An Unusual Case of Extranodal Marginal Zone Lymphoma Mimicking Abdominal Cocoon Syndrome in an Adolescent Patient.

Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is an indolent non-Hodgkin lymphoma rarely seen in pediatric patients. MALT lymphoma most commonly involves the gastrointestinal tract or peri-orbital tissues, potentially as sequela of chronic antigenic stimulation or immune dysregulation. Rare cases of MALT lymphoma arising from the gynecologic tract have been reported in older adult patients. We present the unique case of a 16-year-old postpubescent female with MALT lymphoma localized to the gynecologic tract, who initially presented with abdominal fullness, abnormal uterine bleeding, and obstructive acute kidney injury secondary to urinary outflow obstruction. Intraoperatively, dense fibrosis of the uterus and left fallopian tube was noted which mimicked abdominal cocoon syndrome. She was treated with 6 cycles of bendamustine and rituximab with complete anatomic and metabolic remission. In this report we highlight a very unusual presentation of a rare malignancy in the pediatric population as well as unique treatment considerations given this patient's young age and tumor location.

How Many Tests Does It Take to Diagnose a Triple-Hit B-Lymphoblastic Lymphoma? (Hint, It's A Lot).

B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a precursor B-cell neoplasm that often harbors specific cytogenetic/molecular abnormalities with distinctive clinical, phenotypic, and prognostic characteristics. Subcategorization of B-ALL/LBL therefore requires extensive cytogenetic and/or molecular testing to determine the appropriate classification and therapeutic interventions for these patients. Herein, we present a case of a 17-year-old young woman diagnosed with B-LBL harboring not only an IGH::MYC rearrangement but also BCL2 and BCL6 rearrangements (so-called "triple-hit") and somatic biallelic TP53 inactivation. MYC rearrangements are relatively rare in B-ALL/LBL, and the identification of a "triple-hit" elicited an initial diagnostic dilemma. However, a multimodal approach allowed for the classification of this complex case and helped guide selection of an appropriate therapeutic regimen.

Childhood Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) in children is rare, accounting for < 5% of all childhood hematologic malignancies. With the advent of next-generation sequencing, the etiology of many childhood MDS (cMDS) cases has been elucidated with the finding of predisposing germline mutations in one-quarter to one-third of cases; somatic mutations have also been identified, indicating that cMDS is different than adult MDS. Herein, cMDS classification schema, clinical presentation, laboratory values, bone marrow histology, differential diagnostic considerations, and the recent molecular findings of cMDS are described.

CD43-positive, EWSR1::FLI1 -rearranged Soft Tissue Sarcoma in a Pediatric Patient With History of B-Cell Acute Lymphoblastic Leukemia.

Ewing sarcoma is a small round blue cell tumor typically characterized by an EWSR1 rearrangement and expression of CD99 and NKX2.2, without expression of hematopoietic markers such as CD45. CD43 is an alternative hematopoietic immunohistochemical marker often utilized in the workup of these tumors and its expression typically argues against Ewing sarcoma. We report a 10-year-old with history of B-cell acute lymphoblastic leukemia presenting with an unusual malignant shoulder mass with variable CD43 positivity, but with an EWSR1::FLI1 fusion detected by RNA sequencing. Her challenging workup highlights the utility of next-generation DNA-based and RNA-based sequencing methods in cases with unclear or conflicting immunohistochemical results.

A Malignant Mimicker: Features of Kikuchi-Fujimoto Disease in the Pediatric Population.

BACKGROUND: Kikuchi-Fujimoto disease (KFD) is a rare, benign, and self-limited disease that presents with cervical lymphadenopathy and systemic symptoms. Histologic evaluation is often necessary to differentiate KFD from other entities. METHODS: Electronic medical records and diagnostic material were reviewed for 14 children diagnosed with KFD and 6 children diagnosed with infectious mononucleosis (IM) from 2013-2021. Four cases of KFD were further characterized using targeted DNA-based next-generation sequencing. RESULTS: Systemic symptoms were present in 86% (n = 12/14) of KFD patients, the most common being fever. Laboratory values worrisome for malignancy included cytopenia(s) (n = 9/12), elevated ESR and/or CRP (n = 9/12), elevated ferritin (n = 7/7), and elevated LDH (n = 7/10). Histologically, lymph nodes showed characteristic necrotic foci without neutrophils surrounded by MPO+ "crescentic" histiocytes. Immunoblasts and CD123+ plasmacytoid dendritic cells (pDCs) were also increased surrounding the necrosis. IM lymph nodes showed similar features when necrosis was present but increases in pDCs were patchy and rare neutrophils were seen in the necrotic foci. Molecular analysis of 4 KFD cases did not identify pathogenic variants. CONCLUSION: While the signs/symptoms of KFD are worrisome, there are pathologic features that help differentiate it from potential mimics. We did not identify characteristic molecular features to aid in the work-up of these cases.

Flow cytometric analysis of CD200 expression by pulmonary small cell carcinoma.

BACKGROUND: CD200 is a membrane bound glycoprotein that is expressed by a variety of normal tissues and hematopoietic malignancies. Flow cytometric analysis of CD200 expression has utility in the evaluation of mature B-cell neoplasms, myeloma, and acute leukemia; however, CD200 expression in nonhematopoietic malignancies has not been extensively studied. METHODS: We studied 14 cases of biopsy proven pulmonary small cell carcinoma in which a discrete CD45 negative, CD56 positive abnormal cell population was identified by flow cytometry. We retrospectively evaluated these cases for flow cytometric and immunohistochemical evidence of CD200 expression. RESULTS: Twelve of the 14 cases of pulmonary small cell carcinoma showed convincing expression of CD200 by both immunohistochemistry and flow cytometry. CONCLUSIONS: Pulmonary small cell carcinoma frequently expresses CD200 at a level that can be detected by flow cytometry and immunohistochemistry. CD200 expression therefore may be used to help identify pulmonary small cell carcinoma in flow cytometry specimens and tissue sections. CD200 may also play a role in the biology of pulmonary small cell carcinoma and is a potential target of future therapies. © 2015 International Clinical Cytometry Society.

comparison of clot-based vs chromogenic factor Xa procoagulant phospholipid activity assays.

We compared 2 commercial plasma procoagulant phospholipid activity (PPA) assays, chromogenic, using bound annexin V to capture phosphatidylserine-containing microparticles, and clot-based. In both, anionic phospholipids accelerated activation of prothrombin by factor Xa. PPA levels were lower in the chromogenic vs the clot-based assay, with poor correlation between methods: normal samples, mean ± SD, 27 ± 17 vs 590 ± 414 nmol/L (n = 24; r(2) = 0.29) and patient samples, mean ± SD, 45 ± 44 vs 401 ± 330 nmol/L (n = 51; r(2) = 0.26). Recovery of phosphatidylserine added to normal, heparinized, and warfarin plasma samples averaged 109% ± 39% using the chromogenic assay but was higher and more varied (mean ± SD, 176% ± 59%) in the clot-based assay. Lupus anticoagulants caused low recovery in both assays. Removal of microparticles by 0.22-µm filtration reduced PPA by 91% in the clot-based and 65% in the chromogenic assay. The clot-based assay showed higher correlation (r(2) = 0.82 vs 0.23) with flow cytometric platelet microparticle counts. The 2 assays measure different aspects of PPA in plasma, with the chromogenic assay primarily measuring smaller microparticles.

Fibrillins in adult human ovary and polycystic ovary syndrome: is fibrillin-3 affected in PCOS?

Polycystic ovary syndrome (PCOS) is a common endocrinopathy in women of reproductive age. Although genetic linkage analyses have demonstrated a susceptibility locus for PCOS mapping to the fibrillin-3 gene, the presence of fibrillin proteins in normal and polycystic ovaries has not been characterized. This study compared and contrasted fibrillin-1, -2, and -3 localization in normal and polycystic ovaries. Immunohistochemical stainings of ovaries from 21 controls and 9 patients with PCOS were performed. Fibrillin-1 was ubiquitous in ovarian connective tissue. Fibrillin-2 localized around antral follicles and in areas of folliculolysis. Fibrillin-3 was present in a restricted distribution within the specialized perifollicular stroma of follicles in morphological transition from primordial to primary type [transitional follicles (TFs)]. Fibrillin-1 and -2 stainings of PCOS ovaries were similar to those of the controls. However, in eight of the nine PCOS ovaries, there was a decrease in the number of TFs associated with fibrillin-3, including no staining in five PCOS samples; decreased number of fibrillin-3-associated TFs/mm(2) was confirmed by quantitative analysis. Our findings support a role for fibrillin-3 in the pathogenesis of PCOS and suggest fibrillin-3 may function in primordial to primary follicle transition. We propose that loss of fibrillin-3 during folliculogenesis may be an important factor in PCOS pathogenesis.

Immunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: a potential therapeutic target.

FoxP3 is a marker for immunosuppressive CD25+CD4+ regulatory T cells. These regulatory T cells are thought to play a role in inducing immune tolerance to antigens and may be selectively recruited by carcinomas. We investigated whether breast carcinomas had significant numbers of FoxP3-positive regulatory T cells by immunohistochemistry, and if their presence was associated with other prognostic factors, such as Nottingham grade, hormone receptor immunohistochemical profile, tumor size, or lymph node metastases. Ninety-seven needle core or excisional breast biopsies with invasive breast carcinoma diagnosed at the University of Washington were stained with antibodies to FoxP3, estrogen receptor, and Her2/neu. The numbers of FoxP3-positive cells present within the neoplastic epithelium, and immediately adjacent stroma were counted manually in three high-powered fields (HPFs; x 400) by two independent pathologists. The average scores were then correlated with the parameters of interest. A threshold of >or=15 FoxP3-positive cells/HPF was used to define a FoxP3-positive case in some analyses. Higher average numbers of FoxP3-positive cells present significantly correlated with higher Nottingham grade status (P=0.000229). In addition, the presence of significant numbers (>or=15/HPF) of FoxP3-positive cells in breast carcinoma was positively associated with higher Nottingham grade (P=0.00002585). Higher average numbers of FoxP3-positive cells were also significantly associated with larger tumor size (>2.0 cm; P=0.012824) and trended toward an association with estrogen receptor negativity. Interestingly, 'triple-negative' (estrogen and progesterone receptor negative and Her2/neu negative) Nottingham grade III cases were also significantly associated with high numbers of FoxP3 cells. These results argue that regulatory T cells may play a role in inducing immune tolerance to higher grade, more aggressive breast carcinomas, and are a potential therapeutic target for these cancers.

Analysis of gene expression profile of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) comprises a group of non-Hodgkin lymphomas characterized by the expression of the CD30/Ki-1 antigen. A subset of ALCL is characterized by chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2. While the most common translocation is the t(2;5)(p23;q35) involving the nucleophosmin (NPM) gene on chromosome 5, up to 12 other translocations partners of the ALK gene have been identified. One of these is the t(1;2)(q25;p23) which results in the formation of the chimeric protein TPM3-ALK. While several of the signaling pathways induced by NPM-ALK have been elucidated, those involved in ALCLs harboring TPM3-ALK are largely unknown. In order to investigate the expression profiles of ALCLs carrying the NPM-ALK and TPM3-ALK fusions, we carried out cDNA microarray analysis of two ALCL tissue samples, one expressing the NPM-ALK fusion protein and the other the TPM3-ALK fusion protein. RNA was extracted from snap-frozen tissues, labeled with fluorescent dyes and analyzed using cDNAs microarray containing approximately 9,200 genes and expressed sequence tags (ESTs). Quantitative fluorescence RT-PCR was performed to validate the cDNA microarray data on nine selected gene targets. Our results show a significant overlap of genes deregulated in the NPM-ALK and TPM-ALK positive lymphomas. These deregulated genes are involved in diverse cellular functions, such as cell cycle regulation, apoptosis, proliferation, and adhesion. Interestingly, a subset of the genes was distinct in their expression pattern in the two types of lymphomas. More importantly, many genes that were not previously associated with ALK positive lymphomas were identified. Our results demonstrate the overlapping and unique transcriptional patterns associated with the NPM-ALK and TPM3-ALK fusions in ALCL.

BCL-2 is consistently expressed in hyperplastic marginal zones of the spleen, abdominal lymph nodes, and ileal lymphoid tissue.

BCL-2 is an antiapoptotic protein overexpressed in follicular lymphomas, principally as a result of the t(14;18)(q32;q21), and useful in distinguishing follicular lymphoma (usually BCL-2 positive) from follicular hyperplasia (BCL-2 negative). BCL-2 is also overexpressed in other lymphoma types without the t(14;18), including marginal zone B-cell lymphoma, because of other, poorly understood mechanisms. It has been suggested that BCL-2 immunoreactivity can distinguish between malignant (BCL-2 positive) and reactive (BCL-2 negative) marginal zone B cells. In this study, we evaluated 26 spleen, 10 abdominal lymph node, and 3 ileum specimens with marginal zone B-cell hyperplasia for BCL-2 expression immunohistochemically. We also analyzed these cases using polymerase chain reaction methods to evaluate for the presence of clonal rearrangements of the immunoglobulin heavy chain gene (IgH) using consensus V FRIII and J region primers, and the t(14;18) involving both the major breakpoint and the minor cluster regions of the bcl-2 gene. All (100%) cases of splenic, abdominal lymph node, and ileal marginal zone hyperplasia displayed strong BCL-2 reactivity in the marginal zone B cells. In all cases analyzed, IgH polymerase chain reaction demonstrated a polyclonal pattern, and bcl-2/JH DNA fusion sequences were not detected. Our results indicate that BCL-2 is consistently expressed by reactive marginal zone B cells of the spleen, abdominal lymph nodes, and ileal lymphoid tissue and should not be used as a criterion for discriminating between benign and malignant marginal zone B-cell proliferations involving these sites.

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-Hodgkin's 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 p38beta-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.

Microarray analysis of B-cell lymphoma cell lines with the t(14;18).

The t(14;18) is the most common genetic alteration in follicular lymphoma, and is detectable in a subset of diffuse large B-cell lymphomas (DLBCL), resulting in over-expression of the anti-apoptotic protein BCL-2. Although the t(14;18)-induced over-expression of BCL-2 is an important step in lymphomagenesis, this aberration alone is not sufficient to produce malignant lymphoma. Further analysis of these tumors is needed to identify additional genes that might be involved in the genesis of follicular lymphoma and progression to DLBCL. To address this issue, we analyzed the gene expression profiles of four t(14;18)-positive cell lines and two t(11;14)-positive mantle-cell lymphoma cell lines using cDNA microarrays containing 4364 genes, and compared them to the genetic profile of phenotypically purified B-cells obtained from hyperplastic tonsils. A total of 137 genes were differentially expressed by approximately twofold or more in the t(14;18) cell lines relative to tonsillar B-cells. 68 genes were up-regulated, 69 genes were down-regulated, and approximately 20% of the differentially regulated genes had no known function. The up-regulated genes included a number of genes involved in the promotion of cellular proliferation and survival, as well as cell metabolism. Down-regulated genes included mediators of cell adhesion and negative regulators of cell activation and growth. Hierarchical clustering analysis separated the t(14;18) and mantle-cell lines into distinct groups based on their gene expression profiles. We confirmed the differential expression of approximately 80% of selected up- and down-regulated genes identified by microarray analysis by quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) analysis and/or immunoblotting. This study demonstrates the utility of cDNA microarray analysis for the assessment of global transcriptional changes that characterize t(14;18)-positive cell lines, and also for the identification of novel genes that could potentially contribute to the genesis and progression of non-Hodgkin's lymphomas with this translocation.

Gene expression profiling of cell lines derived from T-cell malignancies.

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated genes (greater than two-fold) in the lymphomas as a group when compared to CD4-positive T-cells. These included genes involved in cytokine signaling, cell adhesion, cytoskeletal elements, nuclear transcription factors, and known oncogenes and tumor suppressor genes. Quantitative fluorescent reverse transcription-polymerase chain reaction analysis demonstrated 70% concordance with the microarray results. While freshly isolated malignant cells may differ in their individual expression patterns relative to established cell lines from the same diagnoses, we feel that the variety of different lymphocytic cell lines that we examined provides a representative picture of the molecular pathogenesis of T-cell malignancies.

Fluorescence PCR quantification of cyclin D1 expression.

We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A beta-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL.