ERK Activity in Immature Leukemic Cells Drives Clonal Selection during Induction Therapy for Acute Myeloid Leukemia.

Selection of resistant clones following intensive chemotherapy is a common obstacle for cure in many cancers, particularly in acute myeloid leukemia (AML). In AML, clone-specific sensitivity to chemotherapy varies even within the same patient. Multiple mutations and genetic aberrations are associated with clones surviving chemotherapy. The current study explored the role of activated signaling pathways in chemoresistance as a function of cell maturation, reflected by CD34 expression. In-vitro, Kasumi-1 leukemic cell line, sorted by CD34 expression, showed increased apoptosis only in the CD34- subpopulation after exposure to cytosine arabinoside (Ara-C) or daunorubicin. The resistant CD34+ subset demonstrated higher expression of ERK1/2 and BCL-2 proteins than CD34- cells. MEK1/2 inhibition elevated Ara-C ability to induce apoptosis in CD34+ cells, suggesting that MEK1/2-ERK1/2 is surviving signaling, which correlates to cell maturation levels and plays a role in chemoresistance. Deep sequencing of sorted CD34+/- populations, both derived from the same patient samples, demonstrated various subclonal distribution of NPM1, DNMT3A and FLT3-ITD mutations. Interestingly, in these samples, p-ERK levels and apoptosis rates following chemotherapy exposure significantly differed between CD34+/- populations. Hence, clones may be selected due to their ability to escape apoptosis rather than a direct effect of chemotherapy on a specific mutated clone.

Follicular eruption with folliculotropic lymphocytic infiltrates associated with anti-tumor necrosis factor alpha therapy: Report and study of 3 cases.

BACKGROUND: We have encountered three cases of follicular eruptions with folliculotropic infiltrates of non-atypical lymphocytes associated with anti-tumor necrosis factor alpha (TNF-α) therapy. METHODS: Three patients aged 15 to 56 years treated with anti-TNF-α therapy (one with adalimumab, and two with infliximab) developed follicular eruptions characterized histopathologically by folliculotropic lymphocytic infiltrates. These were studied clinically, histopathologically, immunophenotypically, and molecularly. RESULTS: All three cases were characterized histopathologically by folliculotropic cell infiltrates of non-atypical T (CD3+) lymphocytes with variable follicular exocytosis. Marked reduction in CD7 staining and marked predominance of CD4+ cells over CD8+ cells were observed in 1 and 2 cases, respectively. T-cell receptor (TCR) gene rearrangement studies were monoclonal in 1 case. Discontinuation of anti-TNF-α therapy in all three cases, with corticosteroid creams in 1 case, led to complete resolution. Rechallenge with adalimumab in 1 case resulted in exacerbation. Replacement of therapy with non-anti-TNF-α biologic agents in 2 cases was not associated with recurrence. CONCLUSION: Follicular eruptions with folliculotropic lymphocytic infiltrates associated with anti-TNF-α therapy may show some immunophenotypical variations and/or monoclonal TCR gene rearrangements but lack sufficient cytomorphological features of folliculotropic MF. They may resolve with discontinuation of anti-TNF-α therapy.

Follicular Eruption With Folliculotropic Lymphocytic Infiltrates Associated With Iatrogenic Immunosuppression: Report and Study of 3 Cases, and Review of the Literature.

BACKGROUND: Several cases of folliculotropic mycosis fungoides, associated with immunosuppressive therapy, including calcineurin inhibitors, have been reported in solid organ transplant patients. We have encountered 3 patients on immunosuppressive therapy who developed follicular eruptions with folliculocentric infiltrates of nonatypical lymphocytes. OBJECTIVE: To characterize these follicular eruptions and review the literature. METHODS: Three patients, aged 7-15 years, who were treated with systemic immunosuppressive therapy developed follicular eruptions characterized histopathologically by folliculocentric lymphocytic infiltrates. These were studied clinically, histopathologically, immunophenotypically, and molecularly for T-cell receptor (TCR) gene rearrangement. RESULTS: All 3 cases were characterized histopathologically by folliculocentric infiltrates of nonatypical CD3 T lymphocytes with variable follicular exocytosis. Two cases also showed follicular mucinosis. Marked reduction in CD7 staining, and marked predominance of CD4 cells over CD8 cells was observed in all 3 cases. The TCR gene rearrangement studies were monoclonal in 2 cases. Oral calcineurin inhibitors (2 cyclosporine A and 1 tacrolimus) were part of the therapeutic regimen in all 3 patients. Their cessation along with local corticosteroid creams in 2 patients, and phototherapy with oral acitretin in one patient, was associated with complete clinical remission. CONCLUSIONS: Patients undergoing systemic immunosuppressive therapy that includes calcineurin inhibitors might develop follicular eruption with some immunophenotypical variations and a monoclonal TCR gene rearrangement but lack sufficient cytomorphological features of folliculotropic mycosis fungoides. Altering the immunosuppressive agent including calcineurin inhibitors may result in regression of the eruptions.

Superior outcome of patients with favorable-risk acute myeloid leukemia using consolidation with autologous stem cell transplantation.

Autologous stem cell transplantation (ASCT), intensifying anti-leukemic effects without significant treatment-related mortality (TRM), is particularly appealing in AML with favorable genetic/molecular profile. This study retrospectively evaluated the outcomes of post-remission treatment in consecutive favorable-risk AML patients. Sixty-six patients were included: 32 had mutated NPM1/wild-type FLT-ITD, 16 had t(8:21) and 18 - inv(16). Forty patients received chemotherapy alone, 26 underwent ASCT upfront. In time-dependent analysis, the ASCT group demonstrated higher relapse-free (RFS) (p = .001) and overall survivals (OS) (p = .0007). The 1-year RFS and OS were 44.2% vs 88% and 71% vs 96% for chemotherapy and ASCT, respectively. The corresponding TRM was 4/40 (10.0%) and 0/26 (0%), with relapse rates of 70.0% and 19.2% (p = .0002). In multivariate analysis, ASCT was associated with superior OS and RFS. In conclusion, ASCT offers significantly superior RFS and OS in favorable-risk AML in first complete remission. These data support the recent resurgence of interest in ASCT for AML.

Relationship Between Pityriasis Lichenoides and Mycosis Fungoides: A Clinicopathological, Immunohistochemical, and Molecular Study.

BACKGROUND: Several cases of pityriasis lichenoides (PL) have been reported to evolve into mycosis fungoides (MF). OBJECTIVE: To elucidate clues to this progression. METHODS: Fifty-eight patients with PL between 2000 and 2013 (follow-up: 3-16 years, average: 8.3). RESULTS: A total of 3 (5.2%) of the 58 patients with PL developed MF after 3-11 years of prolonged clinical course. Papules and small plaques characterized PLs, and patches and larger plaques subsequent MFs. A total of 35 of 41 (85%) followed up non-MF associated patients with PL reported lasting complete remissions. Histopathologically, apoptotic keratinocytes disappeared mostly or completely in subsequent MFs. The presence of epidermotropism, folliculotropism, and epidermal lymphocytic nuclear atypia in PLs was not predictive of MF. CD8 cells were the dominant intraepidermal lymphocytes in the 3 PLs but remained so in only 1 subsequent MF. CD7 lymphocytes decreased substantially in 2 MFs, and lymphocytic nuclear atypia increased markedly in 1. T-cell receptor gene rearrangement studies demonstrated clonal populations in 1 of 2 studied PLs and in all 3 subsequent MFs. CONCLUSIONS: A few PLs may evolve into MF. Prolonged clinical course, appearance of patches and larger plaques, markedly increased lymphocytic nuclear atypia, marked diminution of apoptotic keratinocytes and CD7 and CD8 lymphocytes, and clonal T-cell receptor gene rearrangement may serve as clues.

T-cell receptor gene rearrangement studies using the GeneScan technique as an adjunct to the histopathological diagnosis of mycosis fungoides.

T-cell receptor gene rearrangement studies are considered to be an adjunct to the histopathological diagnosis of mycosis fungoides (MF). Fluorophore-coupled primers in polymerase chain reactions followed by fragment analysis with a capillary electrophoresis device (GeneScan analysis) have been recently advocated and widely used. This study was performed to assess T-cell receptor γ-gene rearrangement GeneScan as an adjunct to the histopathological diagnosis of MF. We studied 163 cases. The rates of clonality using this technique were found to be 22/37 (59.5%) in early patch MF, 30/46 (65.2%) in more advanced stages, 13/34 (38.2%) in inconclusive cases, and 10/46 (21.7%) in benign inflammatory dermatoses. Our results support the histopathological criteria for the diagnosis of early patch MF, because of the close rates of clonality obtained in early patch MF with subtle histopathological findings, and in the more advanced subtypes of MF with more overt and specific histopathological criteria.

Transformation of Sézary syndrome into CD30+ anaplastic large T-cell lymphoma after alemtuzumab therapy with evidence of clonal unity.

Alemtuzumab is a humanized mouse antibody targeting the CD52 cell surface, which has been effective in patients with advanced stage mycosis fungoides (MF) including erythrodermic MF and Sézary syndrome. There are a few descriptions of large cell transformation after its administration. A young patient with an acute onset of Sézary syndrome treated initially unsuccessfully with fludarabine and cyclophosphamide and later on successfully with alemtuzumab has been described. Three weeks after the beginning of therapy, however, she developed transformed T-cell lymphoma indistinguishable from CD30 anaplastic large-cell lymphoma. After bone marrow transplantation, the transformed CD30 cutaneous T-cell lymphoma recurred as a transformed CD30 plaque MF. All 3 types of lesions showed the same T-cell receptor clonal gene rearrangement, which supports the notion that Sézary syndrome, CD30 anaplastic large-cell lymphoma, and MF are interrelated.

Allogeneic stem cell transplantation for patients with chronic myeloid leukemia: risk stratified approach with a long-term follow-up.

The use of allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML) was almost abandoned in recent years for very effective targeted therapy with tyrosine kinase inhibitors (TKIs). However, approximately one third of patients still need another treatment including SCT. 38 consecutive CML patients were treated (most in preimatinib era) with allogeneic SCT, using partial T cell depletion (TCD) and preemptive donor lymphocyte infusion (DLI), without post-transplant graft-versus-host disease (GvHD) prophylaxis. Conditioning included busulfan, cyclophosphamide, antithymocytic globulin, and fludarabine followed by donor stem cell transfusion. With a median follow up of 90.5 months (1-134), 32 patients are alive. 97% engrafted. 5-year leukemia free survival (LFS) and overall survival (OS) were 78.95% and 84.2%, respectively. All patients are in major molecular remission and 78% in complete molecular remission. Transplant-related mortality (TRM) was 13%. Twenty-four patients received DLI for residual disease. Acute GvHD, mostly Grades I-II, occurred in 18% of patients post-transplant and in 24% of patients receiving DLI. In conclusion, the risk-adapted approach using only partial TCD and preemptive escalated dose of DLI precluded the need for immunosuppressive medications and reduced the risk of significant GvHD without compromising engraftment and long-term disease control. Am. J. Hematol. 2012. © 2012 Wiley Periodicals, Inc.

Cutaneous lymphoid hyperplasia presenting as a solitary facial nodule: clinical, histopathological, immunophenotypical, and molecular studies.

OBJECTIVE: To elucidate the clinicopathological, immunophenotypical, and molecular characteristics of cutaneous lymphoid hyperplasia presenting as a solitary facial nodule. DESIGN: Retrospective study. SETTING: University dermatology department. PATIENTS: Three patients with a solitary facial nodule were studied clinically, histologically, immunophenotypically, and molecularly for T-cell receptor and immunoglobulin heavy chain gene rearrangements. MAIN OUTCOME MEASURES: Histological, immunophenotypical, and molecular characteristics in relation to the clinical outcome. RESULTS: Histologically, dense diffuse lymphocytic infiltrates were present throughout the dermis, occasionally extending into the subcutaneous fat and the epidermis and hair follicles. Small lymphocytes predominated, but in 2 cases there were also medium to large atypical lymphocytes, with some blastlike lymphocytes. The lymphocytic population was mixed with more CD3(+) T cells than CD20(+) B cells, without germinal centers. There were more CD4(+) than CD8(+) cells, and some of the T cells stained for the memory T-cell marker CD45RO. Numerous CD68(+) histiocytes were scattered or formed small aggregates, and in 1 case small granulomas and many scattered S100 protein-positive and CD1a(+)dendritic cells were present. In addition, several polytypic plasma cells, eosinophils, and extravasated erythrocytes were found. Immunostaining for CD10, CD21, CD30, CD56, and BCL6 was negative. The Ki-67 proliferation index was relatively low (5%-10%). Results of the T-cell receptor gene rearrangement studies were positive in 2 cases, 1 of which also harbored clonal B cells. Serologic test results for Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii were negative in all 3 cases. Two lesions regressed spontaneously after an incisional biopsy, and none of the cases showed recurrence or extracutaneous spread during a follow-up period of 5.0 to 5.5 years. CONCLUSIONS: Cutaneous lymphoid hyperplasia that presents as a solitary facial nodule may share clinical, cytological, immunophenotypical, and molecular features with both benign reactive lymphocytic infiltrates and cutaneous lymphomas, and therefore a careful clinical and therapeutic approach is warranted.

Mitochondrial pro-apoptotic ARTS protein is lost in the majority of acute lymphoblastic leukemia patients.

Acquired resistance towards apoptosis is the hallmark of most if not all types of cancer. We have previously identified and characterized ARTS, a broadly expressed protein localized to mitochondria. ARTS was initially shown to mediate TGF-beta induced apoptosis. Recently, we have found that high levels of ARTS induce apoptosis without additional pro-apoptotic stimuli. Further, ARTS promotes apoptosis in response to a wide variety of pro-apoptotic stimuli. Here, we report that the expression of ARTS is lost in all lymphoblasts of more than 70% of childhood acute lymphoblastic leukemia (ALL) patients. The loss of ARTS is specific, as the related non-apoptotic protein H5, bearing 83% identity to ARTS, is unaffected. During remission, ARTS expression is detected again in almost all patients. Two leukemic cell lines, ALL-1 and HL-60 lacking ARTS, were resistant to apoptotic induction by ara-C. Transfection of ARTS into these cells restored their ability to undergo apoptosis in response to this chemotherapeutic agent. We found that methylation process contributes to the loss of ARTS expression. We conclude that the loss of ARTS may provide a selective advantage for cells to escape apoptosis thereby contributing to their transformation to malignant lymphoblasts. We therefore propose that ARTS can function as a tumor suppressor protein in childhood ALL.

Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia.

BACKGROUND: Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with combination chemotherapy containing methotrexate (MTX), drug resistance contributes to treatment failure for a substantial fraction of patients. The primary transporter for folates and MTX is the reduced folate carrier (RFC). Impaired drug transport is a documented mechanism of MTX resistance in patients with ALL; however, to the authors' knowledge it is not known whether inactivating RFC mutations are a contributing factor. METHODS: The authors devised a genomic polymerase chain reaction-single strand conformational polymorphism assay followed by sequencing and screened the entire RFC coding region for sequence alterations in DNA from 246 leukemia specimens from patients with diverse ethnic variation, 24 at the time of recurrence and the rest at the time of diagnosis. This cohort was comprised of 203 B-precursor ALL specimens (82.5%), 32 T-lineage ALL specimens (13%), and 11 acute myeloblastic leukemia specimens (4.5%). RESULTS: Of 246 DNA samples, only 3 diagnosis B-precursor ALL specimens (1.2%) were found to harbor alterations in the RFC gene, including heterozygous single nucleotide changes resulting in D56H and D522N substitutions in the first extracellular loop and the C-terminus of this transporter, respectively. The third sample had a sequence alteration in exon 3 that could not be identified because of the lack of availability of DNA. CONCLUSIONS: Whereas inactivating RFC mutations are a frequent mechanism of MTX resistance in human leukemia cell lines and in patients with osteosarcoma, they are not common and do not appear to play any significant role in intrinsic or acquired resistance to MTX in childhood leukemia. This is the first study of RFC mutations in multiple pediatric leukemia specimens.