T/myeloid mixed phenotype acute leukaemia harbouring TLX3::BCL11B with TLX3 activation.

T/myeloid mixed phenotype acute leukaemia (MPAL) is a rare aggressive acute leukaemia with poorly understood pathogenesis. Herein, we report two cases of T/myeloid MPAL harbouring BCL11B-associated structural variants that activate TLX3 (TLX3::BCL11B-TLX3-activation) by genome sequencing and transcriptomic analyses. Both patients were young males with extramedullary involvement. Cooperative gene alterations characteristic of T/myeloid MPAL and T-lymphoblastic leukaemia (T-ALL) were detected. Both patients achieved initial remission following lineage-matched ALL-based therapy with one patient requiring a lineage-switched myeloid-based therapy. Our study is the first to demonstrate the clinicopathological and genomic features of TLX3::BCL11B-TLX3-activated T/myeloid MPAL and provide insights into leukaemogenesis.

Testicular Primary Well-Differentiated Neuroendocrine Tumor: Clinicopathologic, Immunohistochemical, and Molecular Characterization of Two Patients.

Well-differentiated neuroendocrine tumor rarely occurs as a testicular primary tumor, accounting for less than 1% of all testicular cancers, and is rarely reported with sufficient molecular profiles. After searching our departmental database (2003-2023), two testicular primary well-differentiated neuroendocrine tumors were identified in a 35-year-old man and a 23-year-old man, respectively, both of whom had normal serum level of tumor markers. Both tumors grossly exhibited solid, yellow-tan, and homogeneous appearance and histologically displayed a mixture of growth patterns, including organoid, tubular, cribriform, nests, cords, and single cells, were composed of eosinophilic tumor cells with salt-and-pepper chromatin and indistinct cell borders. Immunoreactivity for chromogranin and synaptophysin were detected, with Ki-67 labeling 9% and 2% of tumor cells on counting of 500 tumor cells, respectively. There was no germ cell neoplasia in situ in the background testicular parenchyma. Furthermore, fluorescence in situ hybridization failed to identify the presence of isochromosome 12p in both tumors. A panel-based next-generation sequencing was done in one of tumors and showed no reportable pathogenic variants with a mutation burden of 0.5 mutations per megabase. Although elevated mitotic figures (up to 6 per 10 high power fields), lymphovascular invasion and marked nuclear pleomorphism were present in this tumor, there was no evidence of disease detected in this patient via Dotatate positron emission tomography/computed tomography scan after the surgery. This report expands the spectrum of testicular primary well-differentiated neuroendocrine tumor. Considering its rarity, it may pose a diagnostic challenge or pitfall in certain clinical circumstances. In addition, the literature pertaining to this entity is herein reviewed.

Structural screening and molecular simulation identify potential ligands against the K700E hot spot variant and functional pockets of SF3B1 to modulate splicing in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS), a blood disorder with ineffective hematopoiesis and risk of transformation to acute myeloid leukemia, is characterized by recurring cytogenetic and molecular alterations. By chromosome analysis, approximately 60% of patients, carry chromosome 5 and 7 alterations, trisomy of chromosome 8 and may also present with increasingly complex karyotypes, especially in higher grade MDS (MDS with refractory anemia and increased blasts type 1 and 2). Moreover, somatic pathogenic variants in genes associated with aberrant mRNA splicing are frequently mutated with SF3B1 the most frequently mutated. In the setting of SF3B1, the K700E hot-spot mutation is present in approximately 50% of cases. Since recent studies have highlighted modulation of functional dynamics in SF3B1 by mutant splicing factors, the objective of the study was to identify potential small molecule modulators against the frequently mutated RNA splicing factor SF3B1(K700E) and functional allosteric sites by using a molecular structure-based approach and a molecular dynamic simulation. To identify potential SF3B1 modulators, we collected a series of chemical compounds from the Zinc and Enamine database. An initial screen followed by further molecular analysis and simulation using the Schrödinger suite was performed. Parameters used to monitor the stability and binding of the protein-ligand complex included: RMSF, protein-ligand contacts, electrostatic, Van Der Waals forces and binding energies (MMGBSA). A 100-nanosecond simulation showed strong binding between selected compounds and key amino acid residues, including the mutation hot-spot K700E and functional allosteric amino acid residue R630. Ligand binding energies between compounds and key amino acid residues ranged from -50.67 to -58.04 kcal/mol. In brief, small molecule modulators show strong binding to SF3B1 suggesting these compounds may be used against cells harboring the K700E variant or to modulate splicing by targeting functional allosteric sites.

Retroperitoneal Sarcomatoid Yolk Sac Tumor in a Chemotherapy-Naive Patient With Testicular Postpubertal Type Teratoma: A Rare Case Report With Emphasis on Molecular Features.

Sarcomatoid yolk sac tumor is a very rare histologic type of testicular germ cell tumor and is mainly reported in testicular germ cell tumor patients who receive chemotherapy. Herein, we report an extremely rare concurrent retroperitoneal sarcomatoid yolk sac tumor in a man with a testicular postpuberal teratoma before he received chemotherapy. A 37-year-old man initially presented with a persistent abdominal pain. Subsequent imaging studies revealed a 9.6-cm retroperitoneal mass, and 2 testicular masses (3.1 cm and 0.9 cm in greatest dimension, respectively). His serum tumor markers were within normal ranges. His radical orchiectomy demonstrated a postpubertal type teratoma with an adjacent scarring nodule. Later, his retroperitoneal tumor showed spindle tumor cells embedded in predominantly myxoid and focally fibrous stroma with diffuse and strong immunoreactivity for keratin AE1/AE3, SALL4 and glypican 3. No tumor necrosis or brisk mitotic figures were observed. A diagnosis of sarcomatoid yolk sac tumor was rendered. Fluorescence in situ hybridization analysis of his retroperitoneal sarcomatoid yolk sac tumor revealed polysomy 12 and MYC amplification, whereas no evidence of isochromosome 12p [i(12p)], and DNA sequencing showed 6 mutations per megabase (muts/Mb), and the somatic alterations included ARAF amplification and ATR I774Yfs*5. Considering its rarity, sarcomatoid yolk sac tumor may pose diagnostic challenges. Therefore, relevant clinicoradiologic information and ancillary work up, including immunohistochemistry and molecular studies, may be helpful for the accurate classification. Our tumor further raises awareness of this rare event, expands the spectrum of its clinical presentation, and explores the molecular features.

Clinicopathologic features of relapsed CD19(-) B-ALL in CD19-targeted immunotherapy: Biological insights into relapse and LILRB1 as a novel B-cell marker for CD19(-) B lymphoblasts.

INTRODUCTION: The mechanism of relapsed CD19(-) B-ALL after anti-CD19 immunotherapy (Kymriah [CART-19] and blinatumomab) is under active investigation. Our study aims to assess LILRB1 as a novel B-cell marker for detecting CD19(-) B-lymphoblasts and to analyze the clinicopathologic/genetic features of such disease to provide biological insight into relapse. METHODS: Six patients (3 males/3 females, median age of 14 years) with relapsed CD19(-) B-ALL were analyzed for cytogenetic/genetic profile and immunophenotype. RESULTS: CD19(-) B-ALL emerged after an interval of 5.8 months following anti-CD19 therapy. Five of six patients had B-cell aplasia, indicative of a persistent effect of CART or blinatumomab at relapse. Importantly, LILRB1 was variably expressed on CD19(-) and CD19(+) B lymphoblasts, strong on CD34(+) lymphoblasts and dim/partial on CD34(-) lymphoblasts. Three of six patients with paired B-ALL samples (pre- and post-anti-CD19 therapy) carried complex and different cytogenetic abnormalities, either as completely different or sharing a subset of cytogenetic abnormalities. CONCLUSION: LILRB1 can be used as a novel B-cell marker to identify CD19(-) B lymphoblasts. The emergence of CD19(-) B-ALL appears to be associated with complex cytogenetic evolutions. The mechanism of CD19(-) B-ALL relapse under anti-CD19 immune pressure remains to be explored by comprehensive molecular studies.

RNASeq Analysis for Accurate Identification of Fusion Partners in Tumor Specific Translocations Detected by Standard FISH Probes in Hematologic Malignancies.

Background: Fluorescence labeled DNA probes and in situ hybridization methods had shorter turn round time for results revolutionized their clinical application. Signals obtained from these probes are highly specific, yet they can produce fusion signals not necessarily representing fusion of actual genes due to other genes included in the probe design. In this study we evaluated discordance between cytogenetic, FISH and RNAseq results in 3 different patients with hematologic malignancies and illustrated the need to perform next generation sequencing (NGS) or RNASeq to accurately interpret FISH results. Methods: Bone marrow or peripheral blood karyotypes and FISH were performed to detect recurring translocations associated with hematologic malignancies in clinical samples routinely referred to our clinical cytogenetics laboratory. When required, NGS was performed on DNA and RNA libraries to detect somatic alterations and gene fusions in some of these specimens. Discordance in results between these methods is further evaluated. Results: For a patient with plasma cell leukemia standard FGFR3 / IGH dual fusion FISH assay detected fusion that was interpreted as FGFR3-positive leukemia, whereas NGS/RNASeq detected NSD2::IGH. For a pediatric acute lymphoblastic leukemia patient, a genetic diagnosis of PDGFRB-positive ALL was rendered because the PDGFRB break-apart probe detected clonal rearrangement, whereas NGS detected MEF2D::CSF1R. A MYC-positive B-prolymphocytic leukemia was rendered for another patient with a cytogenetically identified t(8;14) and MYC::IGH by FISH, whereas NGS detected a novel PVT1::RCOR1 not previously reported. Conclusions: These are 3 cases in a series of several other concordant results, nevertheless, elucidate limitations when interpreting FISH results in clinical applications, particularly when other genes are included in probe design. In addition, when the observed FISH signals are atypical, this study illustrates the necessity to perform complementary laboratory assays, such as NGS and/or RNASeq, to accurately identify fusion genes in tumorigenic translocations.

Genomic heterogeneity within B/T mixed phenotype acute leukemia in a context of an immunophenotype.

B/T mixed phenotype acute leukemia (MPAL) is a rare aggressive leukemia. Three cases of B/T MPAL were identified with comprehensive immunophenotypic, cytogenetic, and molecular studies. T-lineage predominant B/T MPAL shares a genetic signature with T-ALL whereas B/T lineage co-dominant B/T MPAL lacks such a T-ALL signature. All three patients were treated with lineage-matched-ALL therapy and alive at the last follow-up. Our study is the first to demonstrate molecular heterogeneity within B/T MPAL in a context of an immunophenotype of T-lineage versus B-lineage predominance. The implication of such a phenotype-genotype association on diagnostic classification is briefly discussed.

The challenges and opportunities of offering and integrating training in clinical molecular genetics and clinical cytogenetics: A survey of LGG Fellowship Program Directors.

Purpose: The specialty of Laboratory Genetics and Genomics (LGG) was created in 2017 in an effort to reflect the increasing convergence in technologies and approaches between clinical molecular genetics and clinical cytogenetics. However, there has not yet been any formal evaluation of the merging of these disciplines and the challenges faced by Program Directors (PDs) tasked with ensuring the successful training of laboratory geneticists under the new model. Methods: An electronic multi-question Qualtrics survey was created and was sent to the PD for each of the Accreditation Council for Graduate Medical Education-accredited LGG fellowship programs at the time. The data were collected, and the responses were aggregated for each question. Results: All of the responding PDs had started training at least 1 LGG fellow. PDs noted challenges with funding, staff shortages, molecular/cytogenetics content integration, limited total training time, increased remote work, increased sendout testing, and a lack of prior cytogenetics knowledge among incoming fellows. Conclusion: This survey attempted to assess the challenges that LGG PDs have been facing in offering and integrating clinical molecular genetics and clinical cytogenetics fellowship training. Common challenges between programs were noted, and a set of 6 concluding comments are provided to facilitate future discussion.