Cross-sectional associations between cutaneous viral infections and regulatory T lymphocytes in circulation.

BACKGROUND: Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood. OBJECTIVES: To examine cross-sectional associations between cutaneous viral infections and circulating forkhead-box P3 (FOXP3)-expressing T-regulatory (Treg) cells, suppressive cells that dampen effective antitumour immunity. MATERIALS AND METHODS: Blood, eyebrow hair (EBH) and skin swab (SSW) samples were collected from 352 patients 60 years and older undergoing skin screening, without prevalent skin cancer, while participating in an ongoing prospective cohort study of cutaneous viral infections and skin cancer. DNA corresponding to 98 cutaneous human papillomavirus (HPV) types and five human polyomaviruses (HPyV) was assessed in EBH and SSW. Distinct classes of circulating Treg-cell subpopulations were defined by flow cytometry including cutaneous lymphocyte antigen (CLA) and CCR4high Treg cells, both previously associated with cutaneous diseases. Age- and sex-adjusted associations between circulating T-cell populations and infection were estimated using logistic regression. RESULTS: Total Treg-cell proportion in peripheral blood was not associated with ß HPV or HPyV infection. However, the proportion of circulating CLA+ Treg cells was inversely associated with γ HPV EBH infection [odds ratio (OR) 0·54, 95% confidence interval (CI) 0·35-0·84]. Interestingly, circulating Treg cells expressing markers indicative of antigen activation (CD27- CD45RA- FOXP3+ CD4+ ) were also inversely associated with γ HPV infection in SSW (OR 0·55, 95% CI 0·30-0·99) and EBH (OR 0·56, 95% CI 0·36-0·86). CONCLUSIONS: Inverse associations between circulating Treg cells and γ HPV infection suggest that localized viral infection may promote immunosuppressive cell migration into skin.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

Intrapatient functional clonality deconvoluted by coupling intracellular flow cytometry and next-generation sequencing in human leukemia.

The interplay between tumor heterogeneity and microenvironmental factors is a critical mechanism for clonal selection in leukemia. Evidence of unique clonal capacities to engraft within patient-derived xenograft (PDX) models suggests that intrapatient genetic architecture may be defined by functional differences at the clonal level. However, methods to detect functional differences assigned to genetically defined clones remain limited. Here, we describe a scalable method to directly measure the functional properties of clones within the same leukemia patient by coupling intracellular flow cytometry and next-generation sequencing (NGS). We provide proof of concept utilizing primary chronic myelmonocytic leukemia (CMML) samples and granulocyte-macrophage colony stimulating factor (GM-CSF) to elucidate the interaction between tumor heterogeneity and microenvironmental factors. Mixtures of human leukemia cell lines, with known response to GM-CSF, were used to validate the accuracy of our methodology. Using this approach, we confirm that our method is capable of discriminating GM-CSF sensitive cell lines, identifies somatic variants in primary leukemia samples, and resolves functional clonal architecture in an illustrative patient. Taken together, our data describes a novel method to determine intrapatient functional clonal heterogeneity and provides proof-of-concept for future investigation aimed at elucidating the clinical relevance of functional clonal differences.

Effect of IL-7 and IL-15 on T cell phenotype in myelodysplastic syndromes.

Aberrant T cell phenotype is one of the characteristics of myelodysplastic syndromes (MDS). In this study, we detected an increased concentration of IL-15 in the plasma of MDS patients (n = 20) compared with that in the plasma of healthy controls (n = 20). In MDS patients, reduced naïve CD4+ and CD8+ T cells [16.11 ± 6.56 vs. 24.11 ± 7.18 for CD4+ T cells (p < 0.001) and 13.15 ± 5.67 vs. 23.51 ± 6.25 for CD8+ T cells (p < 0.001)] were observed. The reduced naïve and increased effector memory T cells were significantly correlated with IL-15 plasma level. Then, the effect of IL-15 and IL-7 was tested in vitro. Peripheral blood mononuclear cells from MDS were treated for 15 days with IL-15. This treatment significantly decreased naïve CD4+ (p < 0.001) and CD8+ (p < 0.001) T cells and correspondingly increased terminal memory CD4+ and CD8+ T cells (p < 0.001). Treatment with IL-7 increased naïve CD4+ (p < 0.05) and CD8+ (p < 0.001) T cells. Our results indicated that exposure to high levels of IL-15 may be involved in the T cell phenotype conversion observed in MDS. IL-7 may be one of the promising therapeutic candidates for recovering the effector immune compartment in MDS patients.

TP53 and MDM2 single nucleotide polymorphisms influence survival in non-del(5q) myelodysplastic syndromes.

P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome.

The relationship of TP53 R72P polymorphism to disease outcome and TP53 mutation in myelodysplastic syndromes.

Nonsynonymous TP53 exon 4 single-nucleotide polymorphism (SNP), R72P, is linked to cancer and mutagen susceptibility. R72P associations with specific cancer risk, particularly hematological malignancies, have been conflicting. Myelodysplastic syndrome (MDS) with chromosome 5q deletion is characterized by erythroid hypoplasia arising from lineage-specific p53 accumulation resulting from ribosomal insufficiency. We hypothesized that apoptotically diminished R72P C-allele may influence predisposition to del(5q) MDS. Bone marrow and blood DNA was sequenced from 705 MDS cases (333 del(5q), 372 non-del(5q)) and 157 controls. Genotype distribution did not significantly differ between del(5q) cases (12.6% CC, 38.1% CG, 49.2% GG), non-del(5q) cases (9.7% CC, 44.6% CG, 45.7% GG) and controls (7.6% CC, 37.6% CG, 54.8% GG) (P=0.13). Allele frequency did not differ between non-del(5q) and del(5q) cases (P=0.91) but trended towards increased C-allele frequency comparing non-del(5q) (P=0.08) and del(5q) (P=0.10) cases with controls. Median lenalidomide response duration increased proportionate to C-allele dosage in del(5q) patients (2.2 (CC), 1.3 (CG) and 0.89 years (GG)). Furthermore, C-allele homozygosity in del(5q) was associated with prolonged overall and progression-free survival and non-terminal interstitial deletions that excluded 5q34, whereas G-allele homozygozity was associated with inferior outcome and terminal deletions involving 5q34 (P=0.05). These findings comprise the largest MDS R72P SNP analysis.

Naive T-cells in myelodysplastic syndrome display intrinsic human telomerase reverse transcriptase (hTERT) deficiency.

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P<0.0001) that correlated with significantly lower hTERT mRNA (P<0.0001), independent of age and disease stratification. hTERT mRNA deficiency affected naive but not memory T-cells, and telomere erosion in MDS occurred without evidence of an hTERT-promoter mutation, copy number variation or deletion. Telomerase insufficiency may undermine homeostatic control within the hematopoietic compartment and promote a change in the T-cell repertoire in MDS.

Lenalidomide promotes p53 degradation by inhibiting MDM2 auto-ubiquitination in myelodysplastic syndrome with chromosome 5q deletion.

Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with myelodysplastic syndrome (MDS) and chromosome 5q deletion (del(5q)). Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger degradation of mouse double minute 2 protein (MDM2), with consequent p53 transactivation. Herein we show that p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. More importantly, we show that lenalidomide (Len) acts to stabilize MDM2, thereby accelerating p53 degradation. Biochemical and molecular analyses showed that Len inhibits the haplodeficient protein phosphatase 2A catalytic domain alpha (PP2Acα) phosphatase resulting in hyperphosphorylation of inhibitory serine-166 and serine-186 residues on MDM2, and displaces binding of RPS14 to suppress MDM2 autoubiquitination whereas PP2Acα overexpression promotes drug resistance. Bone marrow specimens from del(5q) MDS patients resistant to Len overexpressed PP2Acα accompanied by restored accumulation of p53 in erythroid precursors. Our findings indicate that Len restores MDM2 functionality in the 5q- syndrome to overcome p53 activation in response to nucleolar stress, and therefore may warrant investigation in other disorders of ribosomal biogenesis.

Molecular action of lenalidomide in lymphocytes and hematologic malignancies.

The immunomodulatory agent, lenalidomide, is a structural analogue of thalidomide approved by the US Food and Drug Administration for the treatment of myelodysplastic syndrome (MDS) and multiple myeloma (MM). This agent is also currently under active investigation for the treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), as well as in drug combinations for some solid tumors and mantle cell lymphoma (MCL). Although treatment with lenalidomide has translated into a significant extension in overall survival in MM and MDS and has superior safety and efficacy relative to thalidomide, the mechanism of action as it relates to immune modulation remains elusive. Based on preclinical models and clinical trials, lenalidomide, as well as other structural thalidomide derivatives, enhances the proliferative and functional capacity of T-lymphocytes and amplifies costimulatory signaling pathways that activate effector responses and suppress inflammation. This paper summarizes our current understanding of T- and natural killer (NK) cell pathways that are modified by lenalidomide in hematopoietic neoplasms to inform future decisions about potential combination therapies.

Telomere length in myelodysplastic syndromes.

The relationship between telomere length (TL) and predisposition to myelodysplastic syndromes (MDS) remains unclear. We compared peripheral blood leukocyte (PBL) TL among cases of histologically confirmed MDS (n = 65) who were treatment-naive with no prior cancer history to age-matched controls (n = 63). Relative TL was measured in PBLs and saliva by quantitative polymerase chain reaction (PCR) and in CD15+ and CD19+ cells by flow cytometry-fluorescence in situ hybridization (flow-FISH). Human telomerase reverse transcriptase gene (hTERT) mutations were assessed by PCR. After adjustment for age and sex, relative TLs were reduced in PBLs (p = 0.02), CD15+ (p = 0.01), CD19+ (p = 0.25), and saliva (p = 0.13) in MDS cases versus controls, although only the PBL and CD15+ results were statistically significant. Among MDS cases, CD15+ and CD19+ cell TLs were positively correlated (p = 0.03). PBL TL was reduced among those occupationally exposed to paints and pesticides, but was not associated with hTERT genotype. Future studies are needed to further investigate constitutional telomere attrition as a possible predisposing factor for MDS.

A molecular and functional analysis of large granular lymphocyte expansions in patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors.

Tyrosine kinase inhibitor (TKI) therapy has become the standard treatment for chronic myelogenous leukemia (CML). Off-target kinase inhibition has been implicated in the appearance of unique adverse effects, such as colitis and pleural effusions. In addition, some patients present oligoclonal expansions of large granular lymphocytes (LGLs). We sought to further investigate this phenomenon in 64 patients treated with five different TKIs. Clonal expansions of cytotoxic T lymphocytes (CTLs) were identified in all TKI-treated patient groups, but only in dasatinib-treated patients were these expansions characterized as LGLs. Survival factors known to be important in LGL leukemia (interleukin-15 [IL-15] transpresentation, plasma platelet-derived growth factor [PDGF]-BB levels, nuclear factor-κB [NF-κB] and T-bet activation) were found to be associated with TKI-induced LGL expansions. Interestingly, patients with LGL expansions had increased cytotoxicity against non-transformed endothelial cells, which may play a role in observed autoimmune-like side effects. Our results indicate that patients with CML treated with TKIs can develop T cell expansions, which can in certain cases be related to some adverse effects.

A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide.

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases, Cdc25C and PP2Acalpha, which are coregulators of the G(2)-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS.

Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy.

Dasatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), predominantly targets BCR-ABL and SRC oncoproteins and also inhibits off-target kinases, which may result in unexpected drug responses. We identified 22 patients with marked lymphoproliferation in blood while on dasatinib therapy. Clonality and immunophenotype were analyzed and related clinical information was collected. An abrupt lymphocytosis (peak count range 4-20 x 10(9)/l) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy and it persisted throughout the therapy. Fifteen patients had a cytotoxic T-cell and seven patients had an NK-cell phenotype. All T-cell expansions were clonal. Adverse effects, such as colitis and pleuritis, were common (18 of 22 patients) and were preceded by LGL lymphocytosis. Accumulation of identical cytotoxic T cells was also detected in pleural effusion and colon biopsy samples. Responses to dasatinib were good and included complete, unexpectedly long-lasting remissions in patients with advanced leukemia. In a phase II clinical study on 46 Philadelphia chromosome-positive acute lymphoblastic leukemia, patients with lymphocytosis had superior survival compared with patients without lymphocytosis. By inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity associated with good clinical responses and a distinct adverse effect profile.

Altered naive and memory CD4+ T-cell homeostasis and immunosenescence characterize younger patients with myelodysplastic syndrome.

Response to immunosuppressive therapy (IST) in younger patients with myelodysplastic syndrome (MDS) has been linked to a T-cell-dominant autoimmune process that impairs hematopoiesis. Analysis of the age-adjusted CD4:CD8 ratio in 76 MDS patients compared with 54 healthy controls showed that inadequate CD4+, rather than expansion of CD8+ T cells, was associated with a lower ratio in a group that included both lower and higher risk MDS patients defined by the International Prognostic Scoring System. In younger MDS patients, naive and memory phenotypes defined by CD45RA and CD62L display showed depletion of naive CD4+ and CD8+ T cells, suggesting a possible relationship to IST responsiveness. To determine the correlation between T-cell subset distribution, T-cell turnover and autoimmunity, a cohort of 20 patients were studied before and after IST. The CD4:CD8 ratio correlated inversely with the proliferative T-cell index before treatment in IST-responsive patients, suggesting that proliferation may be linked to accelerated CD4+ T-cell turnover and hematopoietic failure. Our data show seminal findings that both CD4+ and CD8+ T-cell subsets are dysregulated in MDS. Association between these T-cell defects and response to IST suggests that aberrant T-cell homeostasis and chronic activation are critical determinants influencing autoimmune hematopoietic suppression in younger patients.

Clinical improvement by farnesyltransferase inhibition in NK large granular lymphocyte leukemia associated with imbalanced NK receptor signaling.

Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)-LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.

Identification of a novel antiapoptotic human protein kinase C delta isoform, PKCdeltaVIII in NT2 cells.

Protein kinase C (PKC) delta plays an important role in cellular proliferation and apoptosis where it is involved in the caspase-3 mediated apoptotic pathway. Cleavage of PKCdeltaI by caspase-3 releases a catalytically active C-terminal fragment that is sufficient to induce apoptosis. In this paper, we identified a novel human PKCdelta isozyme, PKCdeltaVIII (Genbank accession number DQ516383) in human teratocarcinoma (NT2) cells that differentiate into hNT neurons upon retinoic acid (RA) treatment. Expression of PKCdeltaVIII was confirmed by real-time RT-PCR analysis, and we observed that after an initial peak at 24 h following RA treatment, its expression gradually declined with prolonged RA treatment. PKCdeltaVIII is generated via the utilization of an alternative 5' splice site, and this results in an insertion of 31 amino acids in the caspase-3 recognition sequence DMQD. The function of PKCdeltaVIII was examined by subcloning it into an expression vector and raising an antibody specific to PKCdeltaVIII. Using in vivo and in vitro assays, we demonstrated that PKCdeltaVIII is resistant to caspase-3 cleavage. Next, we sought to determine the role of PKCdeltaVIII in apoptosis in NT2 cells. Overexpression of PKCdeltaVIII and knockdown using PKCdeltaVIII siRNA suggest an antiapoptotic function for the PKCdeltaVIII isozyme. We demonstrate that antisense oligonucleotides (ASO) directed toward the 5' splice site I promote the expression of the PKCdeltaVIII isozyme. Our results indicated that ASO mediated PKCdeltaVIII expression rescued NT2 cells from etoposide-induced apoptosis. We conclude that the novel human PKCdeltaVIII splice variant functions as an antiapoptotic protein in NT2 cells.