Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant.

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185DeltaBCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185DeltaBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185DeltaBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185DeltaBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185DeltaBCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185DeltaBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185DeltaBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.

Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis.

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor-dependent myeloid precursor cells, STAT5 activation-deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor-independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor-independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation-defective BCR/ABL SH3+SH2 mutants to induce growth factor-independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor-independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway.

The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.

EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial.

While EBV PCR is used in the management of PTLD, the optimal primer set, relative importance of intracellular versus free plasma EBV, and the baseline profile in an organ transplant population remains unclear. We performed a prospective 2-arm trial utilizing an EBV PCR panel measuring LMP-1, EBER-1 and EBNA-1 in both free plasma as well as intracellular whole blood. Control Arm A consisted of 31 lung transplant patients and Arm B consisted of 35 transplant patients being evaluated for possible PTLD. In Arm A, 1/31 (3%) patients developed a transient plasma EBV load. Thirteen of 31 (42%) had detectable intracellular EBV. In Arm B, 17 (49%) patients were diagnosed with PTLD. Thirteen (76%) had EBV-positive PTLD with 12/13 (92%) having detectable EBV by PCR. The EBV PCR panel had a high sensitivity (92%), specificity (72%), positive predictive value (PPV) (71%) and negative predictive value (NPV) (93%) for diagnosing EBV-positive PTLD and followed patients' clinical course well (p < 0.001). Comparing the individual PCR assays, plasma EBNA PCR was superior with high sensitivity (77%), specificity (100%), PPV (100%) and NPV (86%). We conclude that EBV PCR is a useful test for managing PTLD patients. While plasma EBNA PCR is the best single assay for diagnosing and monitoring PTLD, the complete PCR panel is superior for ruling out its presence.

Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma.

Mantle cell lymphoma (MCL) is a B-cell lymphoma characterized by overexpression of cyclin D1 due to the t(11;14) chromosomal translocation. While expression of cyclin D1 correlates with MCL development, expression of wild-type (WT) cyclin D1 transgene in murine lymphocytes is unable to drive B-cell lymphoma. As cyclin D1 mutants that are refractory to nuclear export display heighten oncogenicity in vitro compared with WT D1, we generated mice expressing FLAG-D1/T286A, a constitutively nuclear mutant, under the control of the immunoglobulin enhancer, Emu. D1/T286A transgenic mice universally develop a mature B-cell lymphoma. Expression of D1/T286A in B lymphocytes results in S phase entry in resting lymphocytes and increased apoptosis in spleens of young premalignant mice. Lymphoma onset correlates with perturbations in p53/MDM2/p19Arf expression and with BcL-2 overexpression suggesting that alterations in one or both of these pathways may contribute to lymphoma development. Our results describe a cyclin D1-driven model of B-cell lymphomagenesis and provide evidence that nuclear-retention of cyclin D1 is oncogenic in vivo.

Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing.

To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16 promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target the epigenetically silenced tumor suppressor genes.

Role of signal transducer and activator of transcription 5 in nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in anaplastic large-cell lymphoma, encodes a M(r) 75,000 hybrid protein that containsthe amino-terminal portion of the nucleolar phosphoprotein nucleophosmin(NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocation. Our studies show that NPM/ALK, similar to other members of this family, activates signal transducer and activator of transcription 5 (STAT5) and that this activation is essential for lymphomagenesis. NPM/ALK-mediated activation of STAT5 was demonstrated by detection of: (a) constitutive tyrosine phosphorylation and enhanced DNA binding ability of STAT5 in NPM/ALK-transformed cells; and (b) NPM/ALK-dependent stimulation of STAT5-mediated transactivation of the beta-casein promoter. Retroviral infection of NPM/ALK+ cells with a dominant-negative STAT5B mutant (STAT5-DNM) inhibited the antiapoptotic activity of NPM/ALK in growth factor and serum-free medium. In addition, STAT5-DNM inhibited proliferation and diminished the clonogenic properties of NPM/ALK-positive cells. Finally, SCID mice injected with NPM/ALK+ cells infected with a virus carrying STAT5-DNM survived significantly longer than mice inoculated with NPM/ALK+ cells infected with the empty virus. Necropsy identified a widespread ALK+ lymphoma in lymph nodes and liver of the affected animals. Together, our data indicate that NPM/ALK-induced activation of STAT5 may play an important role in NPM/ALK-mediated lymphomagenesis.

Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (delta p85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.

The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions--comparison to other cytokines and growth factors.

The role of insulin (INS), and insulin-like growth factor-I (IGF-I) in the regulation of human erythropoiesis is not completely understood. To address this issue we employed several complementary strategies including: serum free cloning of CD34+ cells, RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). In a serum-free culture model, both INS and IGF-I enhanced survival of CD34+ cells, but neither of these growth factors stimulated their proliferation. The influence of INS and IGF-I on erythroid colony development was dependent on a combination of growth factors used for stimulating BFU-E growth. When BFU-E growth was optimally stimulated with erythropoietin (EpO) + kit ligand (KL) the large erythroid colonies developed normally even in the absence of INS or IGF-I. However, the addition of both of these growth factors slightly enhanced colony size. On the other hand, if erythroid colonies were stimulated suboptimally with EpO + IL-3 only, INS or IGF-I increased the number of small erythroid bursts by approximately 30%. Both INS and IGF-I activated signal transduction in maturing human erythropoietic cells as determined by phosphorylation of the insulin receptor substrate-2 (IRS-2) protein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34+, c-kit-R+ marrow cells, and in cells isolated from BFU-E and CFU-GM colonies. Expression of INS-R protein on these cells was subsequently confirmed by cytofluorometry. In contrast, the receptor for insulin-like growth factor-I (IGF-IR) was not detected on CD34+ cells, and was first easily detectable on more differentiated cells derived from day 6 BFU-E and CFU-GM colonies. We conclude that INS and IGF-I may be survival factors for human CD34+ cells, but are not required during early erythropoiesis. In contrast, both growth factors may play some role at the final stages of erythroid maturation.

Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells.

Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Ralphabetagamma) triggers mitogenesis in T cells. IL-2Ralpha expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Ralpha expression, survival, and growth of malignant SS cells.

Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.

Anaplastic large cell lymphoma-propagating cells are detectable by side population analysis and possess an expression profile reflective of a primitive origin.

Cancer stem cells or tumour-propagating cells (TPCs) have been identified for a number of cancers, but data pertaining to their existence in lymphoma so far remain elusive. We show for the first time that a small subset of cells purified from human anaplastic lymphoma kinase (ALK)-positive and -negative, anaplastic large cell lymphoma cell lines and primary patient tumours using the side population (SP) technique have serial tumour-propagating capacity both in vitro and in vivo; they give rise to both themselves and the bulk tumour population as well as supporting growth of the latter through the production of soluble factors. In vivo serial dilution assays utilising a variety of model systems inclusive of human cell lines, primary human tumours and nucleophosmin (NPM)-ALK-induced murine tumours demonstrate the TPC frequency to vary from as many as 1/54 to 1/1336 tumour cells. In addition, the SP cells express higher levels of pluripotency-associated transcription factors and are enriched for a gene expression profile consistent with early thymic progenitors. Finally, our data show that the SP cells express higher levels of the NPM-ALK oncogene and are sensitive to an ALK inhibitor.

Hepatosplenic and subcutaneous panniculitis-like gamma/delta T cell lymphomas are derived from different Vdelta subsets of gamma/delta T lymphocytes.

Gamma/delta T cell lymphomas (gamma/delta TCL) represent rare, often aggressive types of T cell malignancy that are clinically and pathologically diverse. Most gamma/delta TCL occur as a hepatosplenic or subcutaneous type. To date, analysis of the T cell receptor delta (TCRS) gene repertoire of hepatosplenic gamma/delta TCL (gamma/delta HSTCL) and subcutaneous panniculitis-like gamma/delta TCL (gamma/delta SPTCL) has been reported only in a limited number of cases. In this study we analyzed 11 gamma/delta HSTCL and 4 gamma/delta SPTCL by polymerase chain reaction and immunostaining to determine their usage of the Vdelta subtypes (Vdelta1-6). It is noteworthy that 10 of 11 gamma/delta HSTCL expressed the Vdelta1 gene. The remaining case also expressed T cell receptor delta (TCRS) as determined by flow cytometry and TCRdelta rearrangement in Southern blot. However, the Vdelta gene expressed by this lymphoma could not be determined, which suggests usage of an as yet unidentified Vdelta gene. In striking contrast to the gamma/delta HSTCL, all 4 gamma/delta SPTCL expressed the Vdelta2 gene. Our data demonstrate that gamma/delta HSTCL are preferentially derived from the Vdelta1 subset of gamma/delta T lymphocytes, whereas gamma/delta SPTCL are preferentially derived from the Vdelta2 subset. The pattern of Vdelta gene expression in HSTCL and SPTCL corresponds to the respective, predominant gamma/delta T cell subsets normally found in the spleen and skin. This finding suggests that gamma/delta TCL are derived from normal gamma/delta T lymphocytes which reside in the affected tissues. Furthermore, the selective, lymphoma type-specific Vdelta gene segment usage may provide a molecular tool to distinguish better among various types of gamma/delta TCL lymphoma particularly in the clinically advanced, widely disseminated cases.

SHP-1 expression by malignant small B-cell lymphomas reflects the maturation stage of their normal B-cell counterparts.

SHP-1 is a protein phosphotyrosine phosphatase that plays an important role in modulating intracellular signaling, which regulates cell activation, proliferation, differentiation, and migration. It is a negative regulator of signal transduction induced by a number of cell receptors. Our immunohistochemical examination of paraffin-embedded reactive lymph nodes and lymphoid tissues revealed that B lymphocytes in follicle germinal centers do not express SHP-1. A weak staining of the B cells in the germinal center light zones was detected when an ultrasensitive amplification system was used. In contrast, normal B cells in mantle and marginal zones as well as interfollicular B lymphocytes and plasma cells displayed strong immunoreactivity. This pattern of SHP-1 expression was repeated in small B-cell lymphomas. All cases of mantle cell lymphoma (12 of 12), marginal zone lymphoma (10 of 10), and chronic lymphocytic leukemia/small lymphocytic lymphoma (13 of 13) expressed SHP-1 protein. However, only 1 of 30 cases of grade 1 follicle center cell lymphoma expressed SHP-1. Our observations highlight the biologic functions of SHP-1 and demonstrate that the SHP-1 expression pattern by small B-cell lymphomas reflects the maturation stage of their normal cell counterparts. These results indicate that determination of SHP-1 expression may help in the differential diagnosis of small B-cell lymphomas.

Subcutaneous panniculitis-like T-cell lymphoma: clinicopathologic, immunophenotypic, and genotypic analysis of alpha/beta and gamma/delta subtypes.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is an uncommon cutaneous lymphoma that has been proposed as a distinct clinicopathologic entity, but studies of SPTCL are limited. We studied the clinicopathologic, immunophenotypic, and genetic features of 11 SPTCLs. All cases had a variable admixture of pleomorphic small, medium, or large lymphocytes and histiocytes infiltrating the subcutis in a lobular panniculitis-like pattern. A granulomatous reaction was seen in three cases and erythrophagocytosis in four. Karyorrhexis and fat necrosis were present in all cases. Angioinvasion was seen in seven SPTCLs; four had areas of coagulation necrosis. All cases expressed T-cell-associated antigens (CD3epsilon, CD45RO, or CD43) and T-cell receptors (TCR); nine expressed alphabeta TCRs and two expressed gammadelta TCRs. T-cell receptor-gamma, TCRbeta, or TCRdelta genes were clonally rearranged in 8 of 10 cases studied. Both gammadelta SPTCLs expressed Vdelta2+ TCRs and were CD4-, CD8- and CD56+. CD56 was negative in seven of nine alphabeta SPTCLs and inconclusive in the other two. Six of nine alphabeta SPTCLs were CD8+; the CD4/CD8 phenotypes were indeterminate in the other three. Cytolytic granule-associated proteins were expressed by all SPTCLs (11 of 11 were TIA-1+, 4 of 4 were perforin+). In situ hybridization for Epstein-Barr virus-encoded RNA (EBER-1) was negative in all cases. Most patients responded to systemic chemotherapy or local radiation therapy. Seven patients are alive: four without disease (19-73 months) and three with disease (32-72 months); four died: three of disease (3-25 months) and one without disease (42 months). We conclude that SPTCLs are clonal, EBV-, cytotoxic T-cell lymphomas derived from alphabeta T-cells or gammadelta T-cells. The gammadelta SPTCLs appear to be preferentially derived from the Vdelta2+ subset. Subcutaneous panniculitis-like T-cell lymphoma may be rapidly fatal or indolent; local therapy may be appropriate for some patients.

Differences in phosphorylation of the IL-2R associated JAK/STAT proteins between HTLV-I(+), IL-2-independent and IL-2-dependent cell lines and uncultured leukemic cells from patients with adult T-cell lymphoma/leukemia.

To determine activation status of the IL-2R-associated (Jak/STAT) pathway in the HTLV-I infected cells, we examined tyrosine phosphorylation of Jak3, STAT3, and STAT5 in several HTLV-I(+) T-cell lines and in uncultured leukemic T cells isolated from patients with adult T-cell lymphoma/leukemia (ATLL). Constitutive basal phosphorylation of Jak3 and, usually, STAT3 and STAT5 was detected in all four IL-2-independent cell lines tested, but in none of the three IL-2-dependent cell lines. Similarly, there was no detectable basal phosphorylation of Jak3 and STAT5 in the leukemic cells from ATLL patients (0/8 and 0/3, respectively). However, stimulation with IL-2 resulted in Jak3 and STAT5 phosphorylation in both leukemic ATLL cells and IL-2-dependent lines. Furthermore, expression of SHP-1 phosphatase which is a negative regulator of cytokine receptor signaling, was lost in most IL-2 independent cell lines (3/4) but not in the leukemic ATLL cells (0/3). Finally, the HTLV-I(+) T-cell lines (313) but not the control, HTLV-I(-) T-cell lines were resistant to rapamycin and its novel analog RAD. We conclude that (1) HTLV-I infection per se does not result in a constitutive phosphorylation of the Jak3, STAT3, and STAT5 proteins; (2) malignant transformation in at least some cases of ATLL does not require the constitutive, but may require IL-2-induced, activation of the IL-2R Jak/STAT pathway; and (3) there are major differences in T-cell immortalization mechanism(s) which appear to involve SHP-1 and target molecules for rapamycin and RAD.

Cutaneous CD56+ large T-cell lymphoma associated with high serum concentration of IL-2.

We report the case of a patient with a clinically aggressive large cell lymphoma (LCL) which expressed several T-lymphocyte markers and, in addition, CD56 and, to a lesser degree, CD68 antigens. A marked increase in serum concentration of interleukin (IL)-2 was found (490 and 167 pg/0.1 mL in two serum samples collected 6 months apart). This increase in IL-2 appeared unique to this lymphoma because serum concentration of IL-2 was not increased in any of the cases of various types of cutaneous lymphoproliferative disorders tested: mycosis fungoides-related cutaneous T-cell lymphoma (CTCL: 28 patients), granulomatous slack-skin syndrome (GS-SS: 1 patient), anaplastic large cell lymphoma (ALCL: 2 patients), subcutaneous gamma/delta T-cell lymphoma (gamma/delta-TCL: 1 patient), adult-type leukemia/lymphoma (ATLL: 1 patient), and lymphomatoid papulosis (LyP: 4 patients). Furthermore, the increase in IL-2 serum concentration appeared selective in this CD56+ large-cell lymphoma-bearing patient, because concentration of none of the five other cytokines tested (IL-4, IL-6, IFNgamma, GM-CSF, and TNFalpha) was increased. In contrast, soluble receptors for IL-2 and two of the other cytokines (IL-6, and TNFalpha) were markedly increased not only in this patient, but also in most patients with the other cutaneous lymphoproliferative disorders that we examined except for lymphomatoid papulosis. These data indicate that increased IL-2 serum concentration may help to diagnose a unique type of cutaneous CD56(+) large (T-) cell lymphoma and suggest that IL-2 way play a role of an autocrine growth factor for this lymphoma.

The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation.

Immunophenotyping by flow cytometry is widely used in the diagnosis and subclassification of acute myeloid leukemia (AML). CD14 is the monocyte-associated antigen most widely used to identify AML with monocytic differentiation (French-American-British classes M4 and M5); however, we observed that CD14 expression is frequently diminished or absent in such cases. To identify monocyte-associated antigens that might improve recognition of AML M4 and M5, we used 3-color flow cytometry and a panel of antibodies reported to distinguish cells of monocytic lineage in 44 cases of AML. In addition, CD45 vs logarithmic side scatter plots were analyzed in all cases. As expected, CD14 was highly specific but was only moderately sensitive for monocytic differentiation. CD64 had the best-combined sensitivity and specificity for AML M4 and M5. CD45 vs logarithmic side scatter analysis showed a higher percentage of monocytes in AML M4 and M5 compared with nonmonocytic AML. CD64 was expressed in 5 of 5 cases of acute promyelocytic leukemia (AML M3), but the intensity of staining was significantly less in AML M3 than in AML M4 and M5. Our findings show that addition of CD64 and CD45 vs logarithmic side scatter analysis to CD14 greatly improves flow cytometric detection of AML with monocytic differentiation and that CD64, also expressed in AML M3, may help distinguish AML M3 from other subtypes.

Evidence for early hematopoietic progenitor cell involvement in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) represents a subtype of acute myeloid leukemia with characteristic morphologic, molecular, and immunophenotypic features. Previous immunophenotypic analyses have shown that leukemic cells in APL typically express the myeloid markers CD33 and CD13 but lack expression of the early hematopoietic progenitor cell antigens CD34 and HLA-DR. We analyzed selected immunophenotypic features of APL by flow cytometry and showed that 7 (41%) of 17 cases contained significant subsets of CD34+ leukemic cells: CD34+ myeloid cells predominated in 2 APL cases. By using a fluorescence-activated cell sorter-fluorescence in situ hybridization approach, we confirmed that the CD34+ cells harbored the t(15;17) translocation characteristic of APL. By using the same experimental approach, CD34+ populations were stratified into primitive CD34+ CD38- and committed CD34+ CD38+ progenitor cell subpopulations; cells in both subsets contained the t(15;17) translocation. The knowledge that APL may be partly or largely CD34+ is important for proper diagnosis. Furthermore, identification of the t(15;17) translocation in CD34+ CD38- blasts indicates that, in at least some cases, the leukemogenic mutation in APL occurs within primitive hematopoietic progenitor cells.