Kojo S.J. Elenitoba-Johnson

ALK1 and p80 Expression and Chromosomal Rearrangements Involving 2p23 in Inflammatory Myofibroblastic Tumor

Background: Inflammatory myofibroblastic tumor (IMT) is an uncommon tumor of extrapulmonary and pulmonary tissues with an unpredictable clinical course, occasional recurrences, and rare malignant transformation. Clonal abnormalities with rearrangements of chromosome of 2p23 and the ALK gene have been reported in a few cases. The purpose of this study is to investigate whether these are consistent abnormalities among IMTs or represent a distinct subset. Design: Formalin-fixed, paraffin-embedded archival tissue sections from 47 IMTs in 40 patients were immunostained with monoclonal antibodies against ALK and p80. Fluorescence in situ hybridization for ALK rearrangements was done on 22 IMTs from 19 patients. Findings were correlated with clinical features and outcome. Results: ALK positivity was observed in 17 of 47 IMTs (36%) and p80 positivity in 16 of 47 IMTs (34%). Fluorescence in situ hybridization showed ALK rearrangements in nine cases (47%), aneuploidy in three cases (16%), and no rearrangement in seven cases (37%). IMTs with ALK abnormalities by immunohistochemistry and/or fluorescence in situ hybridization originated in the abdomen/pelvis/retroperitoneum, chest, and extremities. The mean age was 6.6 years, with a male/female ratio of 1.3. 64% of patients had no evidence of disease at last follow-up, 45% had one or more recurrences, and 18% displayed histologic evidence of malignant transformation. The IMTs without ALK abnormalities occurred in older children, were more frequent in females, and had fewer recurrences. However, in this group of 40 patients, the differences between the groups with and without ALK abnormalities did not have statistical significance. Aneuploidy without ALK abnormalities was associated with malignant transformation in three of five cases. Conclusions: Abnormalities of ALK and p80 and evidence of chromosomal rearrangements of 2p23 occur in a significant proportion of IMTs. These changes are most frequent in abdominal and pulmonary IMTs in the first decade of life and are associated with a higher frequency of recurrence. These findings confirm the neoplastic nature of a subset IMT with ALK abnormalities and suggest that aneuploid IMT is a subset with more aggressive clinical behavior.

Mechanistic Rationale for Inhibition of Poly(ADP-Ribose) Polymerase in ETS Gene Fusion-Positive Prostate Cancer

Recurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewings sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly (ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS-positive, but not ETS-negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2 deficiency.

Subcutaneous panniculitic T-cell lymphoma is a tumor of cytotoxic T lymphocytes.

Subcutaneous panniculitic T cell lymphoma (SCPTCL) is characterized by primary involvement of the subcutaneous fat in a manner mimicking panniculitis. We studied 16 cases of this lymphoma to define its immunophenotypical profile as well as cellular origin. Involvement of the subcutaneous fat in a lacelike pattern with neoplastic cells rimming individual fat spaces was present in all cases. All 16 cases were of T cell phenotype. Thirteen of the 16 cases were CD8+, whereas three were negative for both CD4 and CD8. Twelve cases were stained for betaF1; of these, eight were betaF1+ and four were betaF1-. Focal staining for CD56 and CD30 was seen in 2 of 13 and two of eight cases, respectively. Intense diffuse positivity for the cytotoxic granular proteins T cell intracellular antigen-1 (TIA-1) and perforin was present in all cases, indicating an origin from cytotoxic T lymphocytes. Ten cases studied for Epstein-Barr viral sequences were negative. Eight of 9 cases with amplifiable DNA showed a clonal TCR gamma gene rearrangement by polymerase chain reaction. Controls included seven cases of benign panniculitis and seven other peripheral T cell lymphomas involving the skin and subcutaneous tissues: two peripheral T cell lymphomas, not otherwise specified (PTL,NOS), four anaplastic large cell lymphomas (ALCL), one T/NK cell lymphoma. The seven cases of panniculitis lacked cytological atypia and were characterized by an admixture of CD4+ and CD8+ cells with interspersed aggregates of L26+ B cells. Only infrequent cells showed staining for TIA-1 and perforin. In the control cases of T cell lymphoma, the infiltrate had a tendency for dermal and sometimes even epidermal involvement, with sheeting out of malignant cells, in contrast to the characteristic subcutaneous localization and rimming of fat spaces noted in SCPTCL. The two PTL, NOS were CD4+ and negative for both TIA-1 and perforin. Although the remaining controls expressed TIA-1 and perforin, in keeping with their cytotoxic T or natural killer (NK) cell origin, histological and other immunophenotypical features allowed distinction from SCPTCL. Five cases of SCPTCL were also stained for apoptosis using a tdt-mediated end labeling kit. All cases showed numerous positive apoptotic bodies, suggesting apoptosis as the mechanism of cell death in these tumors. Our study indicates that SCPTCL constitutes a distinctive clinicopathological entity derived from cytotoxic T lymphocytes and should be differentiated from other benign and malignant lymphoid infiltrates involving the subcutis. The apoptosis seen in these tumors may be mediated by release of cytotoxic granular proteins.

Pathological Findings in Human Autoimmune Lymphoproliferative Syndrome

The defects in lymphocyte apoptosis that underlie the autoimmune lymphoproliferative syndrome (ALPS) are usually attributable to inherited mutations of the CD95 (Fas) gene. In this report, we present the histopathological and immunophenotypic features seen in the lymph nodes (n = 16), peripheral blood (n = 10), bone marrow (n = 2), spleen (n = 3), and liver (n = 2) from 10 patients with ALPS. Lymph nodes showed marked paracortical hyperplasia. Interfollicular areas were expanded and populated by T cell receptor-alphabeta CD3+ CD4-CD8- (double-negative, DN) T cells that were negative for CD45RO. CD45RA+ T cells were increased in all cases studied. The paracortical infiltrate was a result of both reduced apoptosis and increased proliferation, as measured by in situ detection of DNA fragmentation and staining with MIB-1, respectively. The paracortical proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. Many of the paracortical lymphocytes expressed markers associated with cytotoxicity, such as perforin, TIA-1, and CD57. CD25 was negative. In addition, most lymph nodes exhibited florid follicular hyperplasia, often with focal progressive transformation of germinal centers; in some cases, follicular involution was seen. A polyclonal plasmacytosis also was present. The spleens were markedly enlarged, more than 10 times normal size. There was expansion of both white pulp and red pulp, with increased DN T cells. DN T cells also were observed in liver biopsies exhibiting portal triaditis. In the peripheral blood, the T cells showed increased expression of HLA-DR and CD57 but not CD25. CD45RA+ T cells were increased in the four cases studied. Polyclonal B cell lymphocytosis with expansion of CD5+ B cells was a characteristic finding. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS. Of note, two affected family members of one proband developed lymphoma (T-cell-rich B-cell lymphoma and nodular lymphocyte predominance Hodgkins disease, respectively).

Anaplastic large cell lymphoma

Session 8 of the 2005 Society of Hematopathology/European Association for Haematopathology Workshop was devoted to anaplastic large cell lymphoma (ALCL). Most cases submitted were anaplastic lymphoma kinase (ALK)+ ALCL highlighting unusual clinical settings, histologic variants, and variant translocation partners. Cases submitted as ALK- ALCL emphasized the immunohistochemical overlap with classical Hodgkin lymphoma (eg, CD15+/CD30+). It was also clear that consensus histologic and immunohistochemical criteria for the diagnosis of ALK-ALCL are lacking. Many expressed the opinion that ALK-ALCL is not a distinct entity at the immunophenotypic or genetic level and is better designated as peripheral T-cell lymphoma (PTCL), unspecified. Others suggested that the histologic features of ALK-ALCL are distinctive nevertheless and that this diagnosis has meaning that is lost by designating these neoplasms as PTCL, unspecified. This session also included CD30+ anaplastic lymphomas involving skin in which the differential diagnosis included cutaneous ALCL and systemic ALK-ALCL.

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkins lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38β-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.

Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma

NOTCH2 mutations in splenic marginal zone lymphoma are associated with poor prognosis.

The PAF complex synergizes with MLL fusion proteins at HOX loci to promote leukemogenesis

MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis.

Multiple interactions recruit MLL1 and MLL1 fusion proteins to the HOXA9 locus in leukemogenesis

MLL1 fusion proteins activate HoxA9 gene expression and cause aggressive leukemias that respond poorly to treatment, but how they recognize and stably bind to HoxA9 is not clearly understood. In a systematic analysis of MLL1 domain recruitment activity, we identified an essential MLL1 recruitment domain that includes the CXXC domain and PHD fingers and is controlled by direct interactions with the PAF elongation complex and H3K4Me2/3. MLL1 fusion proteins lack the PHD fingers and require prebinding of a wild-type MLL1 complex and CXXC domain recognition of DNA for stable HoxA9 association. Together, these results suggest that specific recruitment of MLL1 requires multiple interactions and is a precondition for stable recruitment of MLL1 fusion proteins to HoxA9 in leukemogenesis. Since wild-type MLL1 and oncogenic MLL1 fusion proteins have overlapping yet distinct recruitment mechanisms, this creates a window of opportunity that could be exploited for the development of targeted therapies.

High prevalence of somatic MAP2K1 mutations in BRAF V600E negative Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) represents a clonal proliferation of Langerhans cells. BRAF V600E mutations have been identified in approximately 50% of cases. To discover other genetic mechanisms underlying LCH pathogenesis, we studied 8 cases of LCH using a targeted next-generation sequencing platform. An E102_I103del mutation in MAP2K1 was identified in one BRAF wild-type case and confirmed by Sanger sequencing. Analysis of 32 additional cases using BRAF V600E allele-specific polymerase chain reaction and Sanger sequencing of MAP2K1 exons 2 and 3 revealed somatic, mutually exclusive BRAF and MAP2K1 mutations in 18 of 40 (45.0%) and 11 of 40 (27.5%) cases, respectively. This is the first report of MAP2K1 mutations in LCH that occur in 50% of BRAF wild-type cases. The mutually exclusive nature of MAP2K1 and BRAF mutations implicates a critical role of oncogenic MAPK signaling in LCH. This finding may also have implications in the use of BRAF and MEK inhibitor therapy.