A new variant in the ZCCHC8 gene: diverse clinical phenotypes and expression in the lung.

Introduction: Pulmonary fibrosis is a severe disease which can be familial. A genetic cause can only be found in ∼40% of families. Searching for shared novel genetic variants may aid the discovery of new genetic causes of disease. Methods: Whole-exome sequencing was performed in 152 unrelated patients with a suspected genetic cause of pulmonary fibrosis from the St Antonius interstitial lung disease biobank. Variants of interest were selected by filtering for novel, potentially deleterious variants that were present in at least three unrelated pulmonary fibrosis patients. Results: The novel c.586G>A p.(E196K) variant in the ZCCHC8 gene was observed in three unrelated patients: two familial patients and one sporadic patient, who was later genealogically linked to one of the families. The variant was identified in nine additional relatives with pulmonary fibrosis and other telomere-related phenotypes, such as pulmonary arterial venous malformations, emphysema, myelodysplastic syndrome, acute myeloid leukaemia and dyskeratosis congenita. One family showed incomplete segregation, with absence of the variant in one pulmonary fibrosis patient who carried a PARN variant. The majority of ZCCHC8 variant carriers showed short telomeres in blood. ZCCHC8 protein was located in different lung cell types, including alveolar type 2 (AT2) pneumocytes, the culprit cells in pulmonary fibrosis. AT2 cells showed telomere shortening and increased DNA damage, which was comparable to patients with sporadic pulmonary fibrosis and those with pulmonary fibrosis carrying a telomere-related gene variant, respectively. Discussion: The ZCCHC8 c.586G>A variant confirms the involvement of ZCCHC8 in pulmonary fibrosis and short-telomere syndromes and underlines the importance of including the ZCCHC8 gene in diagnostic gene panels for these diseases.

New Insights via RNA Profiling of Formalin-Fixed Paraffin-Embedded Lung Tissue of Pulmonary Fibrosis Patients.

In sporadic idiopathic pulmonary fibrosis (sIPF) and pulmonary fibrosis caused by a mutation in telomere (TRG-PF) or surfactant related genes (SRG-PF), there are a number of aberrant cellular processes known that can lead to fibrogenesis. We investigated whether RNA expression of genes involved in these processes differed between sIPF, TRG-PF, and SRG-PF and whether expression levels were associated with survival. RNA expression of 28 genes was measured in lung biopsies of 26 sIPF, 17 TRG-PF, and 6 SRG-PF patients. Significant differences in RNA expression of TGFBR2 (p = 0.02) and SFTPA2 (p = 0.02) were found between sIPF, TRG-PF, and SRG-PF. Patients with low (

Simultaneous Assessment of mTORC1, JAK/STAT, and NLRP3 Inflammasome Activation Pathways in Patients with Sarcoidosis.

The unknown etiology of sarcoidosis, along with the variability in organ involvement and disease course, complicates the effective treatment of this disease. Based on recent studies, the cellular inflammatory pathways involved in granuloma formation are of interest regarding possible new treatment options, such as the mechanistic (formerly mammalian) target of rapamycin complex 1 (mTORC1) pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome pathway. The aim of this study was to explore the potential coexpression of these three inflammatory pathways in patients with sarcoidosis and see whether possible differences were related to disease outcome. The tissue of 60 patients with sarcoidosis was used to determine the activity of these three signaling pathways using immunohistochemistry. The activation of NLRP3 was present in 85% of all patients, and the activation of mTORC1 and JAK/STAT was present in 49% and 50% of patients, respectively. Furthermore, the presence of NLRP3 activation at diagnosis was associated with a chronic disease course of sarcoidosis. Our finding of different new conceptual inflammatory tissue phenotypes in sarcoidosis could possibly guide future treatment studies using the available inhibitors of either NLRP3, JAK-STAT, and mTORC1 inhibitors in a more personalized medicine approach.

Genetic Variant Overlap Analysis Identifies Established and Putative Genes Involved in Pulmonary Fibrosis.

In only around 40% of families with pulmonary fibrosis (PF) a suspected genetic cause can be found. Genetic overlap analysis of Whole Exome Sequencing (WES) data may be a powerful tool to discover new shared variants in novel genes for PF. As a proof of principle, we first selected unrelated PF patients for whom a genetic variant was detected (n = 125) in established PF genes and searched for overlapping variants. Second, we performed WES (n = 149) and identified novel potentially deleterious variants shared by at least two unrelated PF patients. These variants were genotyped in validation cohorts (n = 2748). In 125 unrelated patients, a potentially deleterious variant was detected in known PF genes of which 15 variants in six genes overlapped, involving 51 patients. Overlap analysis of WES data identified two novel variants of interest: TOM1L2 c.421T > C p.(Y141H) and TDP1c.1373dupG p.(S459fs*5), neither gene had been related to pulmonary fibrosis before. Both proteins were present in the alveolar epithelium. No apparent characteristics of telomere disease were observed. This study underlines the potential of searching for overlapping rare potentially deleterious variants to identify disease-associated variants and genes. A previously unreported variant was found in two putative new PF genes, but further research is needed to determine causality.

The Extent of Inflammatory Cell Infiltrate and Fibrosis in Lungs of Telomere- and Surfactant-Related Familial Pulmonary Fibrosis.

Familial pulmonary fibrosis (FPF) is a monogenic disease most commonly involving telomere- (TERT) or surfactant- (SFTP) related mutations. These mutations have been shown to alter lymphocytic inflammatory responses, and FPF biopsies with histological lymphocytic infiltrates have been reported. Recently, a model of a surfactant mutation in mice showed that the disease initially started with an inflammatory response followed by fibrogenesis. Since inflammation and fibrogenesis are targeted by different drugs, we investigated whether the degree of these two features co-localize or occur independently in different entities of FPF, and whether they influence survival. We quantified the number of lymphocyte aggregates per surface area, the extent of diffuse lymphocyte cell infiltrate, the number of fibroblast foci per surface area, and the percentage of fibrotic lung surface area in digitally scanned hematoxylin and eosin (H&E) sections of diagnostic surgical biopsies of patients with TERT-related FPF (TERT-PF; n = 17), SFTP-related FPF (SFTP-PF; n = 7), and sporadic idiopathic pulmonary fibrosis (sIPF; n = 10). For comparison, we included biopsies of patients with cellular non-specific interstitial pneumonia (cNSIP; n = 10), an inflammatory interstitial lung disease with high lymphocyte influx and usually responsive to immunosuppressive therapy. The degree of inflammatory cell infiltrate and fibrosis in TERT-PF and SFTP-PF was not significantly different from that in sIPF. In comparison with cNSIP, the extent of lymphocyte infiltrates was significantly lower in sIPF and TERT-PF, but not in SFTP-PF. However, in contrast with cNSIP, in sIPF, TERT-PF, and SFTP-PF, diffuse lymphocyte cell infiltrates were predominantly present and lymphocyte aggregates were only present in fibrotic areas (p < 0.0001). Furthermore, fibroblast foci and percentage of fibrotic lung surface were associated with survival (p = 0.022 and p = 0.018, respectively), while this association was not observed for lymphocyte aggregates or diffuse lymphocytic infiltration. Inflammatory cells in diagnostic lung biopsies of TERT-PF, SFTP-PF, and sIPF were largely confined to fibrotic areas. However, based on inflammation and fibrosis, no differences were found between FPF and sIPF, substantiating the histological similarities between monogenic familial and sporadic disease. Furthermore, the degree of fibrosis, rather than inflammation, correlates with survival, supporting that fibrogenesis is the key feature for therapeutic targeting of FPF.

A Polymorphism in C-C Chemokine Receptor 5 (CCR5) Associates with Löfgren's Syndrome and Alters Receptor Expression as well as Functional Response.

C-C chemokine receptor 5 (CCR5) and polymorphisms in CCR5 gene are associated with sarcoidosis and Löfgren's syndrome. Löfgren's syndrome is an acute and usually self-remitting phenotype of sarcoidosis. We investigated whether the single nucleotide polymorphism (SNP) rs1799987 is associated with susceptibility for Löfgren's syndrome and has an effect on CCR5 expression on monocytes and function of CCR5. A total of 106 patients with Löfgren's syndrome and 257 controls were genotyped for rs1799987. Expression of CCR5 on monocytes was measured by flowcytometry. We evaluated calcium influx kinetics following stimulation upon N-formylmethionyl-leucyl-phenylalanine (fMLP) and macrophage inflammatory protein-1α (MIP-1α) on monocytes by measuring the median fluorescence intensity (MFI). The frequency of the G allele of rs1799987 was significantly higher in Löfgren's syndrome than in healthy controls (p = 0.0015, confidence interval (CI) 1.22-2.32, odds ratio (OR) 1.680). Patients with a GG genotype showed higher CCR5 expression on monocytes than patients with the AA genotype (p = 0.026). A significantly (p = 0.027) lower count of patients with the GG genotype showed a calcium influx reaction to simulation upon MIP-1 α, compared with patients with the AA genotype. The rs1799987 G allele in CCR5 gene is associated with susceptibility to Löfgren's syndrome and with quantitative and qualitative changes in CCR5, potentially effecting the inflammatory response.

Telomere shortening and DNA damage in culprit cells of different types of progressive fibrosing interstitial lung disease.

Pulmonary fibrosis is strongly associated with telomere shortening and increased DNA damage. Key cells in the pathogenesis involve alveolar type 2 (AT2) cells, club cells and myofibroblasts; however, to what extent these cells are affected by telomere shortening and DNA damage is not yet known. We sought to determine the degree of, and correlation between, telomere shortening and DNA damage in different cell types involved in the pathogenesis of progressive fibrosing interstitial lung disease. Telomere length and DNA damage were quantified, using combined fluorescence in situ hybridisation and immunofluorescence staining techniques, in AT2 cells, club cells and myofibroblasts of controls and patients with pulmonary fibrosis and a telomerase reverse transcriptase mutation (TERT-PF), idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (fHP). In IPF and TERT-PF lungs, AT2 cells contained shorter telomeres and expressed higher DNA damage signals than club cells and myofibroblasts. In fHP lungs, club cells contained highly elevated levels of DNA damage, while telomeres were not obviously short. In vitro, we found significantly shorter telomeres and higher DNA damage levels only in AT2 surrogate cell lines treated with telomerase inhibitor BIBR1532. Our study demonstrated that in IPF and TERT-PF lungs, telomere shortening and accumulation of DNA damage primarily affects AT2 cells, further supporting the importance of AT2 cells in these diseases, while in fHP the particularly high telomere-independent DNA damage signals in club cells underscores its bronchiolocentric pathogenesis. These findings suggest that cell type-specific telomere shortening and DNA damage may help to discriminate between different drivers of fibrogenesis.

From organ to cell: Multi-level telomere length assessment in patients with idiopathic pulmonary fibrosis.

RATIONALE: A subset of patients with idiopathic pulmonary fibrosis (IPF) contains short leukocyte telomeres or telomere related mutations. We previously showed that alveolar type 2 cells have short telomeres in fibrotic lesions. Our objectives were to better understand how telomere shortening associates with fibrosis in IPF lung and identify a subset of patients with telomere-related disease. METHODS: Average telomere length was determined in multiple organs, basal and apical lung, and diagnostic and end-stage fibrotic lung biopsies. Alveolar type 2 cells telomere length was determined in different areas of IPF lungs. RESULTS: In IPF but not in controls, telomere length in lung was shorter than in other organs, providing rationale to focus on telomere length in lung. Telomere length did not correlate with age and no difference in telomere length was found between diagnostic and explant lung or between basal and apical lung, irrespective of the presence of a radiological apicobasal gradient or fibrosis. Fifteen out of 28 IPF patients had average lung telomere length in the range of patients with a telomerase (TERT) mutation, and formed the IPFshort group. Only in this IPFshort and TERT group telomeres of alveolar type 2 cells were extremely short in fibrotic areas. Additionally, whole exome sequencing of IPF patients revealed two genetic variations in RTEL1 and one in PARN in the IPFshort group. CONCLUSIONS: Average lung tissue telomere shortening does not associated with fibrotic patterns in IPF, however, approximately half of IPF patients show excessive lung telomere shortening that is associated with pulmonary fibrosis driven by telomere attrition.

Cell Type-Specific Quantification of Telomere Length and DNA Double-strand Breaks in Individual Lung Cells by Fluorescence In Situ Hybridization and Fluorescent Immunohistochemistry.

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

Short telomere length in IPF lung associates with fibrotic lesions and predicts survival.

Telomere maintenance dysfunction has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). However, the mechanism of how telomere length is related to fibrosis in the lungs is unknown. Surgical lung biopsies of IPF patients typically show a heterogeneous pattern of non-fibrotic and fibrotic areas. Therefore, telomere length (TL) in both lung areas of patients with IPF and familial interstitial pneumonia was compared, specifically in alveolar type 2 (AT2) cells. Fluorescent in situ hybridization was used to determine TL in non-fibrotic and fibrotic areas of 35 subjects. Monochrome multiplex quantitative polymerase chain reaction (MMqPCR) was used for 51 whole lung biopsies and blood TL measurements. For sporadic IPF subjects, AT2 cell TL in non-fibrotic areas was 56% longer than in fibrotic areas. No such difference was observed in the surrounding lung cells. In subjects carrying a telomerase reverse transcriptase (TERT) mutation, AT2 cell TL was significantly shorter than in sporadic subjects. However, no difference in surrounding cell TL was observed between these subject groups. Finally, using biopsy MMqPCR TL measurements, it was determined that IPF subjects with shortest lung TL had a significantly worse survival than patients with long TL. This study shows that shortening of telomeres critically affects AT2 cells in fibrotic areas, implying TL as a cause of fibrogenesis. Furthermore, short lung telomere length is associated with decreased survival.

Effect of Muc5b promoter polymorphism on disease predisposition and survival in idiopathic interstitial pneumonias.

BACKGROUND AND OBJECTIVE: A common polymorphism in the MUC5B gene (rs35705950) is associated with susceptibility to idiopathic pulmonary fibrosis (IPF) and familial interstitial pneumonia (FIP). We investigated predisposition of the MUC5B polymorphism to fibrotic interstitial pneumonias in Dutch Caucasian patient cohorts. Furthermore, we investigated the correlation between MUC5B genotype and survival in these cohorts. METHODS: Sporadic IPF (spIPF, n = 115), FIP (n = 55), idiopathic non-specific interstitial pneumonia (iNSIP, n = 43), connective tissue disease associated interstitial pneumonia (CTD_IP, n = 35) and a control cohort (n = 249) were genotyped for rs35705950. RESULTS: Rs35705950 minor allele frequency (MAF) in controls was 0.09. Case-control analysis showed significant allelic association with spIPF (MAF = 0.27; P = 5.0 × 10(-10)), FIP (MAF = 0.30; P = 2.7 × 10(-9)) and iNSIP (MAF = 0.22; P = 3.4 × 10(-4)). No association was observed in CTD_IP (MAF = 0.07). FIP subgroup analysis revealed an association between MUC5B and telomerase mutated FIP (P = 0.003), and between MUC5B and FIP with unknown genetic cause (P = 1.2 × 10(-8)). In spIPF carriership of MUC5B minor allele did not influence survival. In FIP MUC5B minor allele carriers had better survival (non-carriers 37 vs carriers 53 months, P = 0.01). In iNSIP survival analysis showed an opposite effect. Worse survival was found in iNSIP patients that carried the MUC5B minor allele (non-carriers 118 vs carriers 46 months, P = 0.027) CONCLUSION: This study showed that MUC5B minor allele predisposes to spIPF, FIP and iNSIP. In spIPF, survival is not influenced by MUC5B alleles. In FIP, MUC5B minor allele predicts better survival, pointing towards a subgroup of FIP patients with a milder, MUC5B-driven form of pulmonary fibrosis.

Telomere length in interstitial lung diseases.

BACKGROUND: Interstitial lung disease (ILD) is a heterogeneous group of rare diseases that primarily affect the pulmonary interstitium. Studies have implicated a role for telomere length (TL) maintenance in ILD, particularly in idiopathic interstitial pneumonia (IIP). Here, we measure TL in a wide spectrum of sporadic and familial cohorts of ILD and compare TL between patient cohorts and control subjects. METHODS: A multiplex quantitative polymerase chain reaction method was used to measure TL in 173 healthy subjects and 359 patients with various ILDs, including familial interstitial pneumonia (FIP). The FIP cohort was divided into patients carrying TERT mutations, patients carrying SFTPA2 or SFTPC mutations, and patients without a proven mutation (FIP-no mutation). RESULTS: TL in all cases of ILD was significantly shorter compared with those of control subjects (P range: .038 to < .0001). Furthermore, TL in patients with idiopathic pulmonary fibrosis (IPF) was significantly shorter than in patients with other IIPs (P = .002) and in patients with sarcoidosis (P < .0001). Within the FIP cohort, patients in the FIP-telomerase reverse transcriptase (TERT) group had the shortest telomeres (P < .0001), and those in the FIP-no mutation group had TL comparable to that of patients with IPF (P = .049). Remarkably, TL of patients with FIP-surfactant protein (SFTP) was significantly longer than in patients with IPF, but similar to that observed in patients with other sporadic IIPs. CONCLUSIONS: The results show telomere shortening across all ILD diagnoses. The difference in TL between the FIP-TERT and FIP-SFTP groups indicates the distinction between acquired and innate telomere shortening. Short TL in the IPF and FIP-no mutation groups is indicative of an innate telomere-biology defect, while a stress-induced, acquired telomere shortening might be the underlying process for the other ILD diagnoses.

Association between variations in cell cycle genes and idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined. Rs12951053 and rs12602273, in TP53, were significantly associated with survival in IPF patients. Carriers of a minor allele had a 4-year survival of 22% versus 57% in the non-carrier group (p = 0.006). Rs2395655 and rs733590, in CDKN1A, were associated with an increased risk of developing IPF. In addition, the rs2395655 G allele correlated with progression of the disease as it increased the risk of a rapid decline in lung function. Functional experiments showed that rs733590 correlated significantly with CDKN1A mRNA expression levels in healthy controls. This is the first study to show that genetic variations in the cell cycle genes encoding p53 and p21 are associated with IPF disease development and progression. These findings support the idea that cell cycle control plays a role in the pathology of IPF. Variations in TP53 and CDKN1A can impair the response to cell damage and increase the loss of alveolar epithelial cells.

IL1RN genetic variations and risk of IPF: a meta-analysis and mRNA expression study.

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating lung disease of unknown aetiology. Genetic variations in the IL1RN gene, encoding the interleukin-1 receptor antagonist (IL-1Ra), have been associated with IPF susceptibility. Several studies investigated the variable number tandem repeat (VNTR) or single nucleotide polymorphisms rs408392, rs419598 and rs2637988, with variable results. The aim of this study was to elucidate the influence of polymorphisms in IL1RN on IPF susceptibility and mRNA expression. We performed a meta-analysis of the five case-control studies that investigated an IL1RN polymorphism in IPF in a Caucasian population. In addition, we investigated whether IL1RN mRNA expression was influenced by IL1RN polymorphisms. The VNTR, rs408392 and rs419598 were in tight linkage disequilibrium, with D' > 0.99. Furthermore, rs2637988 was in linkage disequilibrium with the VNTR (D' = 0.90). A haploblock of VNTR*2 and the minor alleles of rs408392and rs419598 was constructed. Meta-analysis revealed that this VNTR*2 haploblock is associated with IPF susceptibility both with an allelic model (odds ratio = 1.42, p = 0.002) and a carriership model (odds ratio = 1.60, p = 0.002). IL1RN mRNA expression was significantly influenced by rs2637988, with lower levels found in carriers of the (minor) GG genotype (p < 0.001). From this meta-analysis, we conclude that the VNTR*2 haploblock is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Ra mRNA expression, suggesting that lower levels of IL-1Ra predispose to developing IPF. Together these findings demonstrate that the cytokine IL-1Ra plays a role in IPF pathogenesis.

A genetic polymorphism in the CAV1 gene associates with the development of bronchiolitis obliterans syndrome after lung transplantation.

BACKGROUND: Caveolin 1 (Cav-1) is the primary structural component of cell membrane invaginations called 'caveolae'. Expression of Cav-1 is implicated in the pathogenesis of pulmonary fibrosis. Genetic polymorphisms in the CAV1 gene influence the function of Cav-1 in malignancies and associate with renal allograft fibrosis. Chronic allograft rejection after lung transplantation, called 'bronchiolitis obliterans syndrome' (BOS), is also characterised by the development of fibrosis.In this study, we investigated whether CAV1 genotypes associate with BOS and whether Cav-1 serum levels are influenced by the CAV1 genotype and can be used as a biomarker to predict the development of BOS. METHODS: Twenty lung transplant recipients with BOS (BOSpos), ninety without BOS (BOSneg) and four hundred twenty-two healthy individuals donated DNA samples. Four SNPs in CAV1 were genotyped. Serial Cav-1 serum levels were measured in a matched cohort of 10 BOSpos patients and 10 BOSneg patients. Furthermore, single-time point Cav-1 serum levels were measured in 33 unmatched BOSneg patients and 60 healthy controls. RESULTS: Homozygosity of the minor allele of rs3807989 was associated with an increased risk for BOS (odds ratio: 6.13; P = 0.0013). The median Cav-1 serum level was significantly higher in the BOSpos patients than in the matched BOSneg patients (P = 0.026). Longitudinal analysis did not show changes in Cav-1 serum levels over time in both groups. The median Cav-1 serum level in the group of 43 BOSneg patients was lower than that in the healthy control group (P = 0.046).In lung transplant recipients, homozygosity of the minor allele of rs3807989 and rs3807994 was associated with increased Cav-1 serum levels. CONCLUSION: In lung transplant recipients, the CAV1 SNP rs3807989 was associated with the development of BOS and Cav-1 serum levels were influenced by the CAV1 genotype.

The long-term impact of in vitro drug sensitivity on risk stratification and treatment outcome in acute lymphoblastic leukemia of childhood (CoALL 06-97).

BACKGROUND: In a study of childhood acute lymphoblastic leukemia (CoALL 06-97 study), the in vitro sensitivity of the patients' cells to prednisolone, vincristine and asparaginase was introduced as a new additional risk parameter for treatment stratification. In parallel in vivo treatment response was assessed by determining the presence and extent of minimal residual disease in a subset of patients (n=224). Here we report the long-term impact of in vitro sensitivity-based risk stratification according to survival and compare the results of in vitro sensitivity with in vivo response. DESIGN AND METHODS: Patients with a sensitive in vitro profile were treated with a reduced intensity protocol (n=167) whereas patients defined as low risk according to conventional parameters but with a resistant in vitro profile were given intensified therapy (n=47). RESULTS: At a median follow-up of 6.8 years event-free survival was 0.80±0.03 for patients with a sensitive profile, 0.73±0.03 for those with an intermediate profile and 0.67±0.08 for those with a resistant profile (P=0.015). Overall, the treatment results of the cases stratified according to in vitro sensitivity were similar to those of the historical control group stratified based on conventional risk factors. Minimal residual disease at the end of induction was a strong predictor of outcome in B-precursor and T-cell acute lymphoblastic leukemia. There was no correlation between in vitro and in vivo treatment response in B-precursor leukemia (Spearman's r=0.13; P=0.15) in contrast to T-cell acute lymphoblastic leukemia (Spearman's r=0.63; P<0.001) CONCLUSIONS: A moderate reduction in treatment intensity for patients with a sensitive in vitro profile was possible without jeopardizing treatment outcome. However, in vitro drug testing was affected by a decrease in risk predictive power over time and was not correlated with in vivo assessment of minimal residual disease in B-precursor acute lymphoblastic leukemia. It was, therefore, abandoned in favor of the assessment of in vivo response in subsequent CoALL trials.

Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells.

Treatment failure in pediatric acute lymphoblastic leukemia (ALL) is related to cellular resistance to glucocorticoids (eg, prednisolone). Recently, we demonstrated that genes associated with glucose metabolism are differentially expressed between prednisolone-sensitive and prednisolone-resistant precursor B-lineage leukemic patients. Here, we show that prednisolone resistance is associated with increased glucose consumption and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cell lines to glucocorticoids. Treatment of prednisolone-resistant Jurkat and Molt4 cells with 2-deoxy-D-glucose (2-DG), lonidamine (LND), or 3-bromopyruvate (3-BrPA) increased the in vitro sensitivity to glucocorticoids, while treatment of the prednisolone-sensitive cell lines Tom-1 and RS4; 11 did not influence drug cytotoxicity. This sensitizing effect of the glycolysis inhibitors in glucocorticoid-resistant ALL cells was not found for other classes of antileukemic drugs (ie, vincristine and daunorubicin). Moreover, down-regulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable with treatment with glycolytic inhibitors. Importantly, the ability of 2-DG to reverse glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary ALL cells from patients. Together, these findings indicate the importance of the glycolytic pathway in glucocorticoid resistance in ALL and suggest that targeting glycolysis is a viable strategy for modulating prednisolone resistance in ALL.

Expression of the outcome predictor in acute leukemia 1 (OPAL1) gene is not an independent prognostic factor in patients treated according to COALL or St Jude protocols.

New prognostic factors may result in better risk classification and improved treatment of children with acute lymphoblastic leukemia (ALL). Recently, high expression of a gene named OPAL1 (outcome predictor in acute leukemia) was reported to be associated with favorable prognosis in ALL. Therefore, we investigated whether OPAL1 expression was of prognostic importance in 2 independent cohorts of children with ALL treated on Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92/97 (n = 180) and St Jude Total 13 protocols (n = 257). We observed a consistently higher (2.8-fold) expression of OPAL1 in TEL-AML1-positive ALL compared with TEL-AML1-negative ALL in both cohorts, but higher OPAL1 expression was not consistently associated with other favorable prognostic indicators such as age and white blood cell count, or ALL genetic subtype. Lower OPAL1 expression was also not associated with increased in vitro drug resistance. Multivariate analyses including known risk factors showed that OPAL1 expression was not independently related to prognosis in either the COALL or St Jude cohorts. In conclusion, OPAL1 expression may not be an independent prognostic feature in childhood ALL, and its previously reported prognostic impact appears to be treatment dependent.