Diagnostic utility of STAT6YE361 expression in classical Hodgkin lymphoma and related entities.

Although the distinction of classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma using morphology and immunostains is straightforward in most instances, occasional cases pose diagnostic challenge. We sought to determine the utility of the novel YE361 STAT6 rabbit monoclonal antibody in Hodgkin lymphoma and diagnostically challenging B- and T-cell non-Hodgkin lymphoma entities with Hodgkin-like features. Cases from seven institutions included: 57 classical Hodgkin lymphomas (31% EBV+), 34 nodular lymphocyte predominant Hodgkin lymphomas, 34 mimicking B- and T-cell non-Hodgkin lymphomas, and 7 reactive lymphoproliferations. After review of histology, STAT6YE361 immunostaining was performed. The intensity and spatial localization of immunopositivity was assessed in neoplastic cells. Additional FISH for programmed death ligand-1 (PD-L1) was performed in one patient in paired treatment-naive and relapse biopsy tissues. Two STAT6YE361 immunopositive cases were examined by whole-exome sequencing after flow sorting to assess mutations in STAT6 pathway genes. Most classical Hodgkin lymphomas showed nuclear staining for STAT6YE361 [46/57 cases (80%)] on Hodgkin cells. Staining was exclusively nuclear in a minority [12/46 (26%)], while dual nuclear and cytoplasmic localization was more common [34/46 (74%)]. In contrast, all nodular lymphocyte predominant Hodgkin lymphomas [0/34 (0%)] were negative for nuclear STAT6YE361 staining on the lymphocyte predominant cells. Within B- and T-cell non-Hodgkin lymphomas, nuclear STAT6YE361 was seen in: B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and in primary mediastinal large B-cell lymphoma. Strong PD-L1 gene amplification was noted in the paired cHL and relapse B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, although STAT6YE361 was negative in both biopsies. Whole-exome sequencing identified mutations in B2M, XPO1, and ITPKB as well CISHP213L (in the STAT pathway) in one classical Hodgkin lymphoma patient positive for nuclear STAT6YE361 although no underlying STAT6 mutations were observed in either sample examined. STAT6YE361 nuclear staining has 100% positive predictive value and 85.7% negative predictive value in confirming or excluding classical Hodgkin lymphoma diagnosis in the distinction from nodular lymphocyte predominant Hodgkin lymphoma and other benign and malignant entities.

Peripheral T-cell lymphomas of follicular helper T-cell type frequently display an aberrant CD3(-/dim)CD4(+) population by flow cytometry: an important clue to the diagnosis of a Hodgkin lymphoma mimic.

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3(-/dim)CD4(+) aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3(-/dim)CD4(+) T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3(-/dim)CD4(+) population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3(-/dim)CD4(+) T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P=0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The high frequency of CD3(-/dim)CD4(+) aberrant T cells is similar in angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas of follicular type, and is a useful feature in distinguishing peripheral T-cell lymphomas of follicular type from morphologic mimics such as reactive hyperplasia or Hodgkin lymphoma.

Real world predictors of response and 24-month survival in high-grade TP53-mutated myeloid neoplasms.

Current therapies for high-grade TP53-mutated myeloid neoplasms (≥10% blasts) do not offer a meaningful survival benefit except allogeneic stem cell transplantation in the minority who achieve a complete response to first line therapy (CR1). To identify reliable pre-therapy predictors of complete response to first-line therapy (CR1) and outcomes, we assembled a cohort of 242 individuals with TP53-mutated myeloid neoplasms and ≥10% blasts with well-annotated clinical, molecular and pathology data. Key outcomes examined were CR1 & 24-month survival (OS24). In this elderly cohort (median age 68.2 years) with 74.0% receiving frontline non-intensive regimens (hypomethylating agents +/- venetoclax), the overall cohort CR1 rate was 25.6% (50/195). We additionally identified several pre-therapy factors predictive of inferior CR1 including male gender (P = 0.026), ≥2 autosomal monosomies (P < 0.001), -17/17p (P = 0.011), multi-hit TP53 allelic state (P < 0.001) and CUX1 co-alterations (P = 0.010). In univariable analysis of the entire cohort, inferior OS24 was predicated by ≥2 monosomies (P = 0.004), TP53 VAF > 25% (P = 0.002), TP53 splice junction mutations (P = 0.007) and antecedent treated myeloid neoplasm (P = 0.001). In addition, mutations/deletions in CUX1, U2AF1, EZH2, TET2, CBL, or KRAS ('EPI6' signature) predicted inferior OS24 (HR = 2.0 [1.5-2.8]; P < 0.0001). In a subgroup analysis of HMA +/-Ven treated individuals (N = 144), TP53 VAF and monosomies did not impact OS24. A risk score for HMA +/-Ven treated individuals incorporating three pre-therapy predictors including TP53 splice junction mutations, EPI6 and antecedent treated myeloid neoplasm stratified 3 prognostic distinct groups: intermediate, intermediate-poor, and poor with significantly different median (12.8, 6.0, 4.3 months) and 24-month (20.9%, 5.7%, 0.5%) survival (P < 0.0001). For the first time, in a seemingly monolithic high-risk cohort, our data identifies several baseline factors that predict response and 24-month survival.

Signals for stress erythropoiesis are integrated via an erythropoietin receptor-phosphotyrosine-343-Stat5 axis.

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+ CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

GATA3 Immunohistochemical Staining in Hodgkin Lymphoma: Diagnostic Utility in Differentiating Classic Hodgkin Lymphoma From Nodular Lymphocyte Predominant Hodgkin Lymphoma and Other Mimicking Entities.

BACKGROUND: Classic Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) are clinically distinct entities, with different prognostic and treatment implications. In addition, several large B-cell lymphomas and some T-cell lymphomas can mimic CHL. Differentiating these entities from CHL is crucial for ensuring appropriate therapy. GATA3 is a T-cell transcription factor involved in T-cell maturation and has been previously shown to be overexpressed in CHL cells via gene expression profiling. We investigated the utility of GATA3 immunostain in differentiating CHL from NLPHL and other mimicking entities. MATERIALS AND METHODS: We accrued 17 NLPHLs, 49 CHLs [23 nodular sclerosis (NS), 3 syncytial variants, 3 lymphocyte rich and 13 mixed cellularity types], 4 primary mediastinal large B-cell lymphomas (PMBLs), 2 Epstein-Barr virus (EBV) positive diffuse large B-cell lymphomas (DLBCLs) (EBV+LBCLs), 2 T-cell/histiocyte-rich large B-cell lymphomas (TCHRBCLs), 1 gray zone lymphoma, and 2 tissue microarrays consisting of 72 DLBCLs. One slide from each was stained with GATA3 and percent positive tumor cells and intensity of nuclear expression was semiquantitatively graded independently by 2 board certified hematopathologists. RESULTS: GATA3 was positive in 80% of CHLs. Both percent positivity and intensity of staining varied greatly. Syncytial variant of NS subtype showed the highest positivity rate (3/3; 100%), followed by NS (20/23; 87%), mixed cellularity (9/13; 70%), and lymphocyte rich (2/3; 67%). GATA3 was negative in all NLPHLs, EBV+LBCLs, TCRBCLs, and DLBCLs stained. The single gray zone lymphoma and 3/4 PMBLs were positive. CONCLUSIONS: Nuclear expression of GATA3 can be used to delineate CHL from NLPHL. GATA3 positivity effectively excludes NLPHL with 100% negative predictive value. However, as 20% of CHL can be negative for GATA3, CHL cannot be ruled out with negative GATA3. Additional findings include GATA3 positivity among PMBLs, whereas all 72 DLBCLs were negative for GATA3. This finding further highlights similarities between CHL and PMBL.

Isolated Ocular Manifestation of Relapsed Chronic Myelogenous Leukemia Presenting as Myeloid Blast Crisis in a Patient on Imatinib Therapy: A Case Report and Review of the Literature.

Blast phase in chronic myelogenous leukemia (CML) has rarely been reported to involve extramedullary sites like skin, lymph nodes, and central nervous system. Clinical history, characteristic hematologic findings (elevated leukocyte counts, myelocytic predominance, and basophilia), and Philadelphia chromosome are of high diagnostic significance especially in isolated extramedullary presentations. We describe a unique case of CML relapse with blast phase involving the eye. A 66-year-old man with a known diagnosis of CML on imatinib and in molecular remission for 3 years presented with a painful blind eye. Histologic examination revealed diffuse involvement of choroid, iris, vitreous humor, and the optic nerve by blast cells. The blasts expressed CD34, aberrant TdT, and a myeloid phenotype (CD13, CD33, and CD117). Fluorescence in situ hybridization (FISH) of vitreous fluid detected BCR-ABL1 gene rearrangement. Additionally, trisomy 8 and gains of 9 and 22 were seen which were not present in the initial diagnostic marrow study 3 years ago. At relapse, the bone marrow, peripheral blood, and the cerebrospinal fluid were not involved by CML. Patient received induction chemotherapy and single dose prophylactic intrathecal methotrexate and was maintained on antityrosine kinase therapy and eventually underwent allogenic stem cell transplantation.

Primary CNS T-cell Lymphomas: A Clinical, Morphologic, Immunophenotypic, and Molecular Analysis.

Primary central nervous system (CNS) lymphomas are relatively rare with the most common subtype being diffuse large B-cell lymphoma. Primary CNS T-cell lymphomas (PCNSTL) account for <5% of CNS lymphomas. We report the clinical, morphologic, immunophenotypic, and molecular characteristics of 18 PCNSTLs. Fifteen cases were classified as peripheral T-cell lymphoma, not otherwise specified, 2 of which were of γδ T-cell derivation and 1 was TCR silent; there was 1 anaplastic large cell lymphoma, ALK-positive and 2 anaplastic large cell lymphoma, ALK-negative. Median age was 58.5 years (range, 21 to 81 y), with an M:F ratio of 11:7. Imaging results showed that 15 patients had supratentorial lesions. Regardless of subtype, necrosis and perivascular cuffing of tumor cells were frequently observed (11/18 cases). CD3 was positive in all cases but 1; 10/17 were CD8-positive, and 5/17 were CD4-positive. Most cases studied had a cytotoxic phenotype with expression of TIA1 (13/15) and granzyme-B (9/13). Polymerase chain reaction analysis of T-cell receptor γ rearrangement confirmed a T-cell clone in 14 cases with adequate DNA quality. Next-generation sequencing showed somatic mutations in 36% of cases studied; 2 had >1 mutation, and none showed overlapping mutations. These included mutations in DNMT3A, KRAS, JAK3, STAT3, STAT5B, GNB1, and TET2 genes, genes implicated previously in other T-cell neoplasms. The outcome was heterogenous; 2 patients are alive without disease, 4 are alive with disease, and 6 died of disease. In conclusion, PCNSTLs are histologically and genomically heterogenous with frequent phenotypic aberrancy and a cytotoxic phenotype in most cases.

The histological and biological spectrum of diffuse large B-cell lymphoma in the World Health Organization classification.

Diffuse large B-cell lymphomas (DLBCLs) are aggressive B-cell lymphomas that are clinically, pathologically, and genetically diverse, in part reflecting the functional diversity of the B-cell system. The focus in recent years has been toward incorporation of clinical features, morphology, immunohistochemistry, and ever evolving genetic data into the classification scheme. The 2008 World Health Organization classification reflects this complexity with the addition of several new entities and variants. The discovery of distinct subtypes by gene expression profiling heralded a new era with a focus on pathways of transformation as well as a promise of more targeted therapies, directed at specific pathways. Some DLBCLs exhibit unique clinical characteristics with a predilection for specific anatomic sites; the anatomic site often reflects underlying biological distinctions. Recently, the spectrum of Epstein-Barr virus (EBV)-driven B-cell proliferations in patients without iatrogenic or congenital immunosuppression has been better characterized; most of these occur in patients of advanced age and include Epstein-Barr virus (EBV)-positive large B-cell lymphoma of the elderly. Human herpesvirus 8 is involved in the pathogenesis of primary effusion lymphoma, which can present as a "solid variant." Two borderline categories were created; one deals with tumors at the interface between classic Hodgkin lymphoma and DLBCL. The second confronts the interface between Burkitt lymphoma and DLBCL, so-called "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma" in the 2008 classification. Most cases harbor both MYC and BCL2 translocations and are highly aggressive. Another interesting entity is anaplastic lymphoma kinase-positive DLBCL, which renders itself potentially targetable by anaplastic lymphoma kinase inhibitors. Ongoing investigations at the genomic level, with both exome and whole-genome sequencing, are sure to reveal new pathways of transformation in the future.

Erythropoietin modulation of podocalyxin and a proposed erythroblast niche.

Epo's erythropoietic capacity is ascribed largely to its antiapoptotic actions. In part via gene profiling of bone marrow erythroblasts, Epo is now shown to selectively down-modulate the adhesion/migration factors chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4) and to up-modulate growth differentiation factor-3 (Gdf3), oncostatin-M (OncoM), and podocalyxin like-1 (PODXL). For PODXL, Epo dose-dependent expression of this CD34-related sialomucin was discovered in Kit(+)CD71(high) proerythroblasts and was sustained at subsequent Kit(-)CD71(high) and Ter119(+) stages. In vivo, Epo markedly induced PODXL expression in these progenitors and in marrow-resident reticulocytes. This was further associated with a rapid release of PODXL(+) reticulocytes to blood. As studied in erythroblasts expressing minimal Epo receptor (EpoR) alleles, efficient PODXL induction proved dependence on an EpoR-PY343 Stat5 binding site. Moreover, in mice expressing an EpoR-HM F343 allele, compromised Epo-induced PODXL expression correlated with abnormal anucleated red cell representation in marrow. By modulating this select set of cell-surface adhesion molecules and chemokines, Epo is proposed to mobilize erythroblasts from a hypothesized stromal niche and possibly promote reticulocyte egress to blood.

Core erythropoietin receptor signals for late erythroblast development.

Critical signals for erythroblast formation are transduced by activated, tyrosine-phosphorylated erythropoietin receptor (EpoR) complexes. Nonetheless, steady-state erythropoiesis is supported effectively by EpoR alleles that are deficient in cytoplasmic phosphotyrosine sites. To better define core EpoR action mechanisms, signaling capacities of minimal PY-null (EpoR-HM) and PY343-retaining (EpoR-H) alleles were analyzed for the first time in bone marrow-derived erythroblasts. Jak2 activation via each allele was comparable. Stat5 (and several Stat5-response genes) were induced via EpoR-H but not via EpoR-HM. Stat1 and Stat3 activation was nominal for all EpoR forms. For both EpoR-HM and EpoR-H, Akt and p70S6-kinase activation was decreased multifold, and JNK activation was minimal. ERKs, however, were hyperactivated uniquely via EpoR-HM. In vivo, Epo expression in EpoR-HM mice was elevated, while Epo-induced reticulocyte production was diminished. In vitro, EpoR-HM erythroblast maturation also was attenuated (based on DNA content, forward-angle light scatter, and hemoglobinization). These EpoR-HM-specific defects were corrected not only upon PY343 site restoration in EpoR-H, but also upon MEK1,2 inhibition. Core EpoR PY site-independent signals for erythroblast formation therefore appear to be Stat5, Stat1, Stat3, p70S6-kinase, and JNK independent, but ERK dependent. Wild-type signaling capacities, however, depend further upon signals provided via an EpoR/PY343/Stat5 axis.

Erythropoietin-dependent erythropoiesis: New insights and questions.

Committed erythroid progenitor cells require exposure to erythropoietin (Epo) for their survival and for their quantitatively regulated transition to red blood cells. With regard to Epo signal transduction mechanisms, much has been learned from analyses in cell line models, fetal liver or spleen-derived primary erythroblasts and human CD34pos progenitor cells from cord blood or mobilized bone marrow. Presently, we have developed an ex vivo system that efficiently supports the expansion and development of murine adult bone-marrow-derived erythroid progenitor cells. This system is outlined together with its demonstrated utility in studying (for the first time) the signaling capacities of two knocked-in phosphotyrosine-deficient Epo receptor alleles (EpoR-H and EpoR-HM). Ways in which these studies advance an understanding of core Epo signal transduction events are outlined. Also introduced are two new putative negative regulators of Epo-dependent erythropoiesis, DYRK3 and DAPK2 kinases.

Attenuated signaling by a phosphotyrosine-null Epo receptor form in primary erythroid progenitor cells.

Signals provided by the erythropoieitin receptor (EpoR) are required for erythroid development beyond the erythroid colony-forming unit (CFU-e) stage and are propagated via the EpoR-tethered Janus kinase, JAK2. JAK2 functions, in part, to phosphorylate 8 conserved EpoR phosphotyrosine (PY) sites for the binding of a diverse set of signaling factors. However, recent studies in transgenic and knock-in mice have demonstrated substantial bioactivity for PY-null EpoR forms. Presently, the activities of a PY-null EpoR-HM form in primary progenitor cells from knock-in mice were further assessed using optimized Epo dose-dependent proliferation, survival, and differentiation assays. As compared with the wild-type (wt)-EpoR, EpoR-HM activity was compromised several-fold in each context when Epo was limited to physiologic concentrations. Possible compensatory increases in serum growth factor levels also were investigated, and as assayed using embryonic stem (ES) cell-derived erythroid G1E2 cells, activities in serum from EpoR-HM mice were substantially elevated. In addition, when challenged with phenylhydrazine-induced anemia, EpoR-HM mice failed to respond with efficient splenic stress erythropoiesis. Thus, the function of this JAK2-coupled but minimal PY-null EpoR-HM form appears to be attenuated in several contexts and to be assisted in vivo by compensatory mechanisms. Roles normally played by EpoR PY sites and distal domains therefore should receive continued attention.