Diagnostic work-up of hematological malignancies with underlying germline predisposition disorders (GPD).

Hematological malignancies with underlying germline predisposition disorders have been recognized by the World Health Organization 5th edition and International Consensus Classification (ICC) classification systems. The list of genes and the associated phenotypes are expanding and involve both pediatric and adult populations. While the clinical presentation and underlying molecular pathogenesis are relatively well described, the knowledge regarding the bone marrow morphologic features, the landscape of somatic aberrations associated with progression to hematological malignancies is limited. These pose challenges in the diagnosis of low-grade myelodysplastic syndrome (MDS) to hematopathologists which carries direct implication for various aspects of clinical management of the patient, donor selection for transplantation, and family members. Here in, we provide a focused review on the diagnostic work-up of hematological malignancies with underlying germline predisposition disorders with emphasis on the spectrum of hematological malignancies associated with each entity, and characteristic bone marrow morphologic, somatic cytogenetic and molecular alterations at the time of diagnosis of hematological malignancies. We also review the key clinical, morphologic, and molecular features, that should initiate screening for these entities.

Diagnostic challenges of renal medullary carcinoma and the role for cytologic assessment: Case report and literature review.

BACKGROUND: Renal medullary carcinoma (RMC) is a diagnostically challenging, aggressive primary renal malignancy associated with abysmal survival. Delays in diagnosis contribute to most patients having diffusely metastatic disease at the time of initial presentation. METHODS: We present the case of a 13-year-old African American male with sickle cell trait who presented with a renal mass and hematuria. Evaluation included imaging, fluid cultures, and cytologic assessment. RESULTS: Patient was diagnosed with RMC based on cytologic assessment of sub-centimeter fluid collections aspirated from the left kidney at the time of cortical biopsy for suspected renal mass. The additional fluid aspiration in conjunction with renal biopsy was an atypical but crucial step in early diagnosis. CONCLUSION: Cytomorphologic evaluation of fluid biospecimens is not currently part of the standard work-up for patients with renal masses but, when available, can provide crucial information that reduces time to diagnosis. Prompt symptom recognition and treatment initiation may improve patient outcomes.

Peripheral Blood Smear Detection of Asymptomatic Central Line Infection in a Patient With Sickle Cell Disease.

Sickle cell disease is a lifelong disorder which may be managed by chronic red cell transfusion including exchange transfusion. Chronic indwelling vascular catheters including ports offer convenient and reliable access for red cell exchange but confer risk of complications including infection and thrombosis. Detection of these complications is essential for preserving vascular access and relies on both clinical and laboratory observation. Here we describe a case of asymptomatic port infection detected by manual screening of a peripheral blood smear.

Quantitative magnetic resonance imaging for determining bone marrow fat fraction at 1.5 T and 3.0 T: a technique to noninvasively assess cellularity and potential malignancy of the bone marrow.

BACKGROUND: Pediatric bone marrow assessment by MRI is challenging and primarily experiential and qualitative, with a paucity of clinically useful quantitative imaging techniques. OBJECTIVE: MRI fat fraction (MRI-FF) is a technique used to quantify the degree of fat in other organ systems. The purpose of this study was to assess whether MRI-FF accurately measures bone marrow composition. MATERIALS AND METHODS: This two-part study included a validation phase, followed by an application phase. For the validation phase, the MRI-FF of piglet bones (6 long bones, 8 axial bones) was performed at 1.5 tesla (T) and 3.0 T, and correlated to the histological fat fraction (H-FF). We used Bland-Altman plots to compare MRI-FF at 1.5 tesla T and 3.0 T. For the application phase, five children with malignant marrow disease were recruited along with seven age- and gender-matched control subjects. The MRI-FF in the children was correlated to the H-FF. Boxplots were used to compare the MRI-FF of patients and control subjects. RESULTS: For the validation animal study, the MRI-FF of piglet bones at both 1.5 T and 3.0 T demonstrated moderate positive correlation to H-FF (r=0.41 and 0.42, respectively). MRI-FF at 1.5 T and 3.0 T were in good agreement, on average 7.7% apart. For the application phase, we included 5 children (4 with leukemia, 1 rhabdomyosarcoma) with median age 7 years, range (3-10 years). All children had MRI-FF and H-FF below 10%. The MRI-FF in patients (3.8±1.2) was significantly lower than that of control subjects (46.1±12.3%) (P<0.01). CONCLUSION: MRI-FF is a valid technique to assess bone marrow fat fraction at both 1.5 T and 3.0 T. The MRI-FF in children with malignant marrow processes is significantly lower than in control subjects with normal marrow.

Ultrasound is superior to palpation for thyroid cancer detection in high-risk childhood cancer and BMT survivors.

PURPOSE: Thyroid cancer is a common subsequent malignant neoplasm in childhood cancer survivors (CCS). Patients who received radiotherapy (RT) to the head, neck, upper thorax, or total body irradiation (TBI) are considered to be at risk for subsequent thyroid cancer. Current Children's Oncology Group screening guidelines recommend annual neck palpation. Our objective was to determine if ultrasound (US) is more sensitive and specific than palpation to detect thyroid cancer in high-risk CCS and bone marrow transplant (BMT) survivors. METHODS: Electronic medical records of patients followed in a longitudinal survivorship clinic from January 1, 2010 to December 31, 2017 were reviewed. Inclusion criteria included history of RT to the head, neck, upper thorax, or TBI for primary therapy or preparation for BMT prior to the age of 20 years. RESULTS: Two hundred and twenty-five patients had documented palpation and 144 (64%) also had US evaluation. Mean radiation dose was 28.6 Gy. Sixteen of 225 patients (7.1%) developed a subsequent thyroid cancer at a mean of 9.7 years from the completion of RT. Sensitivity of US was 100% compared with 12.5% for palpation. US demonstrated higher accuracy, with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.87 versus 0.56 for palpation (P < 0.0001). CONCLUSION: Routine screening with US was more sensitive than palpation for detection of subsequent thyroid cancer after high-risk RT in CCS and BMT survivors. Screening US may lead to earlier detection of thyroid cancer in this population. Earlier diagnosis has the potential to decrease operative complexity, and earlier definitive therapy reduces the likelihood of metastatic disease.

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

Pediatric Chronic Myeloid Leukemia Presenting With Extreme Thrombocytosis Simulating Essential Thrombocythemia.

A 10-year-old boy presented with spontaneous bruising and was found to have extreme thrombocytosis without neutrophilia/shift to immaturity, basophilia or eosinophilia. While the peripheral blood and bone marrow findings initially suggested essential thrombocythemia, BCR-ABL1 translocation was detected and chronic myeloid leukemia, chronic phase, was diagnosed. Apheresis for platelet depletion was performed as a bridge given the delayed effects of medical therapy.

Early mixed T-cell chimerism is predictive of pediatric AML or MDS relapse after hematopoietic stem cell transplant.

Patients with acute myeloid leukemia (AML) who relapse after hematopoietic stem cell transplantation (HCT) have dismal outcomes. Our ability to predict those at risk for relapse is limited. We examined chimerism trends post-HCT in 63 children who underwent HCT for AML or myelodysplastic syndrome (MDS). Mixed T-cell chimerism at engraftment and absence of chronic graft versus host disease (cGVHD) were associated with relapse (P = 0.04 and P = 0.02, respectively). Mixed T-cell chimerism at engraftment was predictive in patients without cGVHD (P = 0.03). Patients with engraftment mixed T-cell chimerism may warrant closer disease monitoring and consideration for early intervention.

Hermansky-Pudlak syndrome: Report of two patients with updated genetic classification and management recommendations.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder caused by mutations in one of nine genes involved in the packaging and formation of specialized lysosomes, including melanosomes and platelet-dense granules. The cardinal features are pigmentary dilution, bleeding diathesis, and accumulation of ceroid-like material in reticuloendothelial cells. Pulmonary fibrosis induced by tissue damage is seen in the most severe forms, and one subtype is characterized by immunodeficiency. We describe two patients with HPS type 1 and review the updated gene-based classification, clinical features, and recommendations for evaluation and follow-up.

Clonality assessment of cutaneous B-cell lymphoid proliferations: a comparison of flow cytometry immunophenotyping, molecular studies, and immunohistochemistry/in situ hybridization and review of the literature.

Cutaneous lymphoid infiltrates are diagnostically challenging. Although ancillary techniques to assess clonality can help distinguish between reactive lymphoid hyperplasia and lymphoma, one of the most widely used techniques in hematopathology, flow cytometry immunophenotyping (FCI), has not been routinely applied to skin specimens. We performed FCI on 73 skin specimens from 67 patients clinically suspected of having a cutaneous B-cell lymphoma (CBCL) and compared the results with those obtained from immunoglobulin heavy chain (IGH) gene molecular studies (58 cases, primarily by polymerase chain reaction) and either immunohistochemistry (IHC) or in situ hybridization to evaluate for light chain restriction (22 and 2 cases, respectively). Sufficient quantity of CD45 (leukocyte common antigen)-positive cells and staining quality were achieved in 88% of cases by FCI, and clonality was detected in 68% of CBCLs versus molecular studies showing sufficient DNA quality in 74% and only 39% clonality detection, and interpretable/contributory IHC results in 84% of cases with 55% clonality detection. Clonality was documented more frequently in secondary rather than primary CBCLs by all 3 techniques. Therefore, FCI is feasible and appears to be more reliable than molecular studies or IHC/in situ hybridization for detecting clonality in CBCLs and can provide additional prognostically and therapeutically relevant information. The exception is cases with plasmacytic differentiation such as marginal zone lymphoma for which IHC might be a superior tool. We have also shown that a large subset of primary cutaneous follicle center lymphomas express CD10 and/or BCL2 by FCI. Recent advances in FCI beg the question of applicability to cutaneous T-cell and NK-cell lymphomas.

Performance of ACR TI-RADS and the Bethesda System in Predicting Risk of Malignancy in Thyroid Nodules at a Large Children's Hospital and a Comprehensive Review of the Pediatric Literature.

While thyroid nodules are less common in children than in adults, they are more frequently malignant. However, pediatric data are scarce regarding the performance characteristics of imaging and cytopathology classification systems validated to predict the risk of malignancy (ROM) in adults and select those patients who require fine-needle aspiration (FNA) and possibly surgical resection. We retrospectively reviewed the electronic medical records of all patients 18 years of age or younger who underwent thyroid FNA at our institution from 1 July 2015 to 31 May 2022. Based on surgical follow-up from 74 of the 208 FNA cases, we determined the ROM for the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) ultrasound risk stratification system and The Bethesda System for Reporting Thyroid Cytopathology and added our results to those of pediatric cohorts from other institutions already published in the literature. We found the following ROMs for 1458 cases using ACR TI-RADS (TR): TR1. Benign: 2.2%, TR2. Not Suspicious: 9.3%, TR3. Mildly Suspicious: 16.6%, TR4. Moderately Suspicious: 27.0%, and TR5. Highly Suspicious 76.5%; and for 5911 cases using the Bethesda system: Bethesda I. Unsatisfactory: 16.8%, Bethesda II. Benign: 7.2%, Bethesda III: Atypia of Undetermined Significance: 29.6%, Bethesda IV. Follicular Neoplasm: 42.3%, Bethesda V. Suspicious for Malignancy: 90.8%, and Bethesda VI. Malignant: 98.8%. We conclude that ACR TI-RADS levels imply higher ROMs for the pediatric population than the corresponding suggested ROMs for adults, and, in order to avoid missing malignancies, we should consider modifying or altogether abandoning size cutoffs for recommending FNA in children and adolescents whose thyroid glands are smaller than those of adults. The Bethesda categories also imply higher ROMs for pediatric patients compared to adults.

Cytochemical Characterization of Cerebrospinal Fluid Macrophage Inclusions in Pediatric Patients Receiving Intrathecal Nusinersen (SPINRAZA®) for Spinal Muscular Atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by biallelic deletion of the SMN1 gene. Nusinersen, an antisense oligonucleotide delivered intrathecally, binds to the pre-mRNA of SMN1's pseudogene, SMN2, to prevent exon skipping and produce functional SMN protein to compensate for the deficiency caused by SMN1 deletion. CASE PRESENTATION: We reviewed 15 cerebrospinal fluid (CSF) cytology specimens from 8 patients receiving nusinersen for SMA. Macrophages with peculiar inclusions ("nusinophages") were seen in 8 specimens from 4 of the patients: 1 infant and 3 children with SMA type 1. This finding has only previously been reported in adults with SMA types 2 and 3 and in 2 infants with SMA type 1. DISCUSSION/CONCLUSION: Specimens containing nusinophages had a significantly higher proportion of macrophages and lower proportion of lymphocytes than those in which nusinophages were not detected. The macrophage inclusions do not represent iron or microorganisms and instead are composed, at least in part, of glycosaminoglycans. Because CSF is a common specimen type, cytotechnologists and cytopathologists need to be aware of these inclusions, so they do not interpret them erroneously as evidence of infection or hemorrhage, especially in light of the fact that oligonucleotide therapy has been approved for a variety of conditions and is currently under investigation for intrathecal delivery in several other neurodegenerative disorders.

High Prevalence of Gene Fusions and Copy Number Alterations in Pediatric Radiation Therapy-Induced Papillary and Follicular Thyroid Carcinomas.

Background: Childhood cancer survivors and bone marrow transplant recipients treated with radiation therapy (RT) are at increased risk for subsequent thyroid cancer. However, the genetic landscape of pediatric thyroid cancer, both primary and RT-induced, remains poorly defined, as pediatric papillary thyroid carcinoma (PTC) has been understudied compared with adults and data on pediatric follicular thyroid carcinoma (FTC) are virtually nonexistent. The objective of this study was to characterize and compare the molecular profiles of pediatric RT-induced PTC and FTC cases with primary pediatric thyroid cancers. Methods: A total of 41 differentiated thyroid carcinomas (11 RT cases and 30 primary cases) from 37 patients seen at Phoenix Children's Hospital between January 1, 2010 and December 31, 2019 were evaluated by targeted next-generation sequencing and/or BRAF immunohistochemistry. Results: Eighty-six percent (6/7) of RT-PTC harbored a gene fusion (GF) compared with 56% (14/25) of primary PTC; 14% (1/7) of RT-PTC had a single-nucleotide variant (SNV; specifically, a point mutation in the DICER1 gene) compared with 44% (11/25) of primary PTC (all of the latter had the BRAFV600E mutation). An exceedingly rare ROS1 fusion was identified in a child with RT-PTC. With respect to FTC, copy number alterations (CNAs) were seen in 75% (3/4) of RT cases compared with 40% (2/5) of primary cases. None of the RT-FTC had SNVs compared with 100% (5/5) of primary FTC. Conclusions: In children, the molecular profile of subsequent RT-induced thyroid cancers appears to differ from primary (sporadic and syndromic) cases, with a high prevalence of GFs in RT-PTC (similar to PTC occurring after the Chernobyl nuclear reactor accident) and CNAs in RT-FTC. A better understanding of the molecular mechanisms underlying these cancers may lead to more accurate diagnosis, prognosis, and treatment, as some of the genomic alterations are potentially targetable.

Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome.

Although Down syndrome (DS), the most common developmental genetic cause of intellectual disability, displays proliferation and migration deficits in the prenatal frontal cortex (FC), a knowledge gap exists on the effects of trisomy 21 upon postnatal cortical development. Here, we examined cortical neurogenesis and differentiation in the FC supragranular (SG, II/III) and infragranular (IG, V/VI) layers applying antibodies to doublecortin (DCX), non-phosphorylated heavy-molecular neurofilament protein (NHF, SMI-32), calbindin D-28K (Calb), calretinin (Calr), and parvalbumin (Parv), as well as ß-amyloid (APP/Aß and Aß1-42) and phospho-tau (CP13 and PHF-1) in autopsy tissue from age-matched DS and neurotypical (NTD) subjects ranging from 28-weeks (wk)-gestation to 3 years of age. Thionin, which stains Nissl substance, revealed disorganized cortical cellular lamination including a delayed appearance of pyramidal cells until 44 wk of age in DS compared to 28 wk in NTD. SG and IG DCX-immunoreactive (-ir) cells were only visualized in the youngest cases until 83 wk in NTD and 57 wk DS. Strong SMI-32 immunoreactivity was observed in layers III and V pyramidal cells in the oldest NTD and DS cases with few appearing as early as 28 wk of age in layer V in NTD. Small Calb-ir interneurons were seen in younger NTD and DS cases compared to Calb-ir pyramidal cells in older subjects. Overall, a greater number of Calb-ir cells were detected in NTD, however, the number of Calr-ir cells were comparable between groups. Diffuse APP/Aß immunoreactivity was found at all ages in both groups. Few young cases from both groups presented non-neuronal granular CP13 immunoreactivity in layer I. Stronger correlations between brain weight, age, thionin, DCX, and SMI-32 counts were found in NTD. These findings suggest that trisomy 21 affects postnatal FC lamination, neuronal migration/neurogenesis and differentiation of projection neurons and interneurons that likely contribute to cognitive impairment in DS.

Identification of KMT2A-ARHGEF12 fusion in a child with a high-grade B-cell lymphoma.

Rearrangements involving KMT2A are common in de novo and therapy-related acute myeloid and lymphoblastic leukemias. There is a diverse recombinome associated with KMT2A involving at least 135 partner genes, with more being discovered due to advances in molecular genetic diagnostics. KMT2A-ARHGEF12 fusion has only rarely been reported, in five cases of acute leukemia and a single case of high-grade B-cell lymphoma. We present a 12-year-old boy with high-grade B-cell lymphoma and KMT2A-ARHGEF12 fusion, whose clinical, morphologic, phenotypic and genotypic profile is strikingly similar to the other case of high grade B cell lymphoma, both otherwise perfectly mimicking Burkitt lymphoma.