Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens.

INTRODUCTION: Tumor mutation profiling is standard-of-care in lung carcinoma patients. However, comprehensive molecular profiling of small specimens, including core needle biopsy (CNB) and fine-needle aspiration (FNA) specimens, may often be inadequate due to limited tissue. Centrifuged FNA supernatants, which are typically discarded, have emerged recently as a novel liquid-based biopsy for molecular testing. In this study, we evaluate the use of lung carcinoma FNA supernatants for detecting clinically relevant mutations. METHODS: Supernatants from lung carcinoma FNA samples (n = 150) were evaluated. Samples were further analyzed using next-generation sequencing (NGS) and ultrasensitive droplet digital PCR (ddPCR). Mutation profiles in a subset of samples were compared with results derived from paired tissue samples from the same patient (n = 67) and available plasma liquid biopsy assay (n = 45). RESULTS: All 150 samples yielded adequate DNA and NGS were carried out successfully on 104 (90%) of 116 selected samples. Somatic mutations were detected in 82% of the samples and in 50% of these patients a clinically relevant mutation was identified that would qualify them for targeted therapy or a clinical trial. There was high overall concordance between the mutation profiles of supernatants and the corresponding tissue samples, with 100% concordance with concurrent FNA and 96% with concurrent CNB samples. Comparison of actionable driver mutations detected in supernatant versus plasma samples showed 84% concordance. CONCLUSIONS: FNA supernatants can provide a valuable specimen source for genotyping lung carcinoma especially in patients with insufficient tumor tissue, thereby reducing multigene mutation profiling failure rates, improving turnaround times, and avoiding repeat biopsies.

Pruritic arthropod bite-like papules in T-cell large granular lymphocytic leukaemia and chronic myelomonocytic leukaemia.

T-cell large granular lymphocytic leukaemia (T-LGLL) is a clinically indolent mature T-cell neoplasm characterized by a monoclonal population of CD3+ CD8+ cytotoxic T cells, which usually presents as neutropenia, anaemia and thrombocytopenia. Chronic myelomonocytic leukaemia (CMML) is a clonal haematopoietic disorder with features of both a myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Patients with CMML exhibit a persistent peripheral blood monocytosis in addition to myelodysplastic features. Because of the rarity of T-LGLL, its cutaneous manifestations are poorly documented, but include vasculitis, vasculopathy, persistent ulcerations, generalized pruritus and disseminated granuloma annulare. Various types of skin lesions have been observed in patients with CMML and reportedly occur in approximately 10% of cases. We report the extraordinary case of a patient with MDS who developed T-LGLL, and subsequently the MDS progressed to CMML. The patient then developed diffuse arthropod bite-like papules and intractable pruritus.

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.

BACKGROUND: In a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array. RESULTS: The average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton. CONCLUSIONS: The Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

Morphologic Spectrum of Lymphadenopathy in Adult-onset Immunodeficiency (Anti-interferon-γ Autoantibodies).

Adult-onset immunodeficiency syndrome (AOIS) caused by anti-interferon-γ autoantibodies is an emerging disease. Affected patients present typically with systemic lymphadenopathy, fatigue, and fever. We studied 36 biopsy specimens, 31 lymph nodes, and 5 extranodal sites, of AOIS confirmed by serum autoantibody or QuantiFERON-TB Gold In-Tube assay. We describe the morphologic features and the results of ancillary studies, including special stains, immunohistochemistry, and molecular testing. The overall median age of these patients was 60.5 years (range, 41 to 83 y) with a male-to-female ratio of 20:16. All biopsy specimens showed nontuberculous mycobacterial infection, and most cases showed the following histologic features: capsular thickening with intranodal sclerosing fibrosis, irregularly distributed ill-formed granulomas or histiocytic aggregates with neutrophilic infiltration, interfollicular expansion by a polymorphic infiltrate with some Hodgkin-like cells that commonly effaces most of the nodal architecture and proliferation of high endothelial venules. In situ hybridization analysis for Epstein-Barr virus-encoded RNA showed scattered (<1%) to relatively more common (4% to 5%) positive cells in 29 of 30 (97%) tested specimens, reflecting immune dysregulation due to an interferon-γ defect. In the 31 lymph node specimens, 23 (74%) cases showed increased immunoglobulin G4-positive plasma cells (4 to 145/HPF; mean, 49.7/HPF) with focal areas of sclerosis reminiscent of immunoglobulin G4-related lymphadenopathy, 4 (13%) cases resembled, in part, nodular sclerosis Hodgkin lymphoma, and 9 (29%) cases mimicked T-cell lymphoma. Among 33 patients with available clinical follow-up, 20 (61%) showed persistent or refractory disease despite antimycobacterial therapy, and 1 patient died of the disease. We conclude that the presence of ill-defined granulomas, clusters of neutrophils adjacent to the histiocytic aggregates, and some Epstein-Barr virus-positive cells are features highly suggestive of AOIS. A high index of clinical suspicion and awareness of the morphologic features and differential diagnosis of AOIS are helpful for establishing the diagnosis.

How I investigate Clonal cytogenetic abnormalities of undetermined significance.

Myelodysplastic syndromes are a group of hematopoietic stem cell diseases characterized by cytopenia(s), morphological dysplasia, and clonal hematopoiesis. In some patients, the cause of cytopenia(s) is uncertain, even after thorough clinical and laboratory evaluation. Evidence of clonal hematopoiesis has been used to support a diagnosis of myelodysplastic syndrome in this setting. In patients with cytopenia(s), the presence of clonal cytogenetic abnormalities, except for +8, del(20q) and -Y, can serve as presumptive evidence of myelodysplastic syndrome. Recent advances in next-generation sequencing have detected myeloid neoplasm-related mutations in patients who do not meet the diagnostic criteria for myelodysplastic syndrome. Various terms have been adopted to describe these cases, including clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Similarly, studies have shown that certain chromosomal abnormalities, including ones commonly detected in myelodysplastic syndrome, may not be associated necessarily with an underlying myelodysplastic syndrome. These clonal cytogenetic abnormalities of undetermined significance (CCAUS) are similar to CHIP and CCUS. Here, we review the features of CCAUS, distinguishing CCAUS from clonal cytogenetic abnormalities associated with myelodysplastic syndrome, and the potential impact of CCAUS on patient management.

Concordant bone marrow involvement of diffuse large B-cell lymphoma represents a distinct clinical and biological entity in the era of immunotherapy.

In diffuse large B-cell lymphoma (DLBCL), the clinical and biological significance of concordant and discordant bone marrow (BM) involvement have not been well investigated. We evaluated 712 de novo DLBCL patients with front-line rituximab-containing treatment, including 263 patients with positive and 449 with negative BM status. Compared with negative BM disease, concordant BM adversely impacted overall and progression-free survival, independent of the International Prognostic Index (IPI) and cell-of-origin classification. Once BM is concordantly involved, poor prognosis was not associated with the extent of BM involvement. Conversely, patients with discordant BM showed favorable overall survival similar to stage I-II DLBCL. A BM-adjusted IPI, using three parameters: concordant BM involvement, age >60 years, and performance status >1, improves the risk stratification for DLBCL with positive BM. Intensive immunochemotherapy seemingly rendered survival benefit for patients with concordant BM, as did rituximab maintenance for the discordant BM group. Frequently revealing adverse clinical and molecular characteristics, patients with concordant BM demonstrated gene expression signatures relevant to tumor cell proliferation, migration and immune escape. In conclusion, clinical and biological heterogeneity is seen in DLBCL with positive BM but concordant BM involvement represents a distinct subset with unfavorable gene signatures, high-risk clinicopathologic features and poor prognosis.

TCL1 shows a regulated expression pattern in chronic lymphocytic leukemia that correlates with molecular subtypes and proliferative state.

Expression of the human oncogene TCL1 in transgenic mice produces B-cell tumors that resemble chronic lymphocytic leukemia (CLL) suggesting its role in B-cell tumorigenesis. To clarify the expression pattern and regulation of TCL1 in CLL, we assessed 213 primary tumors by immunohistochemistry (IHC), flow-cytometry and/or Western blot, using a new monoclonal antibody. TCL1 protein was detectable in the majority of CLL (90% by IHC) but showed marked variations across cases with virtual absence in approximately 10% of tumors. Higher TCL1 levels correlated with markers of the 'pre-germinal center' CLL subtype including unmutated VH status (P=0.005), ZAP70 expression (P=0.007), and presence of chromosome 11q22-23 deletions (P=0.04). Intratumoral heterogeneity in TCL1 levels was also prominent and explained in part by markedly lower TCL1 expression in proliferating tumor cells. In vitro exposure of CLL cells to interleukin-4 (but not other growth factors) produced progressive and irreversible decrease in TCL1 protein levels in association with the onset of proliferation. TCL1 expression patterns in CLL are complex and highly dynamic and appear to reflect both the histogenetic subtypes of the disease and the growth parameters of individual tumors. The observed regulation pattern suggests that TCL1 may exert its effects predominantly in the unmutated/ZAP70-positive tumor subset.

Cytogenetic landscape and impact in blast phase of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy.

The landscape of additional chromosomal alterations (ACAs) and their impact in chronic myeloid leukemia, blast phase (CML-BP) treated with tyrosine kinase inhibitors (TKIs) have not been well studied. Here, we investigated a cohort of 354 CML-BP patients treated with TKIs. We identified +8, an extra Philadelphia chromosome (Ph), 3q26.2 rearrangement, -7 and isochromosome 17q (i(17q)) as the major-route changes with a frequency of over 10%. In addition, +21 and +19 had a frequency of over 5%. These ACAs demonstrated lineage specificity: +8, 3q26.2 rearrangement, i(17q) and +19 were significantly more common in myeloid BP, and -7 more common in lymphoid BP; +Ph and +21 were equally distributed between two groups. Pearson correlation analysis revealed clustering of common ACAs into two groups: 3q26.2 rearrangement, -7 and i(17q) formed one group, and other ACAs formed another group. The grouping correlated with risk stratification of ACAs in CML, chronic phase. Despite the overall negative prognostic impact of ACAs, stratification of ACAs into major vs minor-route changes provided no prognostic relevance in CML-BP. The emergence of 3q26.2 rearrangement as a major-route change in the TKI era correlated with a high frequency of ABL1 mutations, supporting a role for TKI resistance in the changing cytogenetic landscape in CML-BP.

Bendamustine added to allogeneic conditioning improves long-term outcomes in patients with CLL.

Bendamustine has shown a favorable safety profile when included in chemotherapy regimens for several types of lymphoma, including CLL. This study investigated the long-term effect of adding bendamustine to a conditioning regimen on survival, rate of engraftment, immune recovery and GvHD after allogeneic stem cell transplantation (alloSCT) in CLL patients. These outcomes were compared with the fludarabine, cyclophosphamide and rituximab (FCR) conditioning regimen. We reviewed the data for 89 CLL patients treated on three trials at our institution. Twenty-six (29%) patients received bendamustine, fludarabine and rituximab (BFR) and 63 (71%) received FCR. Patient characteristics were similar in both groups. Ten (38%) BFR-treated patients vs only two (3%) FCR-treated patients did not experience severe neutropenia (P=<0.001). The 3-year overall survival estimates for the BFR and FCR groups were 82 and 51% (P=0.03), and the 3-year PFS estimates were 63% and 27% (P=0.001), respectively. The 2-year treatment-related mortality was 8 and 23% and the incidence of grade 3 or 4 GvHD was 4% and 10%, respectively. This study is the first to report that addition of bendamustine to alloSCT conditioning for CLL patients is associated with improved survival and lower mortality, myelosuppression, and GvHD.

Regulation of PI3K signaling in T-cell acute lymphoblastic leukemia: a novel PTEN/Ikaros/miR-26b mechanism reveals a critical targetable role for PIK3CD.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The PTEN, PI3K/AKT and Notch pathways are frequently altered in T-ALL. PTEN is a tumor suppressor that inactivates the PI3K pathway. We profiled miRNAs in Pten-deficient mouse T-ALL and identified miR-26b as a potentially dysregulated gene. We validated decreased expression levels of miR-26b in mouse and human T-ALL cells. In addition, expression of exogenous miR-26b reduced proliferation and promoted apoptosis of T-ALL cells in vitro, and hindered progression of T-ALL in vivo. Furthermore, miR-26b inhibited the PI3K/AKT pathway by directly targeting PIK3CD, the gene encoding PI3Kδ, in human T-ALL cell lines. ShRNA for PIK3CD and CAL-101, a PIK3CD inhibitor, reduced the growth and increased apoptosis of T-ALL cells. Finally, we showed that PTEN induced miR-26b expression by regulating the differential expression of Ikaros isoforms that are transcriptional regulators of miR-26b. These results suggest that miR-26b functions as a tumor suppressor in the development of T-ALL. Further characterization of targets and regulators of miR-26b may be promising for the development of novel therapies.

Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma.

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

p53 gene mutations are uncommon but p53 is commonly expressed in anaplastic large-cell lymphoma.

Anaplastic large-cell lymphoma (ALCL), as defined in the World Health Organization, is a heterogeneous category in which a subset of cases is associated with the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). p53 has not been assessed in currently defined subsets of ALCL tumors. In this study, we assessed ALK+ and ALK- ALCL tumors for p53 gene alterations using PCR, single-strand conformation polymorphism and direct sequencing methods. We also immunohistochemically assessed ALCL tumors for p53 expression. Three of 36 (8%) ALCL tumors (1/14 ALK+, 2/22 ALK-) with adequate DNA showed p53 gene mutations. By contrast, p53 was overexpressed in 36 of 55 (65%) ALCL tumors (16 ALK+, 20 ALK-). p21, a target of p53, was expressed in 15 of 31 (48%) ALCL tumors including seven of 15 (47%) p53-positive tumors. p21 expression in a subset of ALCL suggests the presence of functional p53 protein. Apoptotic rate was significantly higher in p53-positive than p53-negative tumors (mean 2.78 vs 0.91%, P = 0.0003). We conclude that the p53 gene is rarely mutated in ALK+ and ALK- ALCL tumors. Nevertheless, wild-type p53 gene product is commonly overexpressed in ALCL and may be functional in a subset of these tumors.

An overview of the classification of non-Hodgkin's lymphomas: an integration of morphological and phenotypical concepts.

An analysis of trends in the incidence of non-Hodgkin's lymphoma requires an understanding of individual disease entities within this broad group. The non-Hodgkin's lymphomas represent a diverse group of malignancies that have in common an origin from lymphoid cells. Nevertheless, these disorders are heterogeneous in their clinical behavior, morphological appearance, cellular origin, etiology, and pathogenesis. A modern classification of non-Hodgkin's lymphomas must include an integration of morphological, immunophenotypical, and molecular concepts in order to delineate individual diseases within this broad group. Existing classification schemes such as the working formulation, while they may be useful in providing a guide to clinical management, cannot provide this information in the absence of other data. This point is most readily made with the low-grade B-cell lymphomas which include follicular lymphomas, mantle cell lymphomas, small lymphocytic lymphomas, immunosecretory disorders, and lymphomas of mucosa-associated lymphoid tissues. Each of these malignancies has a distinct phenotype and genotype, and indubitably each has a different etiology. The postthymic T-cell tumors are equally diverse. Analysis of epidemiological data from cancer registries must include a recognition that our ability to recognize individual diseases from historical data is limited. Studies of trends in the non-Hodgkin's lymphomas should attempt to delineate biological markers that may be of relevance to pathogenesis in both historical and prospectively accrued cases.

Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Primary testicular diffuse large B-cell lymphoma displays distinct clinical and biological features for treatment failure in rituximab era: a report from the International PTL Consortium.

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a unique subtype of DLBCL. The impact of rituximab on survival and patterns of treatment failure in PT-DLBCL patient remain controversial. We analyzed the clinical and biological feature of 280 PT-DLBCL cases, 64% of which were treated with rituximab-containing regimens. Although most (95%) patients achieved complete remission, a continuous risk of relapse was observed. Rituximab significantly reduced the cumulative risk of relapse (P=0.022) and improved both progression-free survival (PFS, P=0.012) and overall survival (OS, P=0.027) of PT-DLBCL patients (5-year PFS, 56% vs 36%; 5-year OS, 68% vs 48%). Central nervous system and contralateral testis were the most common sites of relapse, but other extranodal and nodal sites of relapse were also observed. Most cases of PT-DLBCL had a non-germinal center B-cell like (84%) immunophenotype and an activated B-cell like (86%) gene expression profile (GEP) subtype. The distinctive GEP signature of primary testicular lymphoma was relevant to tumor cell proliferation, dysregulated expression of adhesion molecules and immune response, likely accounting for the poor outcome. Accordingly, forkhead box P1 transcription factor (FOXP1) and T-cell leukemia/lymphoma 1 (TCL1) oncogenic activation were confirmed and predicted a significant trend of poor survival. This study provides valuable observations for better understanding of both clinical and biological features in PT-DLBCL patients.

Numbers of host "helper" T cells and proliferating cells predict survival in diffuse small-cell lymphomas.

Diffuse small-cell lymphomas of B-lineage comprise a group of immunophenotypically related lymphoid malignancies that display variable clinical aggressiveness. We compared a variety of clinical, pathologic, and immunologic characteristics of 64 B-lineage diffuse small-cell lymphomas to patient survival in an effort to define prognostically relevant subtypes of these neoplasms. Neither clinical parameters nor histological subclassification correlated with patient outcome. In contrast, three immunologic features of these lymphomas showed a statistically significant relationship with actuarial survival. Neoplasms that manifested greater than or equal to 25% Ki-67+ cells (proliferation-associated antigen), less than 25% Leu 4+ cells (pan-T antigen), or less than 15% Leu 3+ cells (helper/inducer T-subset antigen) were associated with significantly decreased patient survival as compared to neoplasms with the reverse phenotype (P = .02, P = .003, P = .0005, respectively). Leu 3 findings were of particular importance in initial biopsies (P = .0007), while the Ki-67 findings were significant regardless of time of biopsy (P = .01 for biopsies at diagnosis and P = .004 for other biopsies). These data indicate that immunologic analysis can demonstrate subsets of diffuse small-cell lymphoma with different biologic potential, and suggest that such analysis be included in the routine work-up of patients with this type of neoplasm.

Primary cutaneous non-Hodgkin's lymphoma of Ann Arbor stage I: preferential cutaneous relapses but high cure rate with doxorubicin-based therapy.

PURPOSE: Establish frequency, presenting features, response and relapse patterns, and outcome of primary cutaneous non-Hodgkin's lymphoma (PCNHL). PATIENTS AND METHODS: Review of untreated patients, older than 16 years, presenting between 1971 and 1993 with cutaneous lymphoma, not mycosis fungoides, and Ann Arbor stage I. RESULTS: We identified 46 patients, 27 males, with median age of 57 years. Treatment was radiotherapy in 10 patients, doxorubicin-based therapy in 33 patients that was followed by radiotherapy in 25 patients, and other combination with radiotherapy in one patient. The complete response rate was 95%. After a median follow-up of 140 months (range, 61 to 284 months), 18 patients have relapsed, and 14 have died from lymphoma. The first failure was exclusively cutaneous in 50% of relapses. For the 44 treated patients, progression-free survival (PFS; actuarial +/- SE) was 61% +/- 7% and survival was 58% +/- 9% at 12 years. For the 18 patients with diffuse large B-cell lymphoma, after doxorubicin-based regimens, PFS was 71% +/- 12% (P = .0003) versus 0% after radiotherapy; survival was 77% +/- 12% versus 25% +/- 22% (P = 004), respectively. For the nine patients with follicular center-cell lymphoma treated with combined modality, the 12-year PFS was 89% +/- 11% and survival 70% +/- 18%. CONCLUSION: PCNHL is rare, and its first relapse is exclusively cutaneous in 50% of patients. Patients with diffuse large B-cell lymphoma are curable with doxorubicin-based regimens but not with radiotherapy. Prospective studies in PCNHL should define the cytogenetics, the basis for cutaneous tropism, the prognosis of histologic subtypes, and the role of radiotherapy.

Trimetrexate in relapsed T-cell lymphoma with skin involvement.

PURPOSE: Methotrexate (MTX) is active against lymphomas, but transport or polyglutamylation mutations confer MTX resistance. Because trimetrexate (TMTX) enters cells by passive diffusion and is not polyglutamylated, its activity in relapsed T-cell lymphoma was investigated. PATIENTS AND METHODS: Eligible patients had histologically confirmed relapsed T-cell lymphoma involving the skin, had received more than one previous regimen, were older than 16 years, had normal organ function, and had no CNS disease or serious infections, including human immunodeficiency virus. TMTX (200 mg/m(2)) was given intravenously every 14 days without topical or systemic corticosteroids. Patients who responded received up to 12 doses. RESULTS: Twenty patients were assessable for response. Median age was 59 years (range, 45 to 87 years); 13 patients were men. Three patients had anaplastic large-cell lymphoma, 15 had mycosis fungoides or Sézary syndrome (14 with large-cell transformation), and two had peripheral T-cell lymphoma. Serum lactate dehydrogenase was high in 35%, and beta-2 microglobulin was more than 3.0 mg/L in 35% of patients. The median number of previous regimens was three (range, two to 15) and included MTX in five patients. Disease was refractory to the regimen immediately preceding TMTX in 85% of patients. Responses were complete in one and partial in eight patients (overall response rate, 45%). Two of five patients previously treated with MTX responded. Grade 3 or 4 mucositis was observed after 4%, infection after 3%, neutropenic fever after 6%, neutrophils less than 100/microL after 4%, and platelets less than 10,000/microL after 3% of TMTX doses. CONCLUSION: TMTX is active with acceptable toxicity in this population and merits further investigation.

Chromosomal translocation t(14;19) as indicated by bcl-3 rearrangement is a rare phenomenon in non-Hodgkin's lymphoma and chronic lymphocytic leukemia: a molecular genetic analysis of 176 cases.

Using a probe for the newly described bcl-3 gene near the breakpoint of the t(14;19), 176 cases of non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia were analyzed. Rearrangement of the bcl-3 gene was found in only one case; a follicular lymphoma involving the salivary gland that had progressed to a diffuse large cell lymphoma. We conclude that rearrangement of the bcl-3 gene as detected by our method occurs rarely in mature B or T cell neoplasms.