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Provides information on a study which examined BK-related polyomavirus vasculopathy in a renal-transplant recipient. Methodology; Results; Conclusion.

The bi- or multinucleated Reed-Sternberg cell (RS) is the diagnostic cornerstone of Epstein-Barr Virus (EBV)-positive and EBV-negative classical Hodgkin lymphoma (cHL). cHL is a germinal center (GC)-derived B-cell disease. Hodgkin cells (H) are the mononuclear precursors of RS. An experimental model has to fulfill three conditions to qualify as common pathogenic denominator: (i) to be of GC-derived B-cell origin, (ii) to be EBV-negative to avoid EBV latency III expression and (iii) to support permanent EBV-encoded oncogenic latent membrane protein (LMP1) expression upon induction. These conditions are unified in the EBV-, diffuse large B-Cell lymphoma (DLBCL) cell line BJAB-tTA-LMP1. 3D reconstructive nanotechnology revealed spatial, quantitative and qualitative disturbance of telomere/shelterin interactions in mononuclear H-like cells, with further progression during transition to RS-like cells, including progressive complexity of the karyotype with every mitotic cycle, due to BBF (breakage/bridge/fusion) events. The findings of this model were confirmed in diagnostic patient samples and correlate with clinical outcomes. Moreover, in vitro, significant disturbance of the lamin AC/telomere interaction progressively occurred. In summary, our research over the past three decades identified cHL as the first lymphoid malignancy driven by a disturbed telomere/shelterin/lamin AC interaction, generating the diagnostic RS. Our findings may act as trailblazer for tailored therapies in refractory cHL.

Background: Skin diseases have been shown to worsen psychological distress, which, in turn, may be detrimental to treatment outcomes. Both the impact of psychological distress on response to treatment in mycosis fungoides (MF) and the effect of treatments on psychological well-being are unclear., Objectives: To evaluate (1) the association between pretreatment psychological morbidity and treatment outcome in early-stage MF and (2) the impact of response to treatment on psychological well-being., Methods: This was a prospective cohort study of patients with early-stage MF who started a new stage-directed treatment for their disease. The response was determined using the modified severity-weighted assessment tool, and psychological distress was assessed using the 12-item General Health Questionnaire (GHQ-12) and Penn State Worry Questionnaire (PSWQ). Participants were followed for 1 year., Results: In all, 24 consecutive patients were recruited. Objective response rate was 71% (17/24), consistent with existing literature. Prior to treatment, 9 patients (38%) had clinically significant psychological distress on the GHQ-12, while 8 (33%) demonstrated high-level worry on the PSWQ. Of these patients, 6 had pathologic scores on both instruments. Patients with significantly less baseline anxiety/depression on the GHQ-12 responded better to treatment than patients with higher levels ( P = .004). In addition, responders' mean GHQ-12 scores decreased by 39% and their PSWQ scores by 17%, whereas nonresponders' GHQ-12 scores increased by 93% ( P = .042) and their PSWQ scores by 11% ( P = .019)., Conclusions: These findings suggest that (1) baseline psychological distress is associated with worse outcomes in patients with early-stage MF and that (2) effective treatment improves psychological morbidity., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: While ultrasound-guided fine-needle aspiration cell block (FNACB) is a cost-effective, expeditious, and reliable procedure used routinely in the initial evaluation of head and neck masses, it has limited efficacy in diagnosing lymphoproliferative disorders such as non-Hodgkin lymphoma (NHL). Flow cytometry performed on an fine-needle aspiration (FNA) sample [ultrasound-guided fine-needle aspirate flow cytometry or flow cytometry performed on an FNA sample (FNAFC)], has been shown to be a valuable adjunct to FNACB in the diagnosis of lymphoproliferative disorders of the spleen, kidney, and thyroid. The objective of this study was to appraise FNAFC's utility as an ancillary tool to detect NHL arising in the head and neck region in adult patients., Methods: This is a retrospective study involving 52 adult patients with head and neck lymphadenopathies and masses suspicious for lymphoproliferative disorders, who underwent ultrasound-guided FNACB and ultrasound-guided FNAFC between January 2017 and November 2022. Patient demographics, FNACB histopathological and immunophenotypic results, postoperative histopathology results (when available), and follow-up information until May 2023 were reviewed., Results: Of the 52 FNACB samples, 23 samples (44.2%) yielded a diagnosis negative for carcinoma, 20 samples (38.5%) were nondiagnostic on account of scant cellularity, 8 samples (15.4%) were suspicious for malignancy, and a single sample (1.9%) was compatible with malignancy. Regarding FNAFC samples, 37 samples (71.2%) were diagnosed as showing no evidence for a lymphoproliferative disorder, 4 samples (7.7%) as nondiagnostic because of insufficient cell count, 4 samples (7.7%) as suspicious for a lymphoproliferative neoplasm, and 7 samples (13.5%) as compatible with a lymphoproliferative neoplasm, most frequently a B-cell lymphoma. 7 of the 11 patients (63.6%) with a suspicious/positive FNAFC result underwent excisional biopsy for additional work up. Postoperative histopathology reports corroborated FNAFC's findings in 6 patients (85.7%), while the remaining patient's (14.3%) suspicious FNAFC result was discordant with postoperative histopathology results. The other 4 patients (36.4%) did not require excisional biopsy as the hemato-oncologist deemed the information provided by the FNAFC as sufficient for the diagnosis and treatment of an NHL in the specific clinical contexts of those patients. All patients with nondiagnostic (due to insufficient cell count), inconclusive, or negative FNAFC (ie, nondiagnostic of a lymphoproliferative disorder) were followed up for a mean follow-up period of 11.9 months (range: 61.2 months; SD: 10.2 months), during which no new lymphadenopathies/masses nor progression was observed., Conclusions: FNAFC is a useful and practical supplementary tool in the diagnosis of lymphoproliferative disorders in the head and neck region, principally B-cell lymphoma. While conventional FNACB offers a valuable insight into the initial work up of head and neck masses, FNAFC can routinely detect small abnormal cell populations. Furthermore, in specific clinical contexts, it can reliably diagnose NHL, thereby averting the need for an excisional biopsy in a subset of patients., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

We report the case of a patient who was referred to our institution with a diagnosis of CD4+ small/medium-sized pleomorphic lymphoma. At the time, the patient showed a plethora of lesions mainly localizing to the legs; thus, we undertook studies to investigate the lineage and immunophenotype of the neoplastic clone. Immunohistochemistry (IHC) showed marked CD4 and CD8 positivity. Flow cytometry (FCM) showed two distinct T-cell populations, CD4+ and CD8+ (+/- PD1), with no CD4/CD8 co-expression and no loss of panT-cell markers in either T-cell subset. FCM, accompanied by cell-sorting (CS), permitted the physical separation of four populations, as follows: CD4+/PD1-, CD4+/PD1+, CD8+/PD1- and CD8+/PD1+. TCR gene rearrangement studies on each of the four populations (by next generation sequencing, NGS) showed that the neoplastic population was of T-cytotoxic cell lineage. IHC showed the CD8+ population to be TIA-1+, but perforin- and granzyme-negative. Moreover, histiocytic markers did not render the peculiar staining pattern, which is characteristic of acral CD8+ T-cell lymphoma (PCACD8). Compared to the entities described in the 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas, we found that the indolent lymphoma described herein differed from all of them. We submit that this case represents a hitherto-undescribed type of CTCL.

Background: Classical Hodgkin lymphoma (cHL) is one of the most frequently diagnosed neoplasms in young adults and is curable even in the relapse setting. Many patients seek advice regarding pregnancy once they have a sustained complete remission (CR). PD1 inhibitors are effective in inducing CRs in relapsed cHL, but little is known about their effects on pregnancy, fetal outcomes, or risk of relapse. The PD1/PDL1 axis is vital in the maintenance of pregnancy, allowing for fetal tolerance. This axis is also a key pathway by which Hodgkin Reed Sternberg cells escape immune surveillance. Thus, exposure to PD1 inhibitors in the context of a pregnant cHL survivor could potentially lead to maternal and fetal complications as well as increase the risk of relapse. Pregnancy and fetal outcomes following PD1 inhibitors have been reported in women with melanoma, but not cHL. Such data may help physicians counsel their patients on this topic., Case: This case describes a 25-year-old woman who was diagnosed with advanced stage cHL that was treated with multiple courses of chemotherapy and autologous stem cell transplant (ASCT) for primary refractory disease. She experienced a relapse eight months following ASCT and was treated with the PD1 inhibitor pembrolizumab. She completed a total of 21 cycles, achieving a CR after cycle five. After 2 years of sustained CR off pembrolizumab, she had an unassisted and uneventful pregnancy. She delivered a healthy baby boy with no significant complications. He reached his normal milestones in his first year. She remains in CR four years following her last dose of pembrolizumab, evoking the possibility of her being cured of cHL., Conclusion: Successful pregnancies and fetal outcomes, while maintaining clinical remissions, are possible in women with relapsed cHL treated with pembrolizumab., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

Hepatic iron overload, a hallmark of hereditary hemochromatosis, triggers progressive liver disease. There is also increasing evidence for a pathogenic role of iron in non-alcoholic fatty liver disease (NAFLD), which may progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular cancer. Mouse models of hereditary hemochromatosis and NAFLD can be used to explore potential interactions between iron and lipid metabolic pathways. Hfe-/- mice, a model of moderate iron overload, were reported to develop early liver fibrosis in response to a high fat diet. However, this was not the case with Hjv-/- mice, a model of severe iron overload. These data raised the possibility that the Hfe gene may protect against liver injury independently of its iron regulatory function. Herein, we addressed this hypothesis in a comparative study utilizing wild type, Hfe-/-, Hjv-/- and double Hfe-/-Hjv-/- mice. The animals, all in C57BL/6J background, were fed with high fat diets for 14 weeks and developed hepatic steatosis, associated with iron overload. Hfe co-ablation did not sensitize steatotic Hjv-deficient mice to liver injury. Moreover, we did not observe any signs of liver inflammation or fibrosis even in single steatotic Hfe-/- mice. Ultrastructural studies revealed a reduced lipid and glycogen content in Hjv-/- hepatocytes, indicative of a metabolic defect. Interestingly, glycogen levels were restored in double Hfe-/-Hjv-/- mice, which is consistent with a metabolic function of Hfe. We conclude that hepatocellular iron excess does not aggravate diet-induced steatosis to steatohepatitis or early liver fibrosis in mouse models of hereditary hemochromatosis, irrespectively of the presence or lack of Hfe., Competing Interests: The authors have declared that no competing interests exist.

Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.

Classical Hodgkin's lymphoma (cHL) is a B-Cell lymphoma comprised of mononuclear Hodgkin cells (H) and bi- to multi-nucleated Reed-Sternberg (RS) cells. Previous studies revealed that H and RS cells express lamin A/C, a component of the lamina of the nuclear matrix. Since no information was available about the three-dimensional (3D) expression patterns of lamin A/C in H and RS cells, we analyzed the 3D spatial organization of lamin in such cells, using 3D fluorescent microscopy. H and RS cells from cHL derived cell lines stained positive for lamin A/C, in contrast to peripheral blood lymphocytes (PBLs), in which the lamin A/C protein was not detected or weak, although its presence could be transiently increased with lymphocyte activation by lipopolysaccharide (LPS). Most importantly, in H and RS cells, the regular homogeneous and spherically shaped lamin A/C pattern, identified in activated lymphocytes, was absent. Instead, in H and RS cells, lamin staining showed internal lamin A/C structures, subdividing the nuclei into two or more smaller compartments. Analysis of pre-treatment cHL patients' samples replicated the lamin patterns identified in cHL cell lines. We conclude that the investigation of lamin A/C protein could be a useful tool for understanding nuclear remodeling in cHL.

Background: Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis disorder that utilizes the RAS-RAF-MEK-ERK pathway. It has a highly variable clinical presentation, where virtually any organ can be involved, thus having the potential of posing a great diagnostic challenge. Over half of the reported cases have the BRAF V600E mutation and have shown a remarkable response to vemurafenib., Case Presentation: We describe herein a patient with a history of stroke-like symptoms and retroperitoneal fibrosis that on initial pathology raised the possibility of IgG4-related disease. However, the patient was refractory to high-dose steroids and progressed further, developing an epicardial soft tissue mass and recurrent neurological symptoms. Integration of the above findings with new information at another hospital about a radiological history of symmetrical lower extremities long bone lesions raised the differential diagnosis of ECD. Molecular analysis of formalin-fixed paraffin-embedded tissue of both of the patient's retroperitoneal biopsies (the second one of which had shown a small focus of foamy histiocytes, CD68+/CD1a-) was positive for BRAF mutation, confirming the diagnosis of ECD. The patient demonstrated a dramatic and sustained metabolic response to vemurafenib on follow-up positron emission tomography scans., Conclusion: This case highlights the need for developing a high index of suspicion for presentations of retroperitoneal fibrosis that could represent IgG4-related disease but fail to respond to steroids. When unusual multisystem involvement occurs, one should consider a diagnosis of a rare histiocytosis. Vemurafenib appears to be an effective treatment for even advanced cases of both ECD and Langerhans histiocytosis bearing the BRAF V600E mutation.

In classical Hodgkin's lymphoma (cHL), specific changes in the 3D telomere organization cause progression from mononuclear Hodgkin cells (H) to multinucleated Reed-Sternberg cells (RS). In a post-germinal center B-cell in vitro model, permanent latent membrane protein 1 (LMP1) expression, as observed in Epstein-Barr virus (EBV)-associated cHL, results in multinuclearity and complex chromosomal aberrations through downregulation of key element of the shelterin complex, the telomere repeat binding factor 2 (TRF2). Thus, we hypothesized that the three-dimensional (3D) telomere-TRF2 interaction was progressively disturbed during transition from H to RS cells. To this end, we developed and applied for the first time a combined quantitative 3D TRF2-telomere immune fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) technique to monolayers of primary H and RS cells, and adjacent benign internal control lymphocytes of lymph node biopsy suspensions from diagnostic lymph node biopsies of 14 patients with cHL. We show that H and RS cells are characterized by two distinct patterns of disruption of 3D telomere-TRF2 interaction. Disruption pattern A is defined by massive attrition of telomere signals and a considerable increase of TRF2 signals not associated with telomeres. This pattern is restricted to EBV-negative cHL. Disruption pattern B is defined by telomere de-protection due to an impressive loss of TRF2 signals, physically linked to telomeres. This pattern is typical of, but is not restricted to, LMP1+EBV-associated cHL. In the disruption pattern B group, so-called 'ghost' end-stage RS cells, void of both TRF2 and telomere signals, were identified, whether or not associated with EBV. Our findings demonstrate that two molecularly disparate mechanisms converge on the level of 3D telomere-TRF2 interaction in the formation of RS cells.

Low grade lymphoma may transform into a more aggressive lymphoma and this transformation is usually associated with a poor outcome. A 65year old man presented with two metabolically active splenic lesions on a staging [18F] fluoro-2-deoxy-d-glucose ( 18 F-FDG) positron emission tomography/computed tomography (PET/CT). Histologic evaluation post splenectomy confirmed the presence of two clonally related lymphomas: a follicular lymphoma (FL) and a diffuse large B-cell lymphoma (DLBCL). Molecular genetic studies confirmed that the DLBCL lesions arose from a pre-existing FL. We present the 18 F-FDG PET/CT imaging characteristics of both lymphoma types which were simultaneously present in the spleen., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although familial aggregation of lymphoproliferative disorders has been described, heredity has not been implicated in the etiology of primary cutaneous B-cell lymphomas (PCBCL). We report herein the first case of 2 young monozygotic twins with PCBCL. The first twin was an 18-year-old woman when she presented with multiple skin nodules on the thorax and head. Histology showed an atypical small B-cell proliferation, consistent with primary cutaneous marginal zone lymphoma (PCMZL). Molecular genetics studies demonstrated B-cell clonality. Seven years later, the second twin developed her first lesion that was histologically similar to that of her twin. She subsequently developed other clinically similar lesions. Histology was consistent with PCMZL and showed B-cell clonality. Occurrence of PCBCL in these monozygotic twins raises the possibility of a genetic risk factor. Further study of such rare cases may offer valuable insights into the molecular basis of the etiology and pathogenesis of this unusual disorder., (© The Author(s) 2016.)

The majority of diffuse large B-cell lymphoma (DLBCL) tumors contain mutations in histone-modifying enzymes (HMEs), indicating a potential therapeutic benefit of histone deacetylase inhibitors (HDIs), and preclinical data suggest that HDIs augment the effect of rituximab. In this randomized phase 2 study, we evaluated the response rate and toxicity of panobinostat, a pan-HDI administered 30 mg orally 3 times weekly, with or without rituximab, in 40 patients with relapsed or refractory de novo (n = 27) or transformed (n = 13) DLBCL. Candidate genes and whole exomes were sequenced in relapse tumor biopsies to search for molecular correlates, and these data were used to quantify circulating tumor DNA (ctDNA) in serial plasma samples. Eleven of 40 patients (28%) responded to panobinostat (95% confidence interval [CI] 14.6-43.9) and rituximab did not increase responses. The median duration of response was 14.5 months (95% CI 9.4 to "not reached"). At time of data censoring, 6 of 11 patients had not progressed. Of the genes tested for mutations, only those in MEF2B were significantly associated with response. We detected ctDNA in at least 1 plasma sample from 96% of tested patients. A significant increase in ctDNA at day 15 relative to baseline was strongly associated with lack of response (sensitivity 71.4%, specificity 100%). We conclude that panobinostat induces very durable responses in some patients with relapsed DLBCL, and early responses can be predicted by mutations in MEF2B or a significant change in ctDNA level at 15 days after treatment initiation. This clinical trial was registered at www.ClinicalTrials.gov (#NCT01238692)., (© 2016 by The American Society of Hematology.)

Background: Numerous treatments are available for cutaneous T-cell lymphoma (CTCL), including systemic retinoids. Very few data are available on topical retinoids., Objectives: The aim of this study was to evaluate the safety and efficiency of tazarotene as monotherapy for early-stage CTCL., Methods: An open-label, prospective study of tazarotene as monotherapy for stages IA to IIA CTCL was conducted. Index lesions on 10 patients were followed for 6 months on treatment, plus at least 6 months off treatment., Results: Six patients (60%) showed complete response (CR). Erythema, scaling, thickness, and lesion area decreased progressively throughout treatment. The mean time to CR was 3.8 months; CR was durable for at least 6 months in 83%. Of the 4 patients (40%) without CR, 2 (20%) had stable disease and 2 (20%) stopped the medication because of local side effects; none showed progression., Conclusions: This is the first Canadian trial providing evidence that topical tazarotene has excellent potential as a monotherapy agent for stages I to IIA CTCL., (© The Author(s) 2016.)

Purpose: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology., Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions., Results: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell-type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes., Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290-300. ©2015 AACR., (©2015 American Association for Cancer Research.)

In this retrospective cohort study of 174 consecutive, newly diagnosed cases of diffuse large B-cell lymphoma (DLBCL), clinical and pathological variables, treatment, response and survival were compared for patients aged 80 and over (n = 40) to those under 80. Eastern Cooperative Oncology Group (ECOG) status and International Prognostic Index (IPI) were significantly worse among older patients. Standard treatment was given to only 32.5% of older versus 86.6% of younger patients, and 65% of the elderly did not receive standard therapy. At 12 months, overall and event-free survival were 51.3% (95% confidence interval [CI]: 35-66%) vs. 93% (CI: 88-97%) and 41.9% (CI: 25-58%) vs. 84.8% (CI: 77-90%), for older versus younger patients, respectively. Choice of therapy was significantly associated with survival in the elderly, and low albumin but not comorbidity score was associated with not receiving standard therapy. Patients with DLBCL aged 80 and over are distinct from all other age groups with regard to treatment tolerance. A minority can receive standard therapy, but for the majority, novel therapeutic options are needed.

Gastrointestinal stromal tumor (GIST) and mantle cell lymphoma involving the appendix are rare as individual disease entities. Their coexistence has not been previously reported in the literature. We describe a 65-year old female who presented with extensive ileocecal mantle cell lymphoma, which extended to the appendix. The appendix was involved by mantle cell lymphoma and an incidental coexistent GIST was noted in the appendiceal wall. The GIST was CD117 positive but did not harbor mutations in the c-kit and PDGFR genes. In addition, it was unusual in showing S-100 immunoreactivity and ultrastructural evidence of autonomic nerve differentiation. This is the first description of the association of a GIST with autonomic nerve differentiation coexisting with mantle cell lymphoma in the appendix.

Background: Angiolymphoid hyperplasia with eosinophilia (ALHE) and Kimura's Disease (KD) share many clinical and histopathological features. Although they were once considered different stages of the same disease, they are now known to represent separate entities. Recently, ALHE is being called epithelioid hemangioma (EH), a term that better describes the possible neoplastic nature of the entity., Case Presentation: An eighteen year-old Asian female presented with a three-month history of fluctuating swelling and ptosis of the left upper eyelid. Computed tomography disclosed a distinct homogeneous lesion in the left superior orbit, molding to the globe and other orbital structures. At histopathological evaluation the lesion was composed of numerous blood vessels lined by plump endothelial cells with oval nuclei protruding into the lumen. Surrounding the vessels, there was a chronic inflammatory infiltrate with a large proportion of eosinophils. Based on clinical and histopathological findings, the diagnosis of EH was made., Conclusion: Although exams like blood count, urinalysis and whole body scans can assist in the differential diagnosis, EH can be diagnosed and differentiated from KD on histopathological grounds. The presence of vascular hyperplasia with plump endothelial cells protruding into the lumen is the most important discriminator in establishing the diagnosis of EH. Such distinction is crucial for the patient because EH is not associated with any of the systemic manifestations present in KD.

Background: CD30-positive cutaneous lymphoproliferative disorders (LPD) represent a spectrum of diseases ranging from low-grade (lymphomatoid papulosis; LyP) to high-grade (pleomorphic and anaplastic large-cell lymphoma; PTL, ALCL) with overlapping morphologic and immunophenotypic features. The common phenotypic hallmark is the expression of CD30-antigen by the tumor cells which morphologically resemble Reed-Sternberg cells. Although LyP is a non-fatal recurring disorder, it is associated with systemic lymphomas including Hodgkin's lymphoma (HL), mycosis fungoides (MF) and ALCL in 5-20% of the cases. Currently there is no marker to predict the development of systemic lymphomas in patients with LyP. Fascin, an actin bundling protein, has recently been shown to be a unique marker found in almost 100% of classical HL., Methods: Because of the association of LyP with HL, fascin expression was analyzed by immunohistochemistry in LyP (n = 45), cutaneous CD30+ ALCL (n = 17) and pleomorphic T-cell lymphoma (n = 9) (PTL) and LyP associated with systemic lymphomas (7 HL, 2 ALCL, 1 MF), with the intent to determine if fascin expression can predict disease progression., Results: Fascin was expressed by tumor cells in 11/45 (24%) cases of LyP, 11/17 (64%) cases of ALCL, 7/9 (77%) cases of PTL and 6/10 (60%) cases of LyP associated with systemic lymphomas. Fascin expression in LyP was significantly less frequent than in ALCL (p < 0.001) and also than in LyP associated with lymphomas (p < 0.05). There was no significant difference of fascin expression within the histological subtypes of LyP. We found no evidence of ALK expression nor of Epstein-Barr virus expression in any case either by in situ hybridization or immunohistochemistry in the LyP cases associated with HL., Conclusions: This is the first study to demonstrate that fascin is expressed in cutaneous CD30+ LPD and that it is a candidate marker of disease progression in LyP.

Lymphomatoid papulosis is part of a spectrum of CD30+ cutaneous lymphoproliferative disorders characterized by spontaneous tumor regression. The mechanism(s) of regression is unknown. In a recent study, a selective increase in CD30 ligand expression in regressing lesions of lymphomatoid papulosis and cutaneous CD30+ anaplastic large cell lymphoma was shown, suggesting that activation of the CD30 signaling pathway may be responsible for tumor regression, whereas no difference in Fas/Fas ligand expression was found between regressing and nonregressing lesions. Therefore we tested the effects of CD30 and Fas activation on three CD30+ cutaneous lymphoma cell lines (Mac-1, Mac-2 A, JK) derived from nonregressing tumors of two patients who had progressed from lymphomatoid papulosis to systemic anaplastic large cell lymphoma. To evaluate the effects of CD30 signaling, the cell lines were incubated with a CD30 agonistic antibody, HeFi-1. Proliferative responses, mitogen-activated protein kinase, and nuclear factor kappa B activities were determined with and without CD30 activation. Mac-1 and Mac-2 A showed increased proliferative responses to incubation with CD30 activating antibody, HeFi-1. Inhibition of the mitogen-activated protein kinase activity caused growth inhibition of the Mac-1, Mac-2 A, and JK cell lines. Activation of the Fas pathway induced apoptosis in all three cell lines. Taken together, these findings suggest that resistance to CD30-mediated growth inhibition provides a possible mechanism for escape of cutaneous anaplastic large cell lymphoma from tumor regression. Mitogen-activated protein kinase inhibitors are potential therapeutic agents for the treatment of advanced cutaneous anaplastic large cell lymphoma. J Invest Dermatol 115:1034-1040, 2000

To develop a model for the biology and treatment of CD30+ anaplastic large cell lymphoma (ALCL), we transplanted leukemic tumor cells from a 22-month-old girl with multiple relapsed ALCL. Tumor cells were inoculated intraperitoneally into a 4-week-old SCID/bg mouse and produced a disseminated tumor within 8 weeks; this tumor was serially transplanted by subcutaneous injections to other mice. Morphology, immunohistochemistry, and molecular genetics which demonstrated the NPM-ALK fusion protein, resulting from the t(2;5)(p23;q35), confirmed the identity of the xenograft with the original tumor. The tumor produced transcripts for interleukin-1alpha, tumor necrosis factor-alpha, and interferon-gamma which could explain the patient's B-symptoms. Treatment of mice with monoclonal antibody (HeFi-1) which activates CD30 antigen administered on day 1 after tumor transplantation prevented tumor growth. Treatment with HeFi-1 after tumors had reached a 0.2 cm(3) volume caused tumor growth arrest and prevention of tumor dissemination. We conclude that transplantation of CD30+ ALCL to SCID/bg mice may provide a valuable model for the study of the biology and design of treatment modalities for CD30+ ALCL.

Using differential display, we sought to identify novel genes expressed in the early stages of 3T3-L1 adipocyte differentiation. A gene which we have named "band25" was identified, and a full-length cDNA sequence was assembled. Sequence analysis revealed that the 2842-bp cDNA encodes a putative 628-amino acid protein product, which is a member of the GTPase-activating protein (GAP) family. This gene may be the murine homolog of the human MgcRacGAP protein, which was identified in male germ cells. Other closely related proteins include the Drosophila protein Rotund, several chimerins, and the human breakpoint cluster region (Bcr) protein. These GAP proteins all specifically inactivate Rac, a member of the Ras-like family of proteins. A consensus sequence for a diacyl glycerol/phorbol ester-binding domain was also found in the Band25 sequence. The expression of band25 mRNA is regulated during the differentiation of both adipocytes and myoblasts. Its mRNA was shown to be expressed at a low level in confluent 3T3-L1 preadipocytes and in differentiated 3T3-L1 adipocytes. Expression of band25 was increased 15.5 fold by 24 h after the induction of differentiation, when 3T3-L1 cells undergo several rounds of postconfluent cell division. Expression was also high in growing 3T3-L1 and C2C12 cells but decreased progressively as C2C12 cells underwent differentiation. These observations suggest that the expression of band25 is growth regulated and that the protein could play a role in the regulation of growth-related processes.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiative insulin signaling pathway, we introduced a leu61-activated ras gene into a Ras-transformable, C3H10T1/2-derived preadipocytic cell line. The results demonstrate that rasleu61 expression in this line blocks differentiation and that this block appears at lower levels than required for full neoplastic transformation. In addition, to examine whether the inability of Rasleu61 to induce differentiation by replacing the insulin signal could be attributed to its transforming effect in this system, we examined the effect of Rasleu61 at levels below the baseline, by expressing rasleu61 in a series of preadipocytes which were rendered deficient in endogenous c-Ras activity. The results show that even very low Rasleu61 levels, insufficient to restore the growth rate of these cells to normal, blocked rather than enhanced differentiation, indicating that rasleu61 expression alone is not sufficient to promote adipocytic differentiation in this system, even in the absence of neoplastic transformation. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the v-Ras effects upon adipocytic differentiation and transformation.

To investigate the role of the cellular ras gene product in neoplastic transformation by the SV40 large tumor antigen (SVLT), murine C3H10T1/2 cells were rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs or through the introduction of the dominant-negative mutant, ras(asn17). Consistent with previous results, SVLT-induced morphological transformation was unaffected by the down-regulation of c-ras gene product activity. On the other hand, colony formation in soft agar and tumorigenicity in nude mice were drastically reduced in c-Ras-deficient cells. In addition, SVLT expression in C3H10T1/2 cells led to increased c-Ras activity, as determined by an increase in the Ras-bound GTP/GTP + GDP ratio. These results suggest that c-Ras is required for full neoplastic transformation by SVLT.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiation-promoting insulin signaling pathway, we introduced a leu61-activated ras gene into the Ras-transformable 3T3 L1 (ATCC CCL92.1) and a number of C3H10T1/2-derived preadipocytic cell lines. The results demonstrate a quantitative reciprocal regulation of differentiation and several transformation-associated properties in response to graded levels of ras gene expression, with the loss of differentiative capacity, morphological transformation, stimulation of proliferation, and anchorage-independent growth requiring increasing levels of Ras(leu61) protein. Furthermore, using novel, tightly regulatable 3T3 L1 transfectants, we demonstrated that Ras(leu61) effectiveness in blocking adipocytic differentiation is strictly dependent on the timing of its expression relative to cell growth arrest, with ras(leu61) expression being ineffective at inhibiting differentiation or inducing morphological transformation once the differentiative process has commenced. Moreover, rasleu61 induction failed to substitute for or enhance the c-Ras-dependent differentiative insulin signal, even under conditions in which it did not induce transformation. Therefore, although necessary for insulin signal transduction, the Ras signal alone is not sufficient to induce adipocytic differentiation in this system. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the Rasleu61 effects on adipocytic differentiation and transformation.

The positive association of the ras oncogene with human cancer and the recognition that malignancy may, in part, represent the imbalance between cell proliferation and differentiation have generated intense interest in the potential role of ras in cell differentiation. We investigated this possibility utilizing as a model system the differentiation of the mesenchymal cell line C3H 10T1/2 (10T1/2) to adipocytes, and a series of transfectants of 10T1/2 cells in which the level of the ras gene product (p21ras; Ras) can be effectively up- or down-modulated. In agreement with previous reports, we found that 10T1/2 cultures, propagated in the resting state for several weeks, spontaneously convert to fat cells at a very low frequency. Downmodulation of endogenous p21ras levels, as a consequence of expression of antisense ras, markedly increased the rapidity and frequency of adipose conversion (6- to 10-fold), which was equivalent in magnitude to that effected by the potent differentiating agent 5-azacytidine. Conversely, overexpression of ras completely inhibited cell differentiation. In addition, adipocytes derived from antisense-ras expressing lines were characterized by a decrease in hormone responsiveness, as well as an apparent deficiency in attaining the terminally differentiated state. These findings suggest that Ras may be a negative regulator of the decision-making step of fibroblast differentiation to adipocytes. In addition, Ras may play an essential positive role in the transduction of hormonal signals necessary for full adipocyte maturation during later progression along the differentiation pathway.

Changes in the natural resistance phenotype were examined for the 2H1, 10T 1/2 cells expressing the activated human H-ras oncogene under the transcriptional regulation of the zinc-inducible mouse metallothionein-I promoter. Culture of the cells in 50 microM ZnSO4 induced an increase in ras protein p21 levels which were maximal within 1 day. Natural-antibody (NAb) binding was significantly increased following 2 days of cell culture in ZnSO4 and continued to increase up to 4 days. The increased NAb binding returned to uninduced levels within 2 days following the removal of added zinc ions from the culture medium. The cells also exhibited a significant increase in natural killer (NK) cell sensitivity following 2 days in ZnSO4. This was maintained as long as the zinc was in the medium, but returned to uninduced levels within 1 day following its removal. The results show that NAb binding and susceptibility to NK cells increased following ras oncogene expression in 10T 1/2 cells and that both parameters were regulated by p21 expression. Repeated i.v. administration of whole-serum NAb prior to tumor inoculation reduced the number of early tumors following s.c. injection of Zn(++)-induced 2Hl cells into Zn(++)-treated C3H/HeN mice, consistent with an in vivo role for NAb in the defense against ras-transformed cells. In contrast, small but statistically significant reductions in NAb binding were observed following v-H-ras transformation of NIH 3T3 cells or v-src transformation of 10T 1/2. The data argue for an NAb- and NK-cell-susceptible phase of ras-induced tumor development which is a prerequisite for these mediators to contribute to a first line of defense against incipient neoplasia, and suggest that characteristics of the recipient cell and the transforming oncogene are important in determining the natural resistance phenotype.

The effect of mutated c-Ha-ras expression on Ca2+ and phospholipid-dependent protein kinase C (PKC) activity during the process of transformation was analysed using an inducible metallothionein-ras hybrid oncogene system. A close correlation was found between the timing of ras expression and the loss of PKC enzymatic activity measured in a cell-free system. Examination of the subcellular distribution of the enzyme in inducible and constitutive ras-transformants revealed that expression of ras was associated with an apparent translocation of PKC to the plasma membrane concomitant with down-regulation of PKC enzymatic activity in particulate as well as cytosolic fractions. Quantitation of PKC protein utilizing a PKC-specific antiserum showed that ras expression was associated with a decrease in the total amount of PKC protein present in the cell. We conclude that transformation by c-Ha-ras is accompanied by down-regulation of PKC activity and that the basis of this effect may, to a large extent, lie in the down-regulation of the amount of PKC protein.

We have isolated a repetitive 1.8 kb KpnI DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag+-Cs2SO4 gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5' AATGG 3' similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed.

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.

Heterogeneous populations of saturable specific high-affinity binding sites for the biologically active subunit of the 7S nerve growth factor complex purified from mouse submaxillary gland (NGF) are detected on human tumor cells of neural crest origin. Detailed studies of the melanoma cell line MeWo demonstrate two populations of binding sites of high affinity with dissociation equilibrium constants of Kd 4 X 10(-11) and 5 X 10(-10) M, respectively. Other cell lines of neural crest origin also show high-affinity binding heterogeneity. Exposure of the cell lines to 1 mM theophylline reversibly reduces the number of available NGF binding sites without influencing the affinities of binding. On the MeWo cell line, this is not related to the stages of the cell cycle or to production and release of a NGF-like molecule by the theophylline-exposed cells. These observations complement earlier studies of theophylline-induced alterations in NK cell sensitivity on the MeWo cell line, providing further evidence for cell surface phenotypic changes induced by a compound that promotes differentiation in melanocytes and melanoma cells. Future studies of cell surface phenomena involved in theophylline-induced NGF binding site disappearance may lead to a better understanding of the NGF receptor and its disappearance from certain embryonic cells of neural crest origin during differentiation.