Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes: A workshop report with focus on novel entities and a literature review including paediatric cases.

Myeloid/lymphoid neoplasms with eosinophilia (M/LN-eo) and tyrosine kinase (TK) gene fusions are a rare group of haematopoietic neoplasms with a broad range of clinical and morphological presentations. Paediatric cases have increasingly been recognised. Importantly, not all appear as a chronic myeloid neoplasm and eosinophilia is not always present. In addition, standard cytogenetic and molecular methods may not be sufficient to diagnose M/LN-eo due to cytogenetically cryptic aberrations. Therefore, additional evaluation with fluorescence in-situ hybridisation and other molecular genetic techniques (array-based comparative genomic hybridisation, RNA sequencing) are recommended for the identification of specific TK gene fusions. M/LN-eo with JAK2 and FLT3-rearrangements and ETV6::ABL1 fusion were recently added as a formal member to this category in the International Consensus Classification (ICC) and the 5th edition of the WHO classification (WHO-HAEM5). In addition, other less common defined genetic alterations involving TK genes have been described. This study is an update on M/LN-eo with TK gene fusions with focus on novel entities, as illustrated by cases submitted to the Bone Marrow Workshop, organised by the European Bone Marrow Working Group (EBMWG) within the frame of the 21st European Association for Haematopathology congress (EAHP-SH) in Florence 2022. A literature review was performed including paediatric cases of M/LN-eo with TK gene fusions.

A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors.

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

Human colon organoids reveal distinct physiologic and oncogenic Wnt responses.

Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.

Netrin-1 expression is an independent prognostic factor for poor patient survival in brain metastases.

The multifunctional molecule netrin-1 is upregulated in various malignancies and has recently been presented as a major general player in tumorigenesis leading to tumor progression and maintenance in various animal models. However, there is still a lack of clinico-epidemiological data related to netrin-1 expression. Therefore, the aim of our study was to elucidate the association of netrin-1 expression and patient survival in brain metastases since those constitute one of the most limiting factors for patient prognosis. We investigated 104 brain metastases cases for netrin-1 expression using in-situ hybridization and immunohistochemistry with regard to clinical parameters such as patient survival and MRI data. Our data show that netrin-1 is strongly upregulated in most cancer subtypes. Univariate analyses revealed netrin-1 expression as a significant factor associated with poor patient survival in the total cohort of brain metastasis patients and in sub-entities such as non-small cell lung carcinomas. Interestingly, many cancer samples showed a strong nuclear netrin-1 signal which was recently linked to a truncated netrin-1 variant that enhances tumor growth. Nuclear netrin-1 expression was associated with poor patient survival in univariate as well as in multivariate analyses. Our data indicate both total and nuclear netrin-1 expression as prognostic factors in brain metastases patients in contrast to other prognostic markers in oncology such as patient age, number of brain metastases or Ki67 proliferation index. Therefore, nuclear netrin-1 expression constitutes one of the first reported molecular biomarkers for patient survival in brain metastases. Furthermore, netrin-1 may constitute a promising target for future anti-cancer treatment approaches in brain metastases.

Diagnosis according to World Health Organization determines the long-term prognosis in patients with myeloproliferative neoplasms treated with anagrelide: results of a prospective long-term follow-up.

OBJECTIVES: During long term follow-up of a cohort of patients with essential thrombocythemia (ET) and polycythemia vera (PV) a higher than expected incidence of myelofibrosis (MF) was noted. In order to test if the explanation could be found in the diagnostic criteria a re-evaluation of diagnosis using the 2008 WHO diagnostic criteria for ET and MF was performed. METHODS: This prospective study of 60 patients with ET and PV was set up in 1998 to evaluate the long-term efficacy and tolerability of anagrelide treatment. Bone marrow trephine biopsies were requested from study start, after 2 and 7 years of follow-up. A blinded re-evaluation of the bone marrow trephines was performed. The 2008 WHO bone marrow criteria were used for diagnosis and fibrosis grading. RESULTS: Of 40 patients with an initial diagnosis of ET, 21 were confirmed as 'true ET' whereas 17 were reclassified as primary myelofibrosis (PMF) (12 PMF-0, 3 PMF-1, 2 PMF-2) and 2 as myeloproliferative neoplasms of uncertain origin. After 7 years of follow-up, 19 of 21 patients with 'true ET' were alive, none had transformed to MF, leukemia, or myelodysplastic syndrome. In contrast, 4/17 patients reclassified as PMF had died, two patients transformed to myelodysplastic syndrome and 7 patients progressed to overt MF. DISCUSSION: We conclude that a blinded re-evaluation of bone marrow trephines from study start and after 7 years of follow-up using 2008 World Health Organization criteria was able to differentiate between true ET and PMF with a marked difference in follow-up outcome.

Pomalidomide is active in the treatment of anemia associated with myelofibrosis.

PURPOSE: Thalidomide and lenalidomide can alleviate anemia in myelofibrosis. However, their value is undermined by their respective potential to cause peripheral neuropathy and myelosuppression. We therefore evaluated the safety and therapeutic activity of another immunomodulatory drug, pomalidomide. METHODS: In a phase II randomized, multicenter, double-blind, adaptive design study, four treatment arms were evaluated: pomalidomide (2 mg/d) plus placebo, pomalidomide (2 mg/d) plus prednisone, pomalidomide (0.5 mg/d) plus prednisone, and prednisone plus placebo. Pomalidomide was administered for up to 12 28-day treatment cycles. Prednisone (30 mg/d) was given in a tapering dose schedule during the first three cycles. Response was assessed by International Working Group criteria. RESULTS: Eighty-four patients with myelofibrosis-associated anemia were randomly assigned to the aforementioned treatment arms: 22, 19, 22, and 21, respectively. Response in anemia was documented in 20 patients, including 15 who became transfusion independent. Response rates in the four treatment arms were 23% (95% CI, 5% to 41%), 16% (95% CI, 0% to 33%), 36% (95% CI, 16% to 56%), and 19% (95% CI, 2% to 36%). The corresponding figures for patients receiving > or = 3 cycles of treatment (n = 62) were 38%, 23%, 40%, and 25%. Response to pomalidomide with or without prednisone was durable (range, 3.2 to 16.9+ months) and significantly better in the absence of leukocytosis (37% v 8%; P = .01); JAK2V617F or cytogenetic status did not affect response. Grade > or = 3 toxicities were infrequent and included (in each treatment arm) neutropenia (9%; 16%; 5%; 5%), thrombocytopenia (14%; 16%; 9%; 5%), and thrombosis (9%; 5%; 0%; 0%). CONCLUSION: Pomalidomide therapy at 0.5 or 2 mg/d with or without an abbreviated course of prednisone is well tolerated in patients with myelofibrosis and active in the treatment of anemia.

Myelofibrosis--what's in a name? Consensus on definition and EUMNET grading.

OBJECTIVE: Myelofibrosis (MF) implies an increase in the bone marrow (BM) fiber content without referring to quantity or quality (reticulin vs. collagen). METHODS: This review on chronic myeloproliferative disorders is based on initial and sequential BM biopsies, clinical data and follow-up examinations. A semiquantitative grading system for MF approved by a panel of experts was applied. RESULTS: In chronic myelogenous leukemia, minimal reticulin to advanced collagen MF is detectable at presentation in about 30% of patients. Significant correlations between BM and clinical features, but especially prognosis, are evident. Chronic idiopathic MF includes a prodromal stage showing no or little reticulin and no relevant MF with myeloid metaplasia (MMM). A stepwise evolution is demonstrable and associated with corresponding clinical data. Usually MMM is the diagnostic guideline for this disorder and consequently early stages with accompanying thrombocytosis may clinically mimic essential thrombocythemia. MF of various degrees may be observed in polycythemia vera depending on the progress of disease. Terminal stages (spent phase) reveal overt collagen corresponding with MMM. If diagnosis of essential thrombocythemia regards characteristic BM features, no relevant MF is seen at presentation and transformation into MMM is neglectable for many years. CONCLUSION: To recognize dynamics of the disease process in chronic myeloproliferative disorders, an easily to reproduce scoring system for MF has been proposed. The clinical diagnosis of MMM does not include initial-early reticulin MF and therefore fails to detect prodromal stages.

A critical reappraisal of the WHO classification of the chronic myeloproliferative disorders.

Following the introduction of the WHO classification of chronic myeloproliferative disorders (MPDs), after approximately 5 years, a critical reappraisal appears to be warranted. Retrospective clinico-pathological evaluations conducted in the meantime, as well as the detection of new biomarkers, may aid in testing the validity of these new criteria. Based on a large series of patients with chronic myeloid leukemia (CML), an analysis of bone marrow (BM) features and risk classifications revealed that the fiber content exerted a most important and independent impact on prognosis. This finding was also supported in a prospective randomized study and therefore myelofibrosis should be included in any staging system in CML related to survival. Moreover, it is important to emphasize the dynamics of the disease process in MPDs, especially in polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF). Latent-stage PV is difficult to recognize when adhering to the proposed limits for hemoglobin (or red cell mass) without regarding the erythropoietin (EPO) level, endogenous erythroid colonies (EECs) or BM histopathology. Initial PV may firstly present with complications and, when accompanied by a high platelet count, mimics essential thrombocythemia (ET). Consequently, BM morphology and EPO level should be entered as major diagnostic criteria for PV. To document more accurately the progress of disease, a simplified scoring system concerning myelofibrosis has to be included in the histological description of CIMF. The diagnostic guidelines of BM features in ET should be improved because, usually, there is neither a significant proliferation nor left-shifting of the granulo- and erythropoiesis detectable and no relevant increase in reticulin. A comparison of clinical data and BM morphology reveals that biomarkers (EPO, EECs, PRV-1, JAK2) show an overlapping pattern of positivity between the different subtypes of MPDs.

Pegylated interferon for the treatment of high risk essential thrombocythemia: results of a phase II study.

BACKGROUND AND OBJECTIVES: Patients with high-risk essential thrombocythemia require cytoreductive therapy in order to normalize the elevated platelet counts. We evaluated the efficacy and toxicity of pegylated interferon in high-risk essential thrombocythemia in a phase II trial. DESIGN AND METHODS: Thirty-six patients with high-risk essential thrombocythemia (median age 54 years; range, 24-72 years) were studied. The dose of pegylated interferon was initially 50 mg per week and could be escalated up to 150 mg per week. RESULTS: During the first three months platelet counts decreased significantly from a median baseline count of 895x10(9)/L (range: 383-1779) to a median count of 485x10(9)/L (range: 211-1283; p<=0.001). A complete response was defined as platelet counts < 450x10(9)/L. The complete response rate was 39%, 47%, 58% and 67% at 3, 6, 9 and 12 months of treatment, respectively. There were 25%, 11%, 8% and 0% poor responders, defined as patients with platelet counts > 600x10(9)/L, at 3, 6, 9 and 12 months of treatment, respectively. After a median time of 23 months (range 3-39 months) 23 of 36 patients (64%) are still receiving pegylated interferon. In ten patients (28%) treatment was stopped due to grade 1 to 2 toxicity, classified according to the WHO standard toxicity scale. One patient, who responded partially to pegylated interferon (platelet count 542x10(9)/L), had a cerebral stroke after 23 months of treatment. INTERPRETATION AND CONCLUSIONS: In high-risk essential thrombocythemia sustained treatment with pegylated interferon is effective and safe in reducing platelet counts with a toxicity comparable to that of conventional interferon.

Hematopathologic findings in chronic idiopathic myelofibrosis.

Until now scant knowledge was available about the dynamics of chronic idiopathic myelofibrosis (CIMF). However, follow-up studies are in keeping with a stepwise evolution starting with a prefibrotic (hypercellular) phase that progressively transforms into the classical fibro-osteosclerotic endstage with myeloid metaplasia. Prefibrotic CIMF is characterized by a granulocytic and megakaryocytic myeloproliferation lacking an increase in reticulin. Most conspicuous are abnormalities of megakaryopoiesis with regard to their histotopography and maturation. There is a more than 65% probability of progression from an early to advanced CIMF accompanied by increasing anemia, splenomegaly, and leuko-erythroblastosis. A significant relationship is recognizable among frequency, tortuosity, and luminal dilation of the microvessels and the extent of myelofibrosis. Quantity of CD34(+) progenitor cells in the bone marrow (BM) reveals a close association with advancement of disease (fibrosis, splenomegaly, anemia, peripheral blasts) and therefore prognosis. Cell kinetic studies show increased proliferation associated with a higher rate of apoptosis in initial (hypercellular) stages, as well as a reduced endoreduplicative activity of megakaryopoiesis and a blocked synthesis phase of the erythroid precursors. It is noteworthy that prefibrotic and early CIMF often present with a marked thrombocytosis mimicking essential thrombocythemia. Regarding prognosis, early CIMF is associated with a significantly more favorable survival than advanced stages.

Acute panmyelosis with myelofibrosis.

Acute panmyelosis with myelofibrosis (APMF) is an ill-defined disorder that may either evolve as a clonal hematopoietic condition or as a sequel of toxic exposure to the bone marrow (BM). Therefore, controversy and discussion continues as to whether APMF may be considered as a hyperfibrotic (de novo) myelodysplastic syndrome (MDS), as acute myeloid leukemia (AML) or as a severe toxic myelopathy with accompanying myelofibrosis. In this context scant knowledge exists about BM findings, but especially evolution of this disorder according to sequential examinations. Clinically patients present with pancytopenia, a very few blasts in the peripheral blood and no or little splenomegaly. Initially BM histopathology is characterized by different degrees of reticulin-collagen fibrosis and wide ranges of cellularity with a prominent left-shifted and often macrocytic erythropoiesis associated with a reduction and maturation defects of the neutrophil series. Most conspicuous are abnormalities of the megakaryocytes including loose clustering, dislocation towards the endosteal border and appearance of atypical microforms with compact nuclei. Moreover, besides myelofibrosis in a number of patients the interstitial compartment displays a remarkable inflammatory reaction with lymphoid nodules, abundant iron-laden macrophages, perivascular plasmacytosis and increase in microvessels. Repeatedly performed BM biopsies reveal an accumulation of dispersed or clustered CD34+ and lysozyme-expressing blasts in keeping with the insidious transformation into acute leukemia. Prognosis is unfavorable with a median survival of less than 1 year. In conclusion, APMF has to be regarded as a condition that shows considerable overlappings with primary hyperfibrotic MDS, AML and toxic myelopathy (secondary MDS) with accompanying myelofibrosis and therefore can not be considered as a definite clinical entity.

Mixed chimerism of bone marrow vessels (endothelial cells, myofibroblasts) following allogeneic transplantation for chronic myelogenous leukemia.

Experimental findings support the hypothesis that within the functional network of the bone marrow (BM) microenvironment the endothelial cells (ECs) exert a pivotal role as gatekeepers by controlling the trafficking and homing of progenitor cells. However, little information is available concerning the origin of ECs after allogeneic bone marrow transplantation (BMT) in CML. To determine the extent of mixed chimerism (MCh) a simultaneous immunohistochemical and fluorescence in-situ hybridization (FISH) study was performed on BM biopsies derived from patients following sex-mismatched BMT with full unmanipulated BM. ECs were identified by their staining with CD34 and the myofibroblasts (MFs) of large vessels were labeled by an antibody against alpha-smooth muscle actin. For sex-typing and demonstration of the bcr/abl fusion product appropriate commercially available probes and detection systems were applied. Contrasting a total congruence of labeling in control samples five patients showed donor type ECs in the early posttransplant period in about 20%. In the remaining four patients the amount of donor type ECs increased slightly after the third month up to 30%. A total of 26 MFs could be identified lining large capillaries and arterioles that exclusively revealed a host origin. Following successful engraftment only very few of the persistent host-derived ECs also displayed the bcr/abl gene. In five patients, a conversion of MCh from donor to host type ECs was recognizable during the evolution of leukemic relapse. This finding was accompanied by a bcr/abl rearrangement of about 10% of these cells. In conclusion, following myelo-ablative therapy, a survival of a considerable number of ECs and MFs of the vessel walls has been found implying persistence of host-derived vascular structures of the BM stroma. However, in only a small proportion bcr/abl+ ECs and thus minimal residual disease was detectable. Evolution of leukemic relapse was characterized by conversion of MCh with almost total loss of donor type ECs and increase in number of bcr/abl+ ECs.