Breast cancer exploits neural signaling pathways for bone-to-meninges metastasis.

The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, which limits the development of effective therapies. In this work, we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral or calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs colocalize with perivascular meningeal macrophages and induce their expression of the prosurvival neurotrophin glial-derived neurotrophic factor (GDNF). Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor neural cell adhesion molecule (NCAM) from BCCs inhibits breast cancer growth within the LM. These data suggest integrin α6 and the GDNF signaling axis as new therapeutic targets against breast cancer LM metastasis.

A Novel Transmembrane CXCR4 Variant That Expands the WHIM Genotype-Phenotype Paradigm.

Warts, Hypogammglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare immunodeficiency disease that results from impaired leukocyte trafficking (myelokathexis) predominately caused by gain-of-function variants in C-X-C chemokine receptor type 4 (CXCR4). Clinical manifestations of WHIM syndrome can differ in familial forms or in people harboring identical CXCR4 variants. All known pathogenic CXCR4 variants associated with WHIM syndrome (CXCR4WHIM) to date are localized in the intracellular C-terminus of CXCR4. We identified 4 unrelated patients with variable WHIM-like clinical presentations harboring a novel heterozygous CXCR4 variant (c.250G>C; p.D84H) localized at a highly conserved position in the transmembrane domain of the receptor outside the C-terminus. Functional characterization of the CXCR4D84Hvariant (CXCR4D84H) using patient-derived peripheral blood mononuclear cells and in vitro cellular assaysshow decreased CXCR4 internalization and increased chemotaxis in response to CXCL12, similar to known CXCR4WHIM, but also revealed unique features of CXCR4D84H signaling to cAMP, Ca2+ mobilization and AKT/ERK pathways. These findings are consistent with molecular dynamics simulations that show disruption of the Na+ binding pocket by D84H, resulting in collapse of the hydrophobic gate above and destabilization of the inactive state of CXCR4. Mavorixafor, a CXCR4 antagonist being evaluated in clinical trials for chronic neutropenia and WHIM syndrome, normalized CXCL12-mediated chemotaxis of CXCR4D84H patient lymphocytes ex vivo and improved WBC and subset counts in 1 patient with CXCR4D84H enrolled in the chronic neutropenia phase 1b clinical trial (NCT04154488). The present study expands the current understanding of CXCR4 function and genotype-phenotype correlations in WHIM syndrome and in people with WHIM-like phenotypes.

Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.

56 Fe ion exposure increases the incidence of lung and brain tumors at a similar rate in male and female mice.

The main deterrent to long-term space travel is the risk of Radiation Exposure Induced Death (REID). The National Aeronautics and Space Administration (NASA) has adopted Permissible Exposure Levels (PELs) to limit the probability of REID to 3% for the risk of death due to radiation-induced carcinogenesis. The most significant contributor to current REID estimates for astronauts is the risk of lung cancer. Recently updated lung cancer estimates from Japan's atomic bomb survivors showed that the excess relative risk of lung cancer by age 70 is roughly four-fold higher in females compared to males. However, whether sex differences may impact the risk of lung cancer due to exposure to high charge and energy (HZE) radiation is not well studied. Thus, to evaluate the impact of sex differences on the risk of solid cancer development post-HZE radiation exposure, we irradiated Rb fl/fl ; Trp53 fl/+ male and female mice infected with Adeno-Cre with various doses of 320 kVp X-rays or 600 MeV/n 56 Fe ions and monitored them for any radiation-induced malignancies. We observed that lung adenomas/carcinomas and esthesioneuroblastomas (ENBs) were the most common primary malignancies in X-ray and 56 Fe ion-exposed mice, respectively. In addition, 1 Gy 56 Fe ion exposure compared to X-rays led to a significantly higher incidence of lung adenomas/carcinomas (p=0.02) and ENBs (p<0.0001). However, we did not find a significantly higher incidence of any solid malignancies in female mice as compared to male mice, regardless of radiation quality. Furthermore, gene expression analysis of ENBs suggested a distinct gene expression pattern with similar hallmark pathways altered, such as MYC targets and MTORC1 signaling, in X-ray and 56 Fe ion-induced ENBs. Thus, our data revealed that 56 Fe ion exposure significantly accelerated the development of lung adenomas/carcinomas and ENBs compared to X-rays, but the rate of solid malignancies was similar between male and female mice, regardless of radiation quality.

Dasatinib-associated follicular lymphoma in a patient with B-cell acute lymphoblastic leukaemia.

The tyrosine kinase inhibitor dasatinib is approved for the treatment of chronic myeloid leukaemia and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL). Patients on dasatinib can rarely develop a form of benign reversible reactive lymphadenopathy termed follicular lymphoid hyperplasia (FLH). Here, we describe a patient with Ph+ ALL who developed follicular lymphoma (FL) after prolonged treatment with dasatinib and who had complete remission of FL after discontinuation of dasatinib. This case suggests that dasatinib-associated FLH could be a premalignant condition that can transform into FL. Moreover, withdrawal of dasatinib may be sufficient for remission of dasatinib-associated FL.

Acute Myeloid Leukemia, Version 3.2023, NCCN Clinical Practice Guidelines in Oncology.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of myeloid blasts in the peripheral blood, bone marrow, and/or other tissues. It is the most common form of acute leukemia among adults and accounts for the largest number of annual deaths from leukemias in the United States. Like AML, blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a myeloid malignancy. It is a rare malignancy characterized by the aggressive proliferation of precursors of plasmacytoid dendritic cells that frequently involves the bone marrow, skin, central nervous system, and other organs and tissues. This discussion section focuses on the diagnosis and management of BPDCN as outlined in the NCCN Guidelines for AML.

A multiple instance learning approach for detecting COVID-19 in peripheral blood smears.

A wide variety of diseases are commonly diagnosed via the visual examination of cell morphology within a peripheral blood smear. For certain diseases, such as COVID-19, morphological impact across the multitude of blood cell types is still poorly understood. In this paper, we present a multiple instance learning-based approach to aggregate high-resolution morphological information across many blood cells and cell types to automatically diagnose disease at a per-patient level. We integrated image and diagnostic information from across 236 patients to demonstrate not only that there is a significant link between blood and a patient's COVID-19 infection status, but also that novel machine learning approaches offer a powerful and scalable means to analyze peripheral blood smears. Our results both backup and enhance hematological findings relating blood cell morphology to COVID-19, and offer a high diagnostic efficacy; with a 79% accuracy and a ROC-AUC of 0.90.

Sequential Development of JAK2V617F Mutation and BCR-ABL1 Fusion in Individual Patients With Myeloproliferative Neoplasms.

CONTEXT.­: Concomitant BCR-ABL1 and JAK2V617F in myeloproliferative neoplasms (MPNs) is rare, and its pathogenesis and clinical significance are unclear. OBJECTIVE.­: To investigate the clonal relationship between the 2 genomic alterations, as well as the clinicopathologic impact. DESIGN.­: Retrospective analysis of MPNs with sequential development of BCR-ABL1 and JAK2V617F. RESULTS.­: Of 6 cases, 5 had JAK2V617F-positive MPN diagnosed before acquiring BCR-ABL1 years later, and 1 had BCR-ABL1+ chronic myeloid leukemia before JAK2V617F-positive myelofibrosis completely replaced the BCR-ABL1+ clone 1 year after tyrosine kinase inhibitor therapy. Among the former group, treatment for the initial MPN involved hydroxyurea, ruxolitinib, and/or supportive care, and the latency to the development of JAK2V617F ranged from 4 to 13 years (median of 9 years). Four cases showed retention of JAK2V617F, whereas BCR-ABL1 emerged as the major clone, including 2 cases that exhibited parallel increases in JAK2V617F and BCR-ABL1 burdens, with both genomic markers exceeding 50%. Three patients received stem cell transplants and demonstrated sustained engraftment, with the genomic markers below detectable levels. CONCLUSIONS.­: Most MPNs with concomitant JAK2V617F and BCR-ABL1 are actually composite MPNs with a "second hit" residing on a different clone. Rare cases demonstrate a subclone harboring a "double-hit" in a background of a JAK2V617F-positive stem line clone. The probability of a "double-hit" with a BCR-ABL1+ stem line clone is probably reduced by effective tyrosine kinase inhibitor treatment. The treatment often involves combined kinase inhibitors and/or hydroxyurea, but the outcome is unpredictable; hematopoietic stem cell transplantation may be the ultimate therapeutic option for this complicated disease.

Plasma cells are essentially absent in the luminal gastrointestinal tract of patients with "complete" 22q11.2 deletion syndrome (DiGeorge syndrome).

Gastrointestinal symptoms are commonly reported in patients with 22q11.2 deletion syndrome or DiGeorge syndrome (DGS) in addition to the dominant cardiac manifestations and immunodeficiency. But literature providing specific morphologic details of the gastrointestinal tract pathology is very limited. Here, we provide the first comprehensive morphologic description of the luminal gastrointestinal tract changes in patients with DGS. Cytogenetically confirmed DGS patients were identified, clinical and laboratory data were reviewed to determine the severity of immunodeficiency, and patients were stratified into mildly immunocompromised, that is, partial DiGeorge anomaly or severely immunosuppressed, that is, complete DiGeorge anomaly groups. Gastrointestinal tract biopsies from these patients were retrospectively reviewed and compared with those from controls without the history of DGS. Patients with immunosuppressed DGS showed a near complete absence of plasma cells in the stomach, duodenum, and colon lamina propria by hematoxylin and eosin evaluation. Immunohistochemistry for CD138 used to highlight plasma cells confirmed this finding. The notable absence of plasma cells adds to the existing knowledge of the pathophysiology underlying DGS and expands the differential diagnostic considerations for this finding, which has been previously described in common variable immunodeficiency. It also provides a useful morphologic marker observable by the readily accessible light microscopy. Second, patients with DGS showed a mild increase in epithelial cell apoptosis in their colon. This finding is significant because of its overlap with morphologic features of gastrointestinal graft versus host disease as thymus transplantation is being used as a treatment option for patients with complete DGS.

A Cautionary Tale: Florid Splenic γδ T-cell Proliferation and False-Positive T-cell Clonality by PCR Leads to a Grave Misdiagnosis.

The discrimination of benign from malignant lymphoproliferative disorders is sometimes difficult because there can be overlap in their histological and immunophenotypic features. In such situations, molecularly based clonality testing is often used to discriminate benign (polyclonal) from malignant (monoclonal) processes. Clonality testing by polymerase chain reaction (PCR) has a number of pitfalls that may result in spurious results. Here we report the case of a woman diagnosed by 2 major academic institutions with hepatosplenic T-cell lymphoma based on a dense infiltration of the spleen by a γδ T-cell population with mild cytologic atypia, resulting in expansion of the splenic red pulp, and a positive T-cell receptor clonality test by PCR. There was likewise mild involvement of the liver and bone marrow by the "atypical" T-cell population. Close attention to her uncharacteristically well clinical appearance led to repeat T-cell receptor clonality testing using next-generation sequencing. Definitive demonstration of polyclonality by this test showed that she in fact did not have hepatosplenic T-cell lymphoma but rather a reactive condition, and allogeneic stem cell transplantation could be safely avoided. As molecular clonality testing is widely used in the practice of hematology, this case brings attention to the pitfalls of clonality testing by PCR that practitioners may encounter. It is therefore a cautionary tale highlighting the need for critical interpretation of test results in full clinical context.

NCCN Guidelines Insights: Acute Myeloid Leukemia, Version 2.2021.

The NCCN Guidelines for Acute Myeloid Leukemia (AML) provide recommendations for the diagnosis and treatment of adults with AML based on clinical trials that have led to significant improvements in treatment, or have yielded new information regarding factors with prognostic importance, and are intended to aid physicians with clinical decision-making. These NCCN Guidelines Insights focus on recent select updates to the NCCN Guidelines, including familial genetic alterations in AML, postinduction or postremission treatment strategies in low-risk acute promyelocytic leukemia or favorable-risk AML, principles surrounding the use of venetoclax-based therapies, and considerations for patients who prefer not to receive blood transfusions during treatment.

Correlation of novel ALKATI with ALK immunohistochemistry and clinical outcomes in metastatic melanoma.

AIMS: Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALKATI mutated melanoma as well as correlation with immunohistochemical (IHC) methods have not yet been described. METHODS AND RESULTS: Clinicopathological characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in-situ hybridisation and RNA-based digital molecular analysis assays were performed on archival tissue from 173 stage III and 192 stage IV tumours. ALKATI was identified in 12.7 and 4.8% stage III and IV tumours, respectively. Discrete presentations of the ALKATI are seen: isolated ALKATI (n = 20) and mixed ALKATI (combined ALKATI and ALKWT ; n = 7). Isolated ALKWT expression (n = 4) was seen with no ALK fusions. Stage III patients showed improved survival with ALKATI expression compared to those with ALKWT or no expression [5-year survival 80, 95% confidence interval (CI) = 57-100% versus 43%, 95% CI = 34-55%, P = 0.013]. Clinicopathological characteristics were not statistically significant. Strong diffuse cytoplasmic staining of ALK IHC (n = 12) has a sensitivity of 52.2%, specificity 100%, PPV of 100% and NPV of 92.5% of detecting isolated ALKATI . CONCLUSION: Presence of ALKATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALKATI for targeted therapy.

Histopathologic assessment of cultured human thymus.

The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.

Sequential development of human herpes virus 8-positive diffuse large B-cell lymphoma and chronic myelomonocytic leukemia in a 59 year old female patient with hemoglobin SC disease.

Hematolymphoid neoplasms, including lymphoma and myeloid neoplasms, can occur in patients with sickle cell disease (SCD) or equivalent hemoglobinopathy, but an underlying connection between the two conditions has yet to be fully determined. Herein, we report a unique case of sequential development of two separate hematolymphoid neoplasms, human herpes virus 8 (HHV8)-positive diffuse large B-cell lymphoma (DLBCL) and chronic myelomonocytic leukemia, in a 59 year-old African American female with hemoglobin SC disease. While etiology of immunodeficiency is unknown, the potential causes include hydroxyurea therapy, disease related immunomodulation, chronic inflammation, and relatively old age. The leukemia cells demonstrated profound trilineage dysplasia and harbored complex cytogenetic abnormalities with loss of chromosome 5q and 7q, which are often observed in therapy-related myeloid neoplasms. Besides the potential causes listed above, we propose that myeloid leukemia in this setting may result from genomic changes due to excessive hematopoietic replication triggered by a hemolysis-induced cytokine storm. While myeloid neoplasms in the setting of SCD seems to herald a dismal clinical outcome per the literature, the HHV8-positive DLBCL in our case was apparently indolent, opposing the current perception of its clinical outcome.

Myeloid neoplasms in the setting of sickle cell disease: an intrinsic association with the underlying condition rather than a coincidence; report of 4 cases and review of the literature.

Myeloid neoplasms occasionally occur in patients with sickle cell disease, and the underlying connection between the two diseases is unclear. Herein, we retrospectively analyzed four cases of sickle cell disease patients who developed myeloid neoplasm. Age at time of diagnosis ranged from 27 to 59 years with a median of 35.5 years. Two patients were treated with hydroxyurea and the other two with supportive care alone, with one out of the four patients receiving additional treatment with hematopoietic stem cell transplant. Three patients presented with leukocytosis, and the remaining patient presented with pancytopenia. Two patients were diagnosed with myelodysplastic syndrome/myeloproliferative neoplasm, one with myelodysplastic syndrome, and the other with acute myeloid leukemia. All four cases demonstrated certain degrees of myelodysplasia and complex cytogenetic abnormalities with - 7/7q- and/or - 5/5q- or with 11q23 (KMT2A) rearrangement. This cytogenetic profile resembles that seen in therapy-related myeloid neoplasm, suggesting that myeloid neoplasm in the setting of sickle cell disease may represent a subcategory of the disease distinct from de novo myeloid neoplasm in general. Extensive literature review further demonstrates this similarity in cytogenetic profile, as well as in other associated pathologic features. Potential etiology includes therapy for sickle cell disease, disease-related immunomodulation, or disease-related chronic inflammation. We hypothesize that constant hematopoietic hyperplasia, stimulated by a hemolysis-induced cytokine storm, may increase the chance of somatic mutations/cytogenetic aberrations, resulting in transformation of myeloid precursors. This group of myeloid neoplasms seems to herald a dismal clinical outcome, with median survival <1 year, although the exact pathogenesis and biology of the disease remain to be investigated by large cohorts in future studies.