliferation index ≥80% was associated with inferior OS in HIV+ DLBCL only (p = 0.03) and cMYC/BCL2 co-expression by IHC was negatively prognostic across the entire cohort (p = 0.01). This study provides among the first molecular characterizations of DLBCL from SSA, demonstrates marked gene expression differences by HIV status, and identifies genomic and immunophenotypic characteristics that can inform future basic and clinical investigations.

Mature outcomes and prognostic indices in diffuse large B-cell lymphoma in Malawi: a prospective cohort.

Outcomes for diffuse large B-cell lymphoma (DLBCL) in sub-Saharan Africa (SSA) are poorly described. We report mature data from one of the first prospective SSA cohorts. Patients aged ≥18 years with DLBCL were enrolled in Malawi 2013-2017. Participants were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy and concurrent antiretroviral therapy (ART) if positive for human immunodeficiency virus (HIV+). Eighty-six participants (mean age 47 years, standard deviation 13) were enrolled: 54 (63%) were male and 51 (59%) were HIV+, of whom 34 (67%) were on ART at DLBCL diagnosis. Median CD4 count was 0·113 cells × 109 /l (interquartile range [IQR] 0·062-0·227) and 25 (49%) had HIV viral load <400 copies/µl. Participants received median six cycles CHOP (IQR 4-6). No patients were lost to follow-up and the 2-year overall survival was 38% (95% confidence interval 28-49). In multivariable analyses, Eastern Cooperative Oncology Group performance status (PS) ≥2 and lactate dehydrogenase (LDH) >2× upper limit of normal (ULN) were associated with mortality. HIV status was not associated with mortality. A simplified prognostic model of LDH >2× ULN and PS ≥2 performed at least as well as the age-adjusted International Prognostic Index. DLBCL can be successfully treated in SSA and outcomes did not differ by HIV status. A simplified prognostic model prognosticates well and may be easier to use in resource-limited settings but requires validation.

Epidemiological and histopathological profile of malignant melanoma in Malawi.

BACKGROUND: Studies on malignant melanoma have largely focused on Caucasian populations due to higher incidence in lighter-skinned individuals. While there is a well developed body of literature describing melanoma in African-Americans, much less is known about melanoma in black Africans. Prior reports have suggested that it is reportedly extremely rare in black Africans who are considered to mostly have the acral lentiginous subtype. However, an accurate understanding of melanoma in this part of the world is hindered by the very limited nature of prior publications. The aim of this study was to determine the epidemiological profile, anatomical distribution and histopathological features of melanoma presenting in Africans at a tertiary referral hospital in Malawi. METHODS: This is a retrospective study that characterized melanoma cases diagnosed from January 2012 to December 2017, at a cancer referral centre in Malawi. All confirmed, malignant melanoma cases during the study period were retrieved. Data abstracted included age, sex, anatomic site and whether it was a primary or metastatic site. Breslow thickness in millimetres, Clark level of invasion, presence of ulceration and melanoma subtype were also evaluated. RESULTS: One hundred thirty-two cases were included in the study, 81 (61%) were female and 26 (20%) were from a metastatic site. The mean age was 57 years (sd = 15) with the majority in the age group 60-69 years. Males presented at an older age than females. Ninety five percent of cutaneous melanomas were located on acral sites, most commonly the foot (87%) and the most common histopathological subtype was acral lentiginous. Eighty four percent presented with a Breslow thickness over 4 mm (median 9 mm). CONCLUSION: Our study shows that malignant melanoma occurs in black people in Malawi and may be an under-appreciated malignancy. While long term clinical follow-up was not available, most patients presented at late stages of the disease, supporting a poor prognosis. These results suggest that increased awareness of melanoma in black Africans and earlier intervention may have meaningful impacts on outcomes and survival.

Plasma Epstein-Barr virus DNA for pediatric Burkitt lymphoma diagnosis, prognosis and response assessment in Malawi.

Point-of-care tools are needed in sub-Saharan Africa (SSA) to improve pediatric Burkitt lymphoma (BL) diagnosis and treatment. We evaluated plasma Epstein-Barr virus (pEBV) DNA as a pediatric BL biomarker in Malawi. Prospectively enrolled children with BL were compared to classical Hodgkin lymphoma (cHL) and nonlymphoma diagnoses. Pediatric BL patients received standardized chemotherapy and supportive care. pEBV DNA was measured at baseline, mid-treatment, and treatment completion. Of 121 assessed children, pEBV DNA was detected in 76/88 (86%) with BL, 16/17 (94%) with cHL, and 2/16 (12%) with nonlymphoma, with proportions higher in BL versus nonlymphoma (p < 0.001) and similar in BL versus cHL (p = 0.69). If detected, median pEBV DNA was 6.1 log10 copies/mL for BL, 4.8 log10 copies/mL for cHL, and 3.4 log10 copies/mL for nonlymphoma, with higher levels in BL versus cHL (p = 0.029), and a trend toward higher levels in BL versus nonlymphoma (p = 0.062). pEBV DNA declined during treatment in the cohort overall and increased in several children before clinical relapse. Twelve-month overall survival was 40% in the cohort overall, and for children with baseline pEBV detected, survival was worse if baseline pEBV DNA was ≥6 log10 copies/mL versus <6 log10 copies/mL (p = 0.0002), and also if pEBV DNA was persistently detectable at mid-treatment versus undetectable (p = 0.041). Among children with baseline pEBV DNA detected, viremia was the only significant risk factor for death by 12 months in multivariate analyses (adjusted hazard ratio 1.35 per log10 copies/mL, 95% CI 1.04-1.75, p = 0.023). Quantitative pEBV DNA has potential utility for diagnosis, prognosis, and response assessment for pediatric BL in SSA.

Extranodal natural killer/T-cell lymphoma in Malawi: a report of three cases.

BACKGROUND: Extranodal NK/T-cell lymphoma (ENKTCL) reports from sub-Saharan Africa (SSA) are remarkably rare, despite early childhood acquisition and high prevalence of the causative infectious agent, Epstein-Barr virus (EBV), and frequent occurrence of other lymphoproliferative disorders causally associated with EBV. CASE PRESENTATIONS: At a national teaching hospital in Malawi, three patients of African descent were seen with ENKTCL between 2013 and 2014. Patients were aged between 29 and 60 years, two with craniofacial involvement and one with a primary abdominal tumor, and all were HIV-negative. All had systemic B symptoms, and two severely impaired performance status. On histologic review, morphology and immunophenotyping demonstrated classical ENKTCL features in all cases, including diffuse proliferations of intermediate-to-large atypical lymphocytes with high mitotic activity and extensive background necrosis, positivity for CD3 and CD56, and negativity for CD20. By in situ hybridization, all three tumors were positive for EBV-encoded RNA (EBER). Baseline plasma EBV DNA was also markedly elevated for all three patients. Due to radiotherapy and chemotherapy limitations, patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) with rapid disease progression. All three patients died from progressive lymphoma within 3 months of initial diagnosis. CONCLUSIONS: Our experience with these three patients in Malawi can highlight that ENKTCL does indeed occur in SSA, increase familiarity with ENKTCL among clinicians and pathologists throughout the region, and emphasize the need for better diagnosis and treatment for this neglected population.

Hodgkin lymphoma, HIV, and Epstein-Barr virus in Malawi: Longitudinal results from the Kamuzu Central Hospital Lymphoma study.

BACKGROUND: Contemporary descriptions of classical Hodgkin lymphoma (cHL) are lacking from sub-Saharan Africa where human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) are prevalent. METHODS: We describe a prospective cHL cohort in Malawi enrolled from 2013 to 2015. Patients received standardized treatment and evaluation, including HIV status and EBV testing of tumors and plasma. RESULTS: Among 31 patients with confirmed cHL, the median age was 19 years (range, 2-51 years) and 22 (71%) were male. Sixteen patients (52%) had stage III/IV, 25 (81%) B symptoms, and 16 (52%) performance status impairment. Twenty-three patients (74%) had symptoms >6 months, and 11 of 29 (38%) had received empiric antituberculosis treatment. Anemia was common with median hemoglobin 8.2 g/dL (range, 3.1-17.1 g/dL), which improved during treatment. No children and 5 of 15 adults (33%) were HIV+. All HIV+ patients were on antiretroviral therapy for a median 15 months (range, 2-137 months), with median CD4 count 138 cells/µL (range, 23-329 cells/µL) and four (80%) having undetectable HIV. EBV was present in 18 of 24 (75%) tumor specimens, including 14 of 20 (70%) HIV- and 4 of 4 (100%) HIV+. Baseline plasma EBV DNA was detected in 25 of 28 (89%) patients, with median viral load 4.7 (range, 2.0-6.7) log10 copies/mL, and subsequently declined in most patients. At 12 months, overall survival was 75% (95% confidence interval [CI], 55%-88%) and progression-free survival 65% (95% CI, 42%-81%). Baseline plasma EBV DNA and persistent viremia during treatment were associated with poorer outcomes. CONCLUSION: cHL in Malawi is characterized by delayed diagnosis and advanced disease. Most cases were EBV associated and one-third of adults were HIV+. Despite resource limitations, 12-month outcomes were good.

Outcomes for paediatric Burkitt lymphoma treated with anthracycline-based therapy in Malawi.

Burkitt lymphoma (BL) is the most common paediatric cancer in sub-Saharan Africa (SSA). Anthracyline-based treatment is standard in resource-rich settings, but has not been described in SSA. Children ≤18 years of age with newly diagnosed BL were prospectively enrolled from June 2013 to May 2015 in Malawi. Staging and supportive care were standardized, as was treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) for six cycles. Among 73 children with BL, median age was 9·2 years (interquartile range 7·7-11·8), 48 (66%) were male and two were positive for human immunodeficiency virus. Twelve (16%) had stage I/II disease, 36 (49%) stage III and 25 (34%) stage IV. Grade 3/4 neutropenia occurred in 17 (25%), and grade 3/4 anaemia in 29 (42%) of 69 evaluable children. Eighteen-month overall survival was 29% (95% confidence interval [CI] 18-41%) overall. Mortality was associated with age >9 years [hazard ratio [HR] 2·13, 95% CI 1·15-3·94], female gender (HR 2·12, 95% CI 1·12-4·03), stage (HR 1·52 per unit, 95% CI 1·07-2·17), lactate dehydrogenase (HR 1·03 per 100 iu/l, 95% CI 1·01-1·05), albumin (HR 0·96 per g/l, 95% CI 0·93-0·99) and performance status (HR 0·78 per 10-point increase, 95% CI 0·69-0·89). CHOP did not improve outcomes in paediatric BL compared to less intensive regimens in Malawi.

Comprehensive genomic profiling reveals molecular subsets of ASXL1-mutated myeloid neoplasms.

A large-scale genomic analysis of patients with ASXL1-mutated myeloid disease has not been performed to date. We reviewed comprehensive genomic profiling results from 6043 adults to characterize clinicopathologic features and co-mutation patterns by ASXL1 mutation status. ASXL1 mutations occurred in 1414 patients (23%). Mutation co-occurrence testing revealed strong co-occurrence (p < 0.01) between mutations in ASXL1 and nine genes (SRSF2, U2AF1, RUNX1, SETBP1, EZH2, STAG2, CUX1, CSF3R, CBL). Further analysis of patients with these co-mutations yielded several novel findings. Co-mutation patterns supported that ASXL1/SF3B1 co-mutation may be biologically distinct from ASXL1/non-SF3B1 spliceosome co-mutation. In AML, ASXL1/SRSF2 co-mutated patients frequently harbored STAG2 mutations (42%), which were dependent on the presence of both ASXL1 and SRSF2 mutation (p < 0.05). STAG2 and SETBP1 mutations were also exclusive in ASXL1/SRSF2 co-mutated patients and associated with divergent chronic myeloid phenotypes. Our findings support that certain multi-mutant genotypes may be biologically relevant in ASXL1-mutated myeloid disease.

A 137-kb deletion within the Potocki-Shaffer syndrome interval on chromosome 11p11.2 associated with developmental delay and hypotonia.

Potocki-Shaffer syndrome (PSS) is a rare disorder caused by haploinsufficiency of genes located on the proximal short arm of chromosome 11 (11p11.2p12). Classic features include biparietal foramina, multiple exostoses, profound hypotonia, dysmorphic features, and developmental delay/intellectual disability. Fewer than 40 individuals with PSS have been reported, with variable clinical presentations due in part to disparity in deletion sizes. We report on a boy who presented for initial evaluation at age 13 months because of a history of developmental delay, hypotonia, subtle dysmorphic features, and neurobehavioral abnormalities. SNP microarray analysis identified a 137 kb deletion at 11p11.2, which maps within the classically defined PSS interval. This deletion results in haploinsufficiency for all or portions of six OMIM genes: SLC35C1, CRY2, MAPK8IP1, PEX16, GYLTL1B, and PHF21A. Recently, translocations interrupting PHF21A have been associated with intellectual disability and craniofacial anomalies similar to those seen in PSS. The identification of this small deletion in a child with developmental delay and hypotonia provides further evidence for the genetic basis of developmental disability and identifies a critical region sufficient to cause hypotonia in this syndrome. Additionally, this case illustrates the utility of high resolution genomic approaches in correlating clinical phenotypes with specific genes in contiguous gene deletion syndromes.

Genome imprinting regulated by the mouse Polycomb group protein Eed.

Epigenetic regulation is essential for temporal, tissue-specific and parent-of-origin-dependent gene expression. It has recently been found that the mouse Polycomb group (PcG) gene Eed (embryonic ectoderm development) acts to maintain repression of the imprinted X chromosome. Here, we investigated whether Eed is also required for regulation of autosomal imprinted loci. Expression analyses showed that transcripts from the silent alleles of a subset of paternally repressed genes were present in Eed(-/-) embryos. Parent-of-origin methylation was preserved in these embryos, but we observed changes in the methylation status of specific CpGs in differentially methylated regions (DMRs) at affected but not at unaffected loci. These data identify Eed as a member of a new class of trans-acting factors that regulate parent-of-origin expression at imprinted loci.

Waldenström macroglobulinemia with secondary pure red cell aplasia in a patient with metastatic castrate resistant prostate cancer receiving an immune checkpoint inhibitor: a case report.

BACKGROUND: Hypoproliferative anemia is a frequently encountered adverse event in cancer patients receiving immune checkpoint inhibitors (ICI). Secondary pure red cell aplasia (PRCA) is a rare but recognized immune related adverse event. With the burgeoning use of ICIs, the association of secondary PRCA with an underlying lymphoproliferative disorder is often overlooked. CASE PRESENTATION: We report a case of a 67-year-old non-Hispanic Caucasian male with metastatic castrate resistant prostate cancer, who developed severe transfusion dependent anemia with reticulocytopenia while receiving treatment with olaparib and pembrolizumab. His bone marrow findings demonstrated erythroid hypoplasia, in addition to a CD5-negative, CD10-negative monotypic B-cell population and a somatic MYD88L265P mutation. With a presence of an IgM-paraprotein, he was diagnosed with Waldenström macroglobulinemia (WM) with secondary PRCA and treated with 6 cycles of bendamustine and rituximab. He achieved a complete response with this regimen and was transfusion independent. CONCLUSION: In this case, underlying WM was uncovered through systematic investigation of anemia caused by ICI therapy. This report highlights the possibility of a lymphoproliferative disorder in patients with concerns for PRCA with prior ICI exposure. If identified, treating the underlying lymphoproliferative disorder is highly efficacious in the management of the secondary PRCA.

A state-of-the-science review and guide for measuring environmental exposure biomarkers in dried blood spots.

BACKGROUND: Dried blood spot (DBS) sampling is a simple, cost-effective, and minimally invasive alternative to venipuncture for measuring exposure biomarkers in public health and epidemiological research. DBS sampling provides advantages in field-based studies conducted in low-resource settings and in studies involving infants and children. In addition, DBS samples are routinely collected from newborns after birth (i.e., newborn dried blood spots, NDBS), with many states in the United States permitting access to archived NDBS samples for research purposes. OBJECTIVES: We review the state of the science for analyzing exposure biomarkers in DBS samples, both archived and newly collected, and provide guidance on sample collection, storage, and blood volume requirements associated with individual DBS assays. We discuss recent progress regarding analytical methods, analytical sensitivity, and specificity, sample volume requirements, contamination considerations, estimating extracted blood volumes, assessing stability and analyte recovery, and hematocrit effects. METHODS: A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted in March 2022. DBS method development and application studies were divided into three main chemical classes: environmental tobacco smoke, trace elements (including lead, mercury, cadmium, and arsenic), and industrial chemicals (including endocrine-disrupting chemicals and persistent organic pollutants). DBS method development and validation studies were scored on key quality-control and performance parameters by two members of the review team. RESULTS: Our search identified 47 published reports related to measuring environmental exposure biomarkers in human DBS samples. A total of 28 reports (37 total studies) were on methods development and validation and 19 reports were primarily the application of previously developed DBS assays. High-performing DBS methods have been developed, validated, and applied for detecting environmental exposures to tobacco smoke, trace elements, and several important endocrine-disrupting chemicals and persistent organic pollutants. Additional work is needed for measuring cadmium, arsenic, inorganic mercury, and bisphenol A in DBS and NDBS samples. SIGNIFICANCE: We present an inventory and critical review of available assays for measuring environmental exposure biomarkers in DBS and NDBS samples to help facilitate this sampling medium as an emerging tool for public health (e.g., screening programs, temporal biomonitoring) and environmental epidemiology (e.g., field-based studies).

Applications of next-generation sequencing in hematologic malignancies.

In the last decade, next-generation sequencing (NGS) has rapidly progressed from a research method to a core component of standard-of-care clinical testing. In oncology, tumor sequencing provides a critical tool to detect somatic driver mutations that not only characterize disease but also impact therapeutic decision-making. Here, we review the important role of NGS in the evaluation of hematopoietic neoplasms. We discusstechnical and practical considerations relevant in somatic mutation testing, emphasizing issues unique to blood cancers. Then, we describe how NGS data is being used to facilitate diagnosis, inform prognosis, guide therapy selection, and even monitor disease. This broad overview highlights the transformative impacts NGS data provides throughout the clinical course of patients with hematologic malignancies.

Identification of high-risk monomorphic post-transplant lymphoproliferative disorder following solid organ transplantation.

Monomorphic post-transplant lymphoproliferative disorder (M-PTLD) occurring after solid organ transplant histologically resembles aggressive non-Hodgkin lymphomas, with diffuse large B-cell lymphoma being the most common. In a cohort of 40 patients with DLBCL-type M-PTLD, inferior progression free survival (PFS) was observed for Revised International Prognostic Index (R-IPI) >2 (p = 0.01) and high-risk pathologic features (p = 0.02), defined by double expressor lymphoma, MYC rearrangement, or increased copy number of either MYC or BCL2. Overall survival (OS) was inferior in R-IPI >2 (p = 0.002) and high-risk pathologic features (p = 0.003). Combining both R-IPI >2 and high-risk pathologic features resulted in well-delineated good, intermediate, and poor risk groups of DLBCL-type M-PTLD with respect to both PFS and OS (p < 0.001). Our results demonstrate a prognostic role for both the R-IPI score and presence of high-risk pathologic features in DLBCL-type M-PTLD.