TFE3 rearrangements characterize histogenetically, topographically, and biologically diverse neoplasms. Besides being a universal defining feature in alveolar soft part sarcoma (ASPS) and clear cell stromal tumor of the lung, TFE3 fusions have been reported in subsets of renal cell carcinoma, perivascular epithelioid cell tumor (PEComa), epithelioid hemangioendothelioma and ossifying fibromyxoid tumors. TFE3 -related neoplasms are rare in the head and neck and may pose diagnostic challenges. We herein describe 22 TFE3 fusion neoplasms affecting 11 males and 11 females aged 4 to 79 years (median, 25) and involving different head and neck sites: sinonasal cavities (n = 8), tongue (n = 4), oral cavity/oropharynx (n = 3), salivary glands (n = 2), orbit (n = 2), and soft tissue or unspecified sites (n = 3). Based on morphology and myomelanocytic immunophenotype, 10 tumors qualified as ASPS, 7 as PEComas (3 melanotic; all sinonasal), and 5 showed intermediate (indeterminate) histology overlapping with ASPS and PEComa. Immunohistochemistry for TFE3 was homogeneously strongly positive in all cases. Targeted RNA sequencing/FISH testing confirmed TFE3 fusions in 14 of 16 successfully tested cases (88%). ASPSCR1 was the most frequent fusion partner in ASPS (4 of 5 cases); one ASPS had a rare VCP::TFE3 fusion. The 6 successfully tested PEComas had known fusion partners as reported in renal cell carcinoma and PEComas ( NONO, PRCC, SFPQ , and PSPC1 ). The indeterminate tumors harbored ASPSCR1::TFE3 (n = 2) and U2AF2::TFE3 (n = 1) fusions, respectively. This large series devoted to TFE3-positive head and neck tumors illustrates the recently proposed morphologic overlap in the spectrum of TFE3 -associated mesenchymal neoplasms. While all PEComas were sinonasal, ASPS was never sinonasal and occurred in diverse head and neck sites with a predilection for the tongue. The indeterminate (PEComa-like) category is molecularly more akin to ASPS but shows different age, sex, and anatomic distribution compared with classic ASPS. We report VCP as a novel fusion partner in ASPS and PSPC1 as a novel TFE3 fusion partner in PEComa (detected in one PEComa). Future studies should shed light on the most appropriate terminological subtyping of these highly overlapping tumors.

• MeSH
• Sarcoma, Alveolar Soft Part * genetics   pathology   MeSH
• Child MeSH
• Adult MeSH
• Phenotype MeSH
• Genetic Predisposition to Disease MeSH
• Gene Rearrangement * MeSH
• In Situ Hybridization, Fluorescence MeSH
• Immunohistochemistry MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Biomarkers, Tumor * genetics   analysis   MeSH
• Head and Neck Neoplasms * genetics   pathology   chemistry   MeSH
• Perivascular Epithelioid Cell Neoplasms * genetics   pathology   chemistry   MeSH
• Child, Preschool MeSH
• Aged MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors * genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

OBJECTIVES: The development of External Quality Assessment Schemes (EQAS) for clinical flow cytometry (FCM) is challenging in the context of rare (immunological) diseases. Here, we introduce a novel EQAS monitoring the primary immunodeficiency Orientation Tube (PIDOT), developed by EuroFlow, in both a 'wet' and 'dry' format. This EQAS provides feedback on the quality of individual laboratories (i.e., accuracy, reproducibility and result interpretation), while eliminating the need for sample distribution. METHODS: In the wet format, marker staining intensities (MedFIs) within landmark cell populations in PIDOT analysis performed on locally collected healthy control (HC) samples, were compared to EQAS targets. In the dry format, participants analyzed centrally distributed PIDOT flow cytometry data (n=10). RESULTS: We report the results of six EQAS rounds across 20 laboratories in 11 countries. The wet format (212 HC samples) demonstrated consistent technical performance among laboratories (median %rCV on MedFIs=34.5 %; average failure rate 17.3 %) and showed improvement upon repeated participation. The dry format demonstrated effective proficiency of participants in cell count enumeration (range %rCVs 3.1-7.1 % for the major lymphoid subsets), and in identifying lymphoid abnormalities (79.3 % alignment with reference). CONCLUSIONS: The PIDOT-EQAS allows laboratories, adhering to the standardized EuroFlow approach, to monitor interlaboratory variations without the need for sample distribution, and provides them educational support to recognize rare clinically relevant immunophenotypic patterns of primary immunodeficiencies (PID). This EQAS contributes to quality improvement of PID diagnostics and can serve as an example for future flow cytometry EQAS in the context of rare diseases.

• MeSH
• Immunophenotyping * standards   methods   MeSH
• Humans MeSH
• Flow Cytometry * standards   methods   MeSH
• Quality Control MeSH
• Immunologic Deficiency Syndromes diagnosis   immunology   MeSH
• Quality Assurance, Health Care MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Auer rods (AuRs) are prominent intracellular structures found almost exclusively in myeloid cell malignancies, such as acute myeloid leukemia (AML), chronic and juvenile myelomonocytic leukemia and myelodysplastic syndrome. Extremely rare AuRs have been reported in patients with acute lymphoblastic leukemia (ALL) or among ambiguous lineage leukemia patients with a dominantly lymphoblastic immunophenotype. PROCEDURE: We report diagnostic and follow-up data of an international cohort of 11 children suffering from leukemias with AuRs and with significant presence of T and myeloid markers, majority of whom categorized as early T-cell precursor (ETP, n = 7); or T-ALL (ETP status unknown, n = 2), ALAL (acute leukemia of ambiguous lineage, n = 1), and AML reclassified from ALAL (n = 1). We described other diagnostic details and treatment types and responses. Moreover, we summarize previously published data. RESULTS: Among the four patients who started and remained on ALL-type therapy, all were in the first complete remission, whereas both patients who started and remained on AML-type therapy relapsed and died. Of the patients who followed either a combined ALL/AML protocol (Interfant 06) or who switched from one of the two types of therapy to the other, one patient died, and the remaining four were in first complete remission at the most recent follow-up. We also searched for similar cases in the literature and found only three additional children with nonmyeloid leukemia and AuRs and 10 adults with this type of leukemia. CONCLUSIONS: Briefly, ALL- or combined ALL/AML-type therapy may be effective for treating AuR-positive leukemia patients with a lymphoid immunophenotype.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology   therapy   immunology   MeSH
• Leukemia, Myeloid, Acute pathology   therapy   immunology   MeSH
• Child MeSH
• Immunophenotyping * MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Follow-Up Studies MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Flow cytometry immunophenotyping is critical for the diagnostic classification of mature/peripheral B-cell neoplasms/B-cell chronic lymphoproliferative disorders (B-CLPD). Quantitative driven classification approaches applied to multiparameter flow cytometry immunophenotypic data can be used to extract maximum information from a multidimensional space created by individual parameters (e.g., immunophenotypic markers), for highly accurate and automated classification of individual patient (sample) data. Here, we developed and compared five diagnostic classification algorithms, based on a large set of EuroFlow multicentric flow cytometry data files from a cohort 659 B-CLPD patients. These included automatic population separators based on Principal Component Analysis (PCA), Canonical Variate Analysis (CVA), Neighbourhood Component Analysis (NCA), Support Vector Machine algorithms (SVM) and a variant of the CA(Canonical Analysis) algorithm, in which the number of SDs (Standard Deviations) varied for each of the comparisons of different pairs of diseases (CA-vSD). All five classification approaches are based on direct prospective interrogation of individual B-CLPD patients against the EuroFlow flow cytometry B-CLPD database composed of tumor B-cells of 659 individual patients stained in an identical way and classified a priori by the World Health Organization (WHO) criteria into nine diagnostic categories. Each classification approach was evaluated in parallel in terms of accuracy (% properly classified cases), precision (multiple or single diagnosis/case) and coverage (% cases with a proposed diagnosis). Overall, average rates of correct diagnosis (for the nine B-CLPD diagnostic entities) of between 58.9 % and 90.6 % were obtained with the five algorithms, with variable percentages of cases being either misclassified (4.1 %-14.0 %) or unclassifiable (0.3 %-37.0 %). Automatic population separators based on CA, SVM and PCA showed a high average level of correctness (90.6 %, 86.8 %, and 86.0 %, respectively). Nevertheless, this was at the expense of proposing a considerable number of multiple diagnoses for a significant proportion of the test cases (54.5 %, 53.5 %, and 49.6 %, respectively). The CA-vSD algorithm generated the smaller average misclassification rate (4.1 %), but with 37.0 % of cases for which no diagnosis was proposed. In contrast, the NCA algorithm left only 2.7 % of cases without an associated diagnosis but misclassified 14.0 %. Among correctly classified cases (83.3 % of total), 91.2 % had a single proposed diagnosis, 8.6 % had two possible diagnoses, and 0.2 % had three. We demonstrate that the proposed AI algorithms provide an acceptable level of accuracy for the diagnostic classification of B-CLPD patients and, in general, surpass other algorithms reported in the literature.

• MeSH
• Algorithms MeSH
• B-Lymphocytes * pathology   MeSH
• Immunophenotyping * methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lymphoproliferative Disorders * diagnosis   classification   MeSH
• Flow Cytometry * methods   MeSH
• Aged MeSH
• Support Vector Machine MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Úvod a cíl studie: Uterinní NK (uNK) buňky jsou specializovanou subpopulací NK (natural killer) lymfocytů nacházejících se v endometriu. Hrají klíčovou roli v regulaci imunitní odpovědi a v procesu implantace embrya. Cílem této studie je retrospektivní analýza výsledků léčby metodou in vitro fertilizace (IVF) v souboru žen, které podstoupily imunofenotypizaci uNK buněk a na základě výsledků tohoto vyšetření byly, nebo nebyly léčeny imunomodulační terapií. Metody: Studie zahrnovala 122 pacientek, které podstoupily imunofenotypizaci uNK buněk v období od dubna do prosince 2023. Imunofenotypizace byla provedena metodou průtokové cytometrie. Pacientky byly roztříděny do čtyř skupin dle fenotypu uNK buněk: normální nálezy, nízké absolutní a relativní počty uNK (LOW-IMMUNE profil), nízké počty uNK v kombinaci s nežádoucím posunem směrem k cytotoxickému uNKdim imunofenotypu (MIXED-IMMUNE profil) a normální počty uNK, ale nežádoucí posun v poměru cytotoxických a regulačních uNK s cytotoxickým fenotypem (OVER-IMMUNE profil). Byly hodnoceny výsledky embryotransferu a výskyt potratů do ukončeného 12. týdne těhotenství v jednotlivých skupinách. Výsledky: Nejvyšší míra dosažení klinické gravidity byla nalezena v léčené skupině OVER- -IMMUNE (70 %), následované skupinou MIXED-IMMUNE (60 %). Skupina LOW-IMMUNE se od neléčené NORMAL skupiny signifikantně nelišila (p = 0,205). Nedostatečná imunitní aktivace (LOW-IMMUNE profil) byla signifikantně nejčastěji sdružena s prvotrimestrální těhotenskou ztrátou (p < 0,0001). Závěr: Tato studie přináší nové poznatky o potenciálu imunofenotypizace uNK buněk a následné imunomodulační terapie v léčbě poruch plodnosti. Ačkoli výsledky naznačují možné klinické přínosy, je zapotřebí dalšího výzkumu k potvrzení těchto zjištění a k objasnění mechanizmů, které vedou ke zlepšení výsledků léčby technikami asistované reprodukce.

Introduction and objective: Uterine NK (uNK) cells, a specialized subpopulation of natural killer (NK) lymphocytes located in the endometrium, play a crucial role in regulating the immune response and in the process of embryo implantation. This study aims to retrospectively analyze the outcomes of in vitro fertilization (IVF) treatment in a cohort of women who underwent uNK cell immunophenotyping with subsequent immunomodulatory therapy applied based on the results. Methods: The study included 122 patients who underwent uNK cell immunophenotyping between April and December 2023. Immunophenotyping was performed using flow cytometry. Patients were categorized into four groups according to their uNK cell phenotypes: normal findings, low absolute and relative numbers of uNK cells (LOW-IMMUNE profile), low numbers of uNK cells combined with the shift towards the cytotoxic uNKc dim immunophenotype (MIXED-IMMUNE profile), and normal numbers of uNK cells, but an undesirable shift in the ratio of cytotoxic to regulatory uNK cells towards the cytotoxic uNK dim phenotype (OVER-IMMUNE profile). Embryo transfer outcomes and the occurrence of miscarriages up to the 12th week of pregnancy were evaluated in each group. Results: The highest clinical pregnancy rate was observed in the treated OVER-IMMUNE group (70%), fol lowed by the MIXED-IMMUNE group (60%). The LOW-IMMUNE group did not differ significantly from the untreated NORMAL group (P = 0.205). Insufficient immune activation (LOW-IMMUNE profile) was significantly associated with first-trimester pregnancy loss (P < 0.0001). Conclusion: This study provides new insights into the potential benefits of uNK cell immunophenotyping and subsequent immunomodulatory therapy in treating fertility disorders. While the results indicate possible clinical advantages, further research is necessary to confirm these findings and elucidate the mechanisms leading to improved outcomes in assisted reproductive techniques.

• Keywords
• uterinní NK buňky, imunofenotypizace lymfocytů, opakované selhání implantace,
• MeSH
• Killer Cells, Natural MeSH
• Endometrium cytology   MeSH
• Fertilization in Vitro * MeSH
• Abortion, Habitual MeSH
• Immunophenotyping MeSH
• Immunomodulation MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Retrospective Studies MeSH
• Infertility, Female * MeSH
• Check Tag
• Humans MeSH
• Female MeSH

OBJECTIVES: Risk-based stratification approaches using measurable residual disease (MRD) successfully help to identify T-acute lymphoblastic leukemia (T-ALL) patients at risk of relapse, whose treatment outcomes are very poor. Because of T-ALL heterogeneity and rarity, a reliable and standardized approach for flow cytometry (FC)-based MRD measurement and analysis is often missing. METHODS: Within the international AIEOP-BFM-ALL-FLOW study group we made a consensus on markers and a standard operating procedure for common 8- and 12-color T-ALL MRD panels. Custom manufactured tubes with dried backbone antibodies were tested in parallel to local FC standards. RESULTS: Altogether, 66 diagnostic and 67 day 15 samples were analyzed. We designed two guided MRD gating strategies to identify blast cells in parallel to expert-based evaluation. We proved that the optimized tubes allowed the correct identification of blast cells in all diagnostic samples. Both, expert and guided analysis of day 15 samples correlated to local standard (Spearman R=0.98 and R=0.94, respectively). Only in 2 (3 %) and 4 (6 %) patients expert gating and guided analysis results were substantially discordant from local standard, respectively. The cases that require an individualized approach may be partially identified at diagnosis through a rare immunophenotype or mixed phenotype acute leukemia status. CONCLUSIONS: Our work shows that standardized operating procedures together with guided analysis are applicable in a great majority of T-ALL cases. Further improvement of MRD detection is needed, as in some cases an individualized analytical approach is still required due to the challenging nature of the T-ALL phenotype.

• MeSH
• Child MeSH
• Infant MeSH
• Consensus MeSH
• Humans MeSH
• Precursor T-Cell Lymphoblastic Leukemia-Lymphoma * diagnosis   pathology   MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Flow Cytometry * standards   methods   MeSH
• Neoplasm, Residual * diagnosis   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH

Vascular neoplasms with epithelioid cytomorphology encompass a wide spectrum of benign and malignant lesions, including epithelioid hemangioma (EH), cutaneous epithelioid angiomatous nodule (CEAN), epithelioid hemangioendothelioma (EHE), and epithelioid angiosarcoma (EAS). Recently, the first case of a cutaneous hemangioma with epithelioid features harboring a TPM3::ALK fusion was reported. Herein, we report 4 additional cases, including 1 case with an alternate TPM4::ALK fusion, and expand on the clinicopathologic and molecular genetic features of these unusual vascular lesions. Including the previously reported case, 5 tumors occurred in 4 male and 1 female patients with a median age of 14 years (range: 2 to 38 y) and involved the shoulder region (2), the lower extremity (1), trunk (1), and head and neck (1). Clinical follow-up (3 patients; 60%) showed no evidence of disease at the last follow-up (median: 5 mo; range: 1 to 16 mo). Histologically, all tumors showed highly similar morphologic features, including an epidermal collarette, well-formed vascular channels composed of epithelioid endothelial cells with intracytoplasmic vacuoles, and admixed inflammatory cells. Immunohistochemically, all tumors were positive for vascular markers such as ERG and CD31, along with strong and diffuse cytoplasmic expression of ALK. RNA sequencing revealed recurrent TPM3 exon 8 :: ALK exon 20 (4) and TPM4 exon 7 :: ALK exon 20 fusions (1). We conclude that cutaneous hemangiomas with epithelioid features harboring TPM3/4::ALK fusions show consistent morphologic, immunophenotypic, and molecular genetic features. It remains to be determined whether this neoplasm represents a distinct entity or a molecular variant of epithelioid hemangioma.

• MeSH
• Anaplastic Lymphoma Kinase * genetics   MeSH
• Child MeSH
• Adult MeSH
• Epithelioid Cells * pathology   MeSH
• Phenotype MeSH
• Gene Fusion MeSH
• Hemangioma * genetics   pathology   MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Biomarkers, Tumor * genetics   analysis   MeSH
• Skin Neoplasms * genetics   pathology   MeSH
• Child, Preschool MeSH
• Tropomyosin * genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH

Persistent selective T-lymphocytopenia is found both in SCID and congenital athymia. Without molecular diagnosis, it is challenging to determine whether HCT or thymus transplantation ought to be performed. Ex vivo T-lymphopoiesis assays have been proposed to assist clinical decision-making for genetically undefined patients. We investigated 20 T-lymphocytopenic patients, including 13 patients awaiting first-line treatment and 7 patients with failed immune reconstitution after previous HCT or thymus transplantation. Whilst developmental blocks in ex vivo T-lymphopoiesis indicated hematopoietic cell-intrinsic defects, successful T-lymphocyte differentiation required careful interpretation, in conjunction with clinical status, immunophenotyping, and genetic investigations. Of the 20 patients, 13 proceeded to treatment, with successful immune reconstitution observed in 4 of the 6 patients post-HCT and 4 of the 7 patients after thymus transplantation, the latter including two patients who had previously undergone HCT. Whilst further validation and standardization are required, we conclude that assessing ex vivo T-lymphopoiesis during the diagnostic pathway for genetically undefined T-lymphocytopenia improves patient outcomes by facilitating corrective treatment choice.

• MeSH
• Cell Differentiation MeSH
• Child MeSH
• Immunophenotyping MeSH
• Infant MeSH
• Humans MeSH
• Lymphopenia * immunology   MeSH
• Lymphopoiesis * genetics   MeSH
• Adolescent MeSH
• Child, Preschool MeSH
• Primary Immunodeficiency Diseases therapy   genetics   immunology   MeSH
• T-Lymphocytes * immunology   MeSH
• Thymus Gland immunology   MeSH
• Hematopoietic Stem Cell Transplantation * methods   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

PURPOSE: The IMMUNOSARC trial combined an antiangiogenic agent (sunitinib) with a PD1 inhibitor (nivolumab) in advanced sarcomas. Here, we present the first correlative studies of the soft-tissue sarcoma cohort enrolled in this trial. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded and peripheral blood samples were collected at baseline and week 13. Formalin-fixed paraffin-embedded samples were used for transcriptomics and multiplex immunofluorescence, whereas peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients. RESULTS: The density of intratumoral CD8+ T cells, measured by multiplexed immunophenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in the gene expressions of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1 and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at week 13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated 2 groups with distinct progression-free survival (PFS): 4.1 months [95% confidence interval, 3.5-not reached (NR)] versus 17 months (95% confidence interval, 12.0-NR), P = 0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples. CONCLUSIONS: Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T-cell density and the expression of several genes/proteins with relevance in the response to PD1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of antiangiogenics plus PD1 inhibitor therapy.

• MeSH
• Programmed Cell Death 1 Receptor antagonists & inhibitors   MeSH
• CD8-Positive T-Lymphocytes immunology   drug effects   metabolism   MeSH
• Adult MeSH
• Angiogenesis Inhibitors therapeutic use   administration & dosage   MeSH
• Immune Checkpoint Inhibitors therapeutic use   pharmacology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Biomarkers, Tumor MeSH
• Tumor Microenvironment drug effects   immunology   MeSH
• Nivolumab therapeutic use   administration & dosage   MeSH
• Prognosis MeSH
• Antineoplastic Combined Chemotherapy Protocols therapeutic use   MeSH
• Sarcoma * drug therapy   pathology   genetics   MeSH
• Aged MeSH
• Lymphocytes, Tumor-Infiltrating immunology   metabolism   drug effects   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Classification MeSH
• Neoplasms diagnosis   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• patologie
• onkologie