Atypical thymomas with squamoid and spindle cell features: clinicopathologic, immunohistochemical and molecular genetic study of 120 cases with long-term follow-up.

Thymomas are rare tumors characterized by a broad range of morphologic appearances that can sometimes give rise to difficulties for classification. We have studied a series of 120 thymoma patients in whom the tumors were characterized by sheets of atypical epithelial cells with squamoid and/or spindle cell features. They occurred in 63 men and 57 women and presented as a discrete mass in the anterior mediastinum measuring 2-23 cm (mean: 8.2 cm). Patients' ages ranged from 14 to 86 years (mean: 57.8) and most had symptoms referable to a mass lesion. 20 patients had myasthenia gravis or other autoimmune disorder. 76 cases were characterized by a predominant population of round to polygonal tumor cells while 32 cases were characterized by atypical oval or spindle cells. 12 cases showed mixed features and 16 cases showed the development of thymic carcinoma arising from thymoma. All cases were positive for p40/p63 and cytokeratin AE1/AE3. 23 cases were positive for CD5 (25%), and 13 for CD117 (14%). MIB1 showed a significant increase in proliferative activity (mean = 11.6%). Next generation sequencing in 47 cases did not disclose any variants amenable to current targeted therapies. Clinical follow up ranging from 2 to 29 years showed a progressive increase in aggressive behavior and fatality rate with advancing stage. Overall survival was 87% at 5 years, 67% at 10 years, and 23% at 20 years. Completeness of resection and staging were the most significant parameters for survival. The more aggressive tumors followed a protracted clinical course with multiple recurrences and metastases over a long period of time (mean = 19.8 years from time of initial relapse to death). Atypical thymomas are a distinct category of thymic epithelial neoplasm characterized by a slowly progressive clinical course with increased potential for metastases, transformation to a higher-grade malignancy, and fatal outcome in some cases.

Contribution of clonal hematopoiesis to adult-onset hemophagocytic lymphohistiocytosis.

Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease of immune hyperactivation. Unlike pediatric HLH, adult HLH is rarely driven by germline genetic variants. Although numerous precipitating etiologies have been identified, the reason that HLH occurs in only a subset of individuals and how other factors contribute to the disease remains unknown. We hypothesized that clonal hematopoiesis (CH), a state in which somatic mutations in blood cells cause an expanded population of mutant hematopoietic cells and drive an aberrant inflammatory state, could contribute to adult-onset HLH. In a highly annotated cohort of older adults with HLH we found that CH was more prevalent than in control cohorts. Using the adult-onset HLH mouse model in which repeated treatments of the TLR9 agonist, ODN1826, was delivered to the mouse, we observed that macrophages carrying mutations in Tet2, one of the most commonly mutated genes in CH, have an enhanced inflammatory response to TLR9 agonism. Finally, mice carrying Tet2 mutations in the hematopoietic compartment (a common model for CH) displayed an exaggerated response to TLR9 agonism, including worse splenomegaly and anemia. Our data suggest that CH is more common in individuals with adult-onset HLH and can contribute to the pathophysiology of this disease.

Expression patterns for Bcl-2, EMA, ß-catenin, E-cadherin, PAX8, and MIB1 in thymomas.

The expression of immunohistochemical markers has been extensively investigated in thymomas to assist in the differential diagnosis. We have studied six select markers to determine their utility in the evaluation of these tumors. A series of 126 thymomas including 33 type A, 27 type AB, 20 type B1, 22 type B2, and 24 type B3, were examined utilizing a tissue microarray (TMA) technique with antibodies to e-cadherin, ß-catenin, PAX8, bcl-2, EMA, and MIB-1. Keratin AE1/AE3 and p63 were used for quality control. A significant finding was strong and consistent positivity for bcl-2 in type A (90%) and type AB (88.8%) thymoma, while 100% of B1, B2, and B3 were negative. The distribution of e-cadherin and ß-catenin was not useful for differential diagnosis. E-cadherin and ß-catenin were expressed in a high proportion of all the tumors (92-100%), except for B2 thymoma which showed only 45% expression. A significant increase in the expression of the MIB-1 proliferation marker (mean: 12.8% nuclear positivity) was also observed in B3 thymoma compared with the other histologic types. Statistical significance was confirmed using Kruskal's non-parameterized test for distribution. EMA was generally negative except for spindle cells in the fibrous septa in types A and AB thymoma. PAX8 showed less consistent nuclear staining than p63 and was only widely expressed in 55.7% of cases. Bcl-2 may serve as a useful marker to separate spindle cell thymomas (Type A and AB) from the other types, and the MIB1 proliferation index may be of use to differentiate type B2 from type B3 thymoma.

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Outcomes after Allogeneic Stem Cell Transplantation in Patients with Double-Hit and Double-Expressor Lymphoma.

Double-hit lymphomas (DHLs) and double-expressor lymphomas (DELs) are associated with resistance to frontline and salvage immunochemotherapy, as well as autologous stem cell transplantation (SCT). We hypothesized that allogeneic SCT (alloSCT) could overcome the chemoresistance associated with DEL/DHL. We retrospectively studied the impact of DEL/DHL status in a multicenter cohort of patients who underwent alloSCT for relapsed/refractory (rel/ref) aggressive B cell non-Hodgkin lymphoma (B-NHL). Seventy-eight patients transplanted at 3 centers in whom tumor tissue was available for immunohistochemistry and fluorescence in situ hybridization were enrolled; 47% had DEL and 13% had DHL. There were no significant differences in 4-year progression-free (PFS) or overall survival (OS) between patients with DEL compared with patients without DEL (PFS 30% versus 39%, P = .24; OS 31% versus 49%, P = .17) or between patients with DHL compared with patients without DHL (PFS 40% versus 34%, P = .62; OS 50% versus 38%, P = .46). The lack of association between DEL or DHL and outcome was confirmed in multivariable models, although inadequate sample size may have limited our ability to detect significant differences. In our cohort alloSCT produced durable remissions in patients with rel/ref aggressive B-NHL irrespective of DEL and DHL status, justifying its consideration in the treatment of patients with rel/ref DEL/DHL.

Expression of PD-L1/PD-1 in lymphoepithelioma-like carcinoma of the thymus.

Poorly differentiated non-keratinizing squamous cell carcinoma of the thymus, also known as lymphoepithelioma-like carcinoma, is a rare primary malignant neoplasm of thymic origin. The mainstay of treatment for these tumors is surgical and they tend to respond poorly to chemotherapy. The checkpoint programmed cell death ligand-1 protein (PD-L1) bound to its receptor (PD-1) has been demonstrated to be an important therapeutic target for many different tumors. Expression of PD-L1/PD-1 in lymphoepithelioma-like carcinoma of the thymus may indicate that these tumors are potential targets for inhibitor therapy. Twenty-one cases of lymphoepithelioma-like carcinoma of the thymus were collected and reviewed. Tissue microarrays were created using triplicate 2 mm cores for each case. PD-L1/PD-1 staining pattern (neoplastic cells versus tumor infiltrating lymphocytes) was documented for each case. Out of 21 cases, 15 (71.4%) showed various degrees of membranous PD-L1 staining. Of the positive cases, 48% showed high expression of PD-L1 (>50% of tumor cells) and 24% showed low expression (<50%). PD-1 staining showed focal positivity in 12/20 (60%) cases among tumor infiltrating lymphocytes. PD-L1/PD-1 inhibitor therapy has been applied successfully in other solid malignant tumors with high expression of PD-L1/PD-1. The high level of PD-L1 expression in our cases indicates that PD-L1 may play a role in the pathogenesis of these tumors and that PD-L1/PD-1 blockade may be a viable therapeutic option for patients with lymphoepithelioma-like carcinoma of the thymus who have failed other first-line therapies.

Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease.

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.

Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

Survival signaling mediated by c-Jun NH(2)-terminal kinase in transformed B lymphoblasts.

The c-Jun NH(2)-terminal kinase (JNK) is implicated in the apoptotic response of cells exposed to stress, but the JNK signal transduction pathway may not act exclusively in apoptosis. In some studies of tumor cells, JNK has been implicated in signaling cell survival. The possibility that JNK might mediate a survival signal in tumor cells is consistent with the observation that it is activated in response to some oncogenes, such as the leukemogenic oncogene BCR-ABL, which is created by a reciprocal translocation between human chromosomes 9 and 22 (ref. 2). The BCR-ABL protein activates the JNK signaling pathway in hematopoietic cells and increases transcriptional activity mediated by the transcription factor AP1 (ref. 3). Also, inhibition of c-Jun or JNK prevents BCR-ABL-induced cell transformation in vitro. Although this implicates the JNK signaling pathway in transformation by BCR-ABL, the possible role of JNK in this process is unclear. We find that disruption of the JNK ortholog Mapk8 (also known as Jnk1) in mice causes defective transformation of pre-B cells by BCR-ABL in vitro and in vivo. The Jnk1 protein is required for the survival of the transformed cells in the absence of stromal support. Failure to survive is associated with decreased expression of Bcl2, and the effect of Jnk1 deficiency can be rescued by transgenic expression of Bcl2. Our results show that Jnk1 signals cell survival in transformed B lymphoblasts and suggest that it may contribute to the pathogenesis of some proliferative diseases.

Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.

Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

Myeloid sarcoma with NPM1 mutation may be clinically and genetically distinct from AML with NPM1 mutation: a study from the Bone Marrow Pathology Group.

Myeloid sarcoma (MS) is currently considered equivalent to de novo acute myeloid leukemia (AML); however, the relationship between these entities is poorly understood. This retrospective multi-institutional cohort study compared 43 MS with NPM1 mutation to 106 AML with NPM1 mutation. Compared to AML, MS had more frequent cytogenetic abnormalities including complex karyotype (p = .009 and p = .007, respectively) and was enriched in mutations of genes involved in histone modification, including ASXL1 (p = .007 and p = .008, respectively). AML harbored a higher average number of gene mutations (p = .002) including more frequent PTPN11 mutations (p < .001) and mutations of DNA-methylating genes including DNMT3A and IDH1 (both p < .001). MS had significantly shorter overall survival (OS) than AML (median OS: 44.9 vs. 93.2 months, respectively, p = .037). MS with NPM1 mutation has a unique genetic landscape, and poorer OS, compared to AML with NPM1 mutation.

First study comparing genetic profiles of MS and AML with a common disease-defining lesion.NPM1^Mut MS may be genetically distinct from NPM1^Mut AML.NPM1^Mut MS may have inferior overall survival compared to NPM1^Mut AML.

Primary epidural lymphocyte-depleted Hodgkin's lymphoma of the thoracic spine - presentation of a rare disease variant.

BACKGROUND: Lymphocyte-depleted Hodgkin's lymphoma is the rarest form of classical Hodgkin's lymphoma, accounting for < 1% of all cases. Patients often have advanced-stage disease at the time of presentation with an aggressive clinical course. Even more uncommon is primary extranodal disease and rarely it will be presenting with spinal cord compression. CASE PRESENTATION: An 88-year-old Caucasian female presented with a history of upper back pain for several months and new onset bilateral leg numbness and weakness. MRI of the spine showed a dorsal epidural lesion with cord compression at T1-T4 with involvement of the paraspinal muscles. The patient received urgent surgical decompression, with final histopathology showing a lymphocyte-depleted Hodgkin's lymphoma. Systemic work-up did not show evidence of nodal disease. Following surgery, she received a course of radiotherapy with good outcome. CONCLUSION: To the best of our knowledge, this is the first reported case of primary lymphocyte-depleted Hodgkin lymphoma presenting as epidural spinal cord compression. Our report, in conjunction with a review of the literature, suggests that surgical intervention is clearly indicated in de novo disease followed by radiotherapy.

Mutations and aneuploidy: co-conspirators in cancer?

The role of intragenic point mutations in human cancer is well established. However, the contribution of massive genomic changes collectively known as aneuploidy is less certain. Recent experimental work suggests that aneuploidy is required for sporadic carcinogenesis in mice and that it may collaborate with intragenic mutations during tumorigenesis. The genomic plasticity afforded by aneuploidy could facilitate emergence of protumorigenic gene dosage changes and accelerate accumulation of oncogenes and loss of tumor suppressor genes. These new findings force us to rethink the pathogenesis of carcinoma in ways that have significant implications for diagnosis and therapy.

Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue.

Wnt5a is a member of the Wnt family of secreted glycoproteins that play essential organizing roles in development. Similar to other Wnt members, Wnt5a can upregulate cell proliferation and has been proposed to have oncogenic function. Here we report that Wnt5a signals through the noncanonical Wnt/Ca++ pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation in a cell-autonomous manner. Wnt5a hemizygous mice develop myeloid leukemias and B cell lymphomas that are clonal in origin and display loss of Wnt5a function in tumor tissues. Furthermore, analysis of human primary leukemias reveals deletion of the WNT5A gene and/or loss of WNT5A expression in a majority of the patient samples. These results demonstrate that Wnt5a suppresses hematopoietic malignancies.

Lymphocyte-Rich Spindle Cell Thymoma: Clinicopathologic, Immunohistochemical, Ultrastructural and Molecular Genetic Study of 80 Cases.

A study of 80 cases of spindle cell thymoma in which the spindle cell component was overshadowed by massive numbers of stromal lymphocytes is presented. The patients were 38 women and 42 men, aged 8 to 81 years (mean=54 y). All tumors presented as an anterior mediastinal mass; 5 patients had myasthenia gravis and one had Good syndrome. The tumors were well-circumscribed, encapsulated, and measured 2.9 to 26.0 cm in greatest diameter (mean=7.3 cm). Using modified Masaoka staging, 66 tumors were stage I, 10 were stage IIa, 2 were stage III and 1 was stage IV. Histologically the tumors were characterized by a predominant lymphocytic population admixed with scattered small spindle epithelial cells. The neoplastic spindle cells in these tumors demonstrated 2 major growth patterns: in 33 cases, the tumors were exclusively composed of dense sheets of lymphocytes containing scattered spindle cells resembling a lymphocyte-rich thymoma (WHO type B1); in the remaining cases the tumors showed admixtures of a predominantly lymphocytic component with areas that were lymphocyte-poor and contained a pure spindle cell population similar to WHO type A. Immunohistochemical stains and electron microscopy corroborated the spindle cell morphology in both types. The GTF2I p.L424H variant was identified in 53 of 63 (84%) cases analyzed. Clinical follow-up in 27 cases showed that most of the tumors behaved in an indolent manner. Our study expands the spectrum of spindle cell thymoma by demonstrating the existence of cases that are predominantly composed of lymphocyte-rich elements and lack areas with a pure (lymphocyte poor) spindle cell morphology.

GCB-type is a favorable prognostic factor in primary CNS diffuse large B-cell lymphomas.

Primary CNS lymphomas (PCNSLs) are aggressive diffuse large B-cell lymphomas (DLBCLs) limited to the CNS that generally have a poor prognosis. Classification of DLBCL into germinal center B-cell (GCB) and activated B-cell (non-GCB) subtypes has prognostic value in systemic DLBCL, with GCB-type having a better prognosis. The aim of this study was to determine whether GCB versus non-GCB classification in PCNSLs has similar prognostic value. We analyzed clinical, radiological and histologic data from 24 patients with biopsy confirmed DLBCL of the CNS with classification into GCB versus non-GCB subtypes. We found that after a median follow-up of 15 months, only 39% of patients with non-GCB-type PCNS DLBCL were alive, whereas all patients with GCB-type were alive. Non-GCB-type had a median survival of 11 months, whereas all GCB-type patients were alive after a median follow-up of 22 months. As previously reported, we also found that patients younger than 70 years had longer survival (median 29 months) compared to older patients (median 8.8 months). There was no statistically significant difference between the ages, gender, focality, size or location of lesions, or treatment of non-GCB and GCB-type patients. Our findings suggest that classifying PCNSLs into GCB versus non-GCB subtype using the Hans algorithm may help stratify patients into two groups with different prognosis.

Rosai-Dorfman disease: Ultrasonography and histopathology study of a soft tissue mass in the forearm.

Rosai-Dorfman disease (RDD) is uncommon in daily practice, but needs to be ruled out in rheumatologic conditions to elucidate a wide differential diagnosis. Beside its typical presentation, soft tissue masses can be easily seen in our Rheumatology clinics. Ultrasonography widely extended in our specialty, could also play a role in the diagnosis, to end up with the histological confirmation of the disease.