Unmutated Genes Research Articles

A stochastic programming approach to the antibiotics time machine problem

Antibiotics Time Machine is an important problem to understand antibiotic resistance and how it can be reversed. Mathematically, it can be modeled as follows: Consider a set of genotypes, each of which contain a set of mutated and unmutated genes. Suppose that a set of growth rate measurements of each genotype under a set of antibiotics is given. The transition probabilities of a ‘realization’ of a Markov chain associated with each arc under each antibiotic are computable via a predefined function given the growth rate realizations. The aim is to maximize the expected probability of reaching to the genotype with all unmutated genes given the initial genotype in a predetermined number of transitions, considering the following two sources of uncertainties: (i) the randomness in growth rates, (ii) the randomness in transition probabilities, which are functions of growth rates. We develop stochastic mixed-integer linear programming and dynamic programming approaches to solve static and dynamic versions of the Antibiotics Time Machine Problem under the aforementioned uncertainties. We adapt a Sample Average Approximation approach that exploits the special structure of the problem and provide accurate solutions that perform very well in an out-of-sample analysis.

Prognostication in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western countries. CLL is a highly heterogeneous disease: some patients may never require therapy and others relapse several times after different therapeutic strategies. Therefore, in CLL, prognostic markers are essential to capture high-risk patients for different clinical endpoints including early treatment requirement, early progression after BTK or BCL2 inhibitors and Richter transformation. In early stage CLL, different biological and clinical biomarkers have been identified to predict time to treatment requirement that could be used to identify the most appropriate population for early intervention clinical trial. However, at the moment, the standard of care for early stage CLL remains watch & wait since no survival benefit has been identified in clinical trials with chemoimmunotherapy and with BTK inhibitors. In patients requiring treatment TP53 disruptions identify high-risk patients who benefit the most from long-term continuous therapy with BTKi. On the opposite side of the spectrum, IGHV mutated patients devoid of TP53 disruption benefit the most from fixed-duration therapy with venetoclax-obinutuzumab. In between, the highly heterogenous subgroup of patients with IGHV unmutated genes represents the group in which further efforts are needed to identify additional prognostic biomarkers aimed at selecting patients who can benefit from fixed-duration and patients who can benefit from long term BTKi therapy. In the context of the aggressive transformation of CLL, namely Richter syndrome, the clonal relationship to the CLL counterpart represents the strongest prognostic biomarker. Clonally related Richter syndrome still represents an unmet clinical need which requires further efforts to identify new therapeutic strategies.

Immunopeptidomics-Guided Warehouse Design for Peptide-Based Immunotherapy in Chronic Lymphocytic Leukemia

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches.

An Update on Gene Therapy for Inherited Retinal Dystrophy: Experience in Leber Congenital Amaurosis Clinical Trials

Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene mutations that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye’s accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understanding the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs.

MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy.

An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRAS(G12C)-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action. MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1.

MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL.

BackgroundMicroRNAs are known to inhibit gene expression by binding to the 3′UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients.MethodsBy applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3′UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed.ResultsHSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome.ConclusionsHSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1212-2) contains supplementary material, which is available to authorized users.

Serum Tumor Necrosis Factor-α and Interleukin-10 Levels as Markers to Predict Outcome of Patients with Chronic Lymphocytic Leukemia in Different Risk Groups Defined by the IGHV Mutation Status

Tumor necrosis factor (TNF)-α and interleukin (IL)-10 are cytokines involved in the balance between cell-mediated and humoral immunity. We investigated whether serum TNF-α and IL-10 levels have any impact on clinical outcome of patients with chronic lymphocytic leukemia (CLL). TNF-α and IL-10 levels were determined in the serum of 160 CLL patients at the time of diagnosis. The cytokine low-risk group consisted of patients with either TNF-α and IL-10 levels below their medians or those with only one elevated parameter. Both TNF-α and IL-10 levels greater than or equal to their medians defined the cytokine high-risk group. The high-risk patients presented a shorter 3-year treatment-free survival (TFS) than low-risk subjects (15 vs. 69.6%; p<0.0001). The high-risk group (p=0.0002) along with high leukocyte count (p<0.0001) and unmutated immunoglobulin heavy-chain variable region genes (p<0.0001) independently predict the risk of progression in patients with Rai stage 0-II. Furthermore, the high-risk group had an independent prognostic impact on shorter TFS both in patients with mutated (24.3 vs. 78.2%; p<0.0001) and unmutated (8.2 vs. 49%; p=0.004) immunoglobulin heavy-chain variable region genes (IGHV) as compared to the low-risk group. The estimated 5-year overall survival (OS) of high-risk patients was shorter than those in the low-risk group (83.3 vs. 97.1%; p=0.003). Multivariate analysis demonstrated the cytokine high-risk group (p=0.02) followed by Rai stage III-IV (p=0.048) to be independent factors predicting shorter OS. At diagnosis, TNF-α and IL-10 may predict the outcome of patients with CLL.

The prognostic impact of clinical and molecular features in hairy cell leukaemia variant and splenic marginal zone lymphoma

Hairy cell leukaemia variant (HCL-variant) and splenic marginal zone lymphoma (SMZL) are disorders with overlapping features. We investigated the prognostic impact in these disorders of clinical and molecular features including IGH VDJ rearrangements, IGHV gene usage and TP 53 mutations. Clinical and laboratory data were collected before therapy from 35 HCL-variant and 68 SMZL cases. End-points were the need for treatment and overall survival. 97% of HCL-variant and 77% of SMZL cases required treatment (P = 0·009). Survival at 5 years was significantly worse in HCL-variant [57% (95% confidence interval 38-73%)] compared with SMZL [84% (71-91%); Hazard Ratio 2·25 (1·20-4·25), P = 0·01]. In HCL-variant, adverse prognostic factors for survival were older age (P = 0·04), anaemia (P = 0·01) and TP 53 mutations (P = 0·02). In SMZL, splenomegaly, anaemia and IGHV genes with >98% homology to the germline predicted the need for treatment; older age, anaemia and IGHV unmutated genes (100% homology) predicted shorter survival. IGHV gene usage had no impact on clinical outcome in either disease. The combination of unfavourable factors allowed patients to be stratified into risk groups with significant differences in survival. Although HCL-variant and SMZL share some features, they have different outcomes, influenced by clinical and biological factors.

Immune Thrombocytopenia in Patients with Chronic Lymphocytic Leukemia Is Associated with Stereotyped B-cell Receptors

To assess biologic features related to the development of immune thrombocytopenia (ITP) in patients with chronic lymphocytic leukemia (CLL). We retrospectively analyzed 463 patients with CLL with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration [heavy-chain complementary-determining region 3 (HCDR3)], of whom thirty-six developed ITP, according to previously defined criteria. Most of them had available cytogenetic analysis. We observed a significant association between ITP occurrence and IGHV unmutated gene status (P < 0.0001), unfavorable cytogenetic lesions (P = 0.005), and stereotyped HCDR3 (P = 0.006). The more frequent stereotyped HCDR3 subsets were #1 (IGHV1-5-7/IGHD6-19/IGHJ4; 16 of 16 unmutated) and #7 (IGHV1-69 or IGHV3-30/IGHD3-3/IGHJ6; 13 of 13 unmutated), both being significantly more represented among patients developing ITP (P = 0.003 and P = 0.001, respectively). Moreover, restricting the analysis to unmutated patients, subset #7 confirmed its independent significant association with the occurrence of ITP (P = 0.013). Both unmutated IGHV mutational status, del(11)(q23) and stereotyped BCR were significantly associated with shorter time to ITP development (P < 0.0001, P = 0.02, and P = 0.005, respectively) than other patients. Our data suggest that patients with CLL and peculiar BCR conformations are at higher risk of developing secondary ITP and that stereotyped BCR may be involved in the pathogenesis of this complication.

Highlights of This Issue

Hypoxia is important in many human cancers and influences clinical behavior, development of metastases, and patient survival. This prospective study by Milosevic and colleagues in 247 men with intermediate-risk prostate cancer demonstrates, for the first time, that tumor hypoxia measured prior to treatment is associated with early biochemical recurrence after escalated-dose radiotherapy. This pattern of recurrence is compatible with the presence of hypoxia-driven occult metastases. The results of this study provide new insight about the biologic behavior of prostate cancer after radiotherapy and have important implications for combining radiotherapy with hypoxia-targeted therapeutics or other systemic anticancer treatments.Immune thrombocytopenia (ITP) is one of the most frequently observed autoimmune complications in patients with chronic lymphocytic leukemia (CLL). Visco and colleagues provide insights into the pathobiology of this event by showing a significant association between ITP occurrence and immunoglobulin heavy-chain variable (IGHV) unmutated gene status, unfavorable cytogenetic lesions, and stereotyped conformation of the B-cell receptor (BCR). In particular, they show that stereotyped IGHV subsets #1 and #7 were significantly the most represented among patients developing ITP. Moreover, they show a prognostic implication for stereotyped BCR subset #7, which was significantly associated with shorter time to ITP development independently of IGHV mutational status. These findings strongly support a role for specific BCR triggering in the pathogenesis of this immune complication in CLL.A distinct need exists for novel risk stratification markers to improve outcome in childhood ependymoma. Using interphase FISH, Kilday and colleagues evaluated a prognostic role for 1q25 copy number gain across 3 pediatric European clinical trial cohorts defined according to patient age and adjuvant therapy administered. Gain of 1q25 was shown to be an independent marker of tumor progression for all patients, particularly for younger children treated with primary postoperative chemotherapy. Moreover, incorporating tumor 1q25 status with the degree of surgical resection enabled a novel patient risk-group classification system that was reproducible across all trial groups, thereby advocating the prospective evaluation of 1q25 gain in future international trials of pediatric ependymoma.Ma and colleagues present their findings from an evaluation of the dual inhibitor of polo-like kinase 1 and phosphoinositide 3-kinase, ON-01910.Na (rigosertib), in combination with gemcitabine in advanced cancer patients with a focus on pancreatic adenocarcinoma. This phase I trial represents the translation of preclinical work that identified rigosertib as a rational agent to modulate the vulnerability of pancreatic cancer to gemcitabine. This clinical study assessed the combination of rigosertib and gemcitabine, its pharmacology, and its preliminary efficacy in patients with pancreatic cancer, finding acceptable tolerability and hints of activity. This study serves as an example of rapid translation of solid preclinical findings to the clinic.

Prognostic Significance of Telomere Length in Chronic Lymphocytic Leukemia Patients in Early Stage Disease,

Abstract 3890Chronic lymphocytic leukemia (CLL) is a genetically heterogeneous disease with a variable outcome. The identification of factors that could predict the clinical course of early-stage CLL represents a crucial objective. Although previous studies indicated that telomere length may be a useful independent prognostic factor in the risk stratification of CLL, limited information has been reported in asymptomatic early stage patients (Binet stage A). We investigate the association of telomere length with the major biological and cytogenetic markers known to predict clinical outcome in CLL. The global DNA methylation levels of Alu and LINE sequences, was also investigated. Correlation with disease progression, measured as the time elapsed from diagnosis to first treatment, was evaluated. We measured relative telomere length (RTL) by real-time PCR in a panel of highly purified (>90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 untreated CLLs. All the cases were characterized by FISH for the most frequent chromosomal aberrations (Fabris et al. GCC, 2008). Molecular markers including mutation status of the heavy chain variable regions of immunoglobulin genes (IGVH), the expression of the 70-kd zeta-chain T-cell receptor-associated protein kinase (ZAP-70) and CD38 cell surface antigen protocols were previously reported (Cutrona et al., Haematologica, 2008). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu and LINE-1. We found a significantly lower RTL values in CLLs (median RTL=0.4 IQR 0.3–0.6) as compared with controls (median RTL=1.0 IQR 0.9–1.3) (P <0.001). A progressive and significant RTL decrease in low (13q- and normal karyotype), intermediate (+12) and high (11q- and 17p-) cytogenetic risk categories (P for trend =0.008) was observed. Patients with IGVH mutated genes had longer telomeres than patients with unmutated genes (P <0.001). No significant association between telomere length and either CD38 or ZAP-70 expression was found. Telomere shortening was significantly correlated with hypomethylation of Alu (P =0.048) and LINE-1 (P =0.001), indicating a contribution to chromosome instability. Finally, follow-up analysis showed a significantly higher risk of starting treatment for patients with shorter telomeres (P =0.0037). Our results extended previous evidence that telomere length could be used as marker for the identification of CLLs with a different prognostic risk. Disclosures:No relevant conflicts of interest to declare.

Abstract 1229: Prognostic significance of telomere length in B-chronic lymphocytic leukemia patients in early stage disease

Abstract BACKGROUND: Cell chronic lymphocytic leukemia (CLL) is a genetically heterogeneous disease with a variable outcome. The identification of factors that could predict the clinical course of early-stage CLL represents a crucial objective in this malignancy. Although previous studies indicated that telomere length may be a useful independent prognostic factor in the risk stratification of CLL patients, limited information has been reported in asymptomatic early stage patients (Binet stage A). OBJECTIVES: The present study was aimed at investigating the association of telomere length with the major biological and cytogenetic markers known to predict clinical outcome in CLL. The global DNA methylation of repeat sequences, Alu and LINE-1, both associated with chromosomal instability, was also studied. Finally, correlation with disease progression, measured as the time elapsed from diagnosis to first treatment, was evaluated. METHODS: We measured relative telomere length (RTL) by real-time PCR in a panel of highly purified (&amp;gt;90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 untreated CLL patients. All cases were characterized by FISH for the most frequent chromosomal aberrations, namely trisomy of chromosome 12 and deletions at 17p13.1, 11q22.3 and 13q14.3 loci (Fabris et al. GCC, 2008). Molecular markers including mutation status of the heavy chain variable regions of immunoglobulin genes (IGVH), the expression of the 70-kd zeta-chain T-cell receptor-associated protein kinase (ZAP-70) and CD38 cell surface antigen protocols were previously reported (Cutrona et al. Haematologica, 2008). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu and LINE-1. RESULTS: We found a significantly lower RTL values in CLLs (median RTL=0.4 IQR 0.3-0.6) as compared with controls (median RTL=1.0 IQR 0.9-1.3) (P &amp;lt; 0.001). A progressive and significant RTL decrease in low (13q- and normal karyotype), intermediate (+12) and high (11q- and 17p-) cytogenetic risk categories (P for trend =0.008) was observed. Patients with IGVH mutated genes had longer telomeres than patients with unmutated genes (P&amp;lt;0.001). No significant association between telomere length and either CD38 or ZAP70 expression was found. In line, telomere shortening was significantly correlated with hypomethylation of Alu (P=0.048) and LINE-1 (P=0.001), indicating a contribution to chromosome instability. Finally, follow-up analysis, available for 63 patients, showed a significantly higher risk of starting treatment for patients with shorter telomeres (P=0.037) CONCLUSIONS: Our results extended previous evidence that telomere length could be used as marker for the identification of different CLL prognostic subgroups. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2011-1229

Filamentous phage as a model immunogen for tracking B-cell responses to infection and vaccination (154.30)

Abstract Humoral immunity to infection can be categorized into four response stages: (i) innate-like, (ii) early-induced, (iii) adaptive, and perhaps (iv) the response to chronic infection (ChI). The first three stages are mediated by B-cell lineages distinguishable by phenotypic, genetic and functional characteristics. Our analysis of antibodies (Abs) produced during ChI indicates that “ChI Abs” have unique genetic features including high somatic mutation (SM) and usage of distal germline VH genes; ChI is also associated with aberrations in B-cell subsets. Here, we use filamentous phage as a model antigen to determine the genetic features and cellular origins of “ChI Abs” in the mouse. The phage’s surface consists of ~3,000 copies of the major coat protein, pVIII, and a majority of the anti-phage Ab response cross-reacts with a 12-aa peptide comprising the N-terminus of pVIII, allowing easy isolation of phage-specific B cells. After a single immunization, two “waves” of Ab-secreting cells (ASCs) appeared: first, IgM and IgG ASCs expressing proximal unmutated genes, followed by IgG ASCs expressing more distal genes bearing SMs. Phage-specific ASCs and serum IgG increased with repeated immunization, peaking after the fifth immunization and declining thereafter. Thus, we have validated the approach of combining cellular, flow cytometric, serological and immunogenetic analyses to characterize the dynamics of the B-cell response after initial and repeated encounter with antigen.

Somatic Hypermutation In Stereotyped Subset 4 BCRs/mAbs of CLL Patients, Expressing IGHV4-34 gene, Edit Anti-DNA Reactivity

AbstractAbstract 2444Extensive analysis of immunoglobulin gene sequences from chronic lymphocytic leukemia (CLL) patients reveals that ~30% carry stereotyped B-cell antigen receptors (BCRs) with highly homologous HCDR3s, over-represented in selected IGHV/IGHD/IGHJ rearrangements involving such IGHVs as 1–69, 4–34, and 3–21. This finding along with other structurally unique features of CLL BCRS suggests that antigens or superantigens or both may play an active role in the disease.The mutational status of IGHV genes is one of the most powerful prognostic molecular markers in CLL. Patients with IGHV genes with less than 2% differences from the germ line (“unmutated”; U-CLL) have a more aggressive clinical course compared to those with more than 2% differences (“mutated”; M-CLL). In subsets of sequences with stereotyped HCDR3s certain amino acid changes appear to be “CLL-biased”. One of the most distinctive SHM patterns has been described in sequences expressing stereotyped IGHV4-34 BCRs belonging to Subset 4, that are also characterized by long HCDR3s enriched in basic residues (especially arginine).The IGHV4-34 gene encodes antibodies which are autoreactive due to their recognition of the N-acetyllactosamine (NAL) antigenic determinant of the I/i blood group antigen, also expressed on a 220-kDa CD45 B cell-specific isoform; this recognition is mediated in a superantigenic fashion via a motif in HFR1 of the IGHV4-34 sequence. In addition, IGHV4-34 antibodies with HCDR3 sequence motifs enriched in basic (electropositive) amino acids have been shown to react against B cells and DNA, although HCDR3 electropositivity per se does not universally endow IGHV4-34 B cells with this property.Along these lines, we wanted to investigate if the modifications of subset 4 IGHV4-34 by SHM in precursors of the CLL clones could have affected the binding properties if the respective IG receptors, especially against B cells and DNA. We therefore recombinantly expressed 4 mAbs with IGHV4-34 rearrangements, from our CLL patients sample library; mAbs CLL183, CLL240, CLL342 belong to stereotypic subset 4 and have mutated heavy chain immunoglobulin genes, whereas CLL141 expresses an unmutated gene without belonging to any described subset.As previously reported, we tested reactivity of these mAbs with apoptotic and viable B and T cells. All 4 mAbs reacted significantly only with viable cells; the 3 mAbs belonging to subset 4 bound only viable B cells, with a preference for naïve B cells. We also tested the reactivity of these mAbs with single stranded DNA (ssDNA) and double stranded (dsDNA). We used common assays for identifying nuclear autoantigens in autoimmune disorders: 1) for binding total anti-nuclear antigens, human epithelial cell line HEp-2, and 2) for binding dsDNA, Crithidia Luciliae (CL). None of these tests had a strong positive result.To identify a role played by SHM in editing the anti-DNA reactivity, we chose to revert to germline 9 amino acid positions mutated in the IGHV and 1 in the IGKV sequences of CLL240. We either made revertant antibodies retromutated at single positions or at all mutated positions (completely unmutated and germline-like). Enzyme-linked assay (ELISA) for the semi-quantitative detection of ssDNA and dsDNA showed that the complete revertant antibody regained the ability to strongly bind ssDNA and partially dsDNA. The light chain did not appear to play a crucial role in the reacquisition of the binding, because the recombinant mAb expressing only the IGKV in the germline sequence did not bind ssDNA or dsDNA, as did the original recombinant mAb. Some of the revertants also showed a stronger binding to normal human B cells, with a few of them acquiring a partial ability to also bind T cells. The role of the single revertants is being further analyzed.Few IGHV4-34 CLL sequences are altered in the specific HFR1 motif responsible for NAL binding. This suggests that these IGHV4-34 expressing CLL cells could still bind and be stimulated for clonal expansion by NAL epitopes present in self and exogenous antigens. However, the findings of the present study also indicate that the distinctive modifications introduced by SHM in the stereotyped IGHV4-34 BCRs of subset 4 CLL likely represent a censoring mechanism for avoiding intense self-reactivity, mediated by the arginine-rich HCDR3s, and subsequent clonal deletion. Disclosures:No relevant conflicts of interest to declare.

Scientific Review: A new insight on predicting tumour malignancies using synergistic computational intelligence and bioinformatics approaches

Recently, the National Human Genome Research Institute and National Cancer Institute, both part of NIH, US Department of Health and Human Services, have launched The Cancer Genome Atlas (TCGA). Based on the mission of TCGA, we have proposed a further parallel paradigm on cancer: it is not only the genetic changes (i.e., mutations of genes) but also changes of gene expressions and regulatory networks that are ultimately responsible for cancer development. Under this parallel paradigm, un-mutated genes with differential expressions and alternative splicing may also induce changes in the differential regulatory networks that also cause cancer when cells are subjected to unusual environments. We developed a novel synergistic computational intelligence and bioinformatics approach to predict malignancies of neuroendocrine tumours that are particularly important to discover the mechanisms of human genome mechanisms relating malignant transformation.

Autoimmune or Chronic Infectious Disease in B-CLL at Diagnosis: Association with Unmutated VH Gene Status and Unfavorable Cytogenetics.

It is well known that chronic stimulation of the immune system (chronic infection, autoimmune disease) confers an increased risk of developing cancer. B-CLL represents an interesting model for the association of chronic antigenic stimulation with neoplastic transformation. We investigated the incidence of pre-existing chronic stimulation in patients with B-CLL at diagnosis. Chronic stimulation (CS) was defined as suffering from autoimmune disease or chronic or recurrent infection. The B-CLL control group consisted of patients without known chronic stimulation or concomitant diseases (non-CS). 187 unselected patients with known VH status diagnosed at our department were included in the study. Baseline characteristics were typical for a representative CLL patient cohort. Median age was 63 ranging from 25 to 83 years. Female:male ratio was 1:1,7. 87% of patients were staged Binet A, 13% Binet B or C. 63% of patients had mutated IgVH genes, 37% had unmutated genes (cut-off ≤ 98%). Normal karyotype was found by FISH in 25.8% of patients, 13q− in 46,1%, 11q− in 21,3%, +12 in 18%, and 17p− in 7,9% of patients. Of these 187 patients, 23,5% had a history of chronic stimulation (10,7% AI, 12,8% Inf). Age and Binet stage were similar between CS and non-CS patients. CS patients included a higher percentage of women (43.3 vs. 33.3%), and a higher percentage of CD38 positive (cut-off = 30%) patients (43,8 vs. 27,9%) compared to the non-CS subgroup, although both not statistically different. CS patients had a significantly higher percentage of unmutated clones (53%) compared to non-CS patients (28%) (p=0,002). Unfavorable cytogenetics (11q−, 17p−, +12) were more pronounced in CS patients compared to the non-CS group (77,8% vs 26,4%). The 4 most frequently used VH genes were 1–69, 3–30, 4–34, and 5–51 for the CS group, and 1–69, 2–5, 3–23, and 3–7 for the non-CS group. VH 1-69, the most frequently used VH gene overall, was unmutated in all CS patients, in non-CS patients several mutated cases were found. Several VH genes (2–26, 2–5, 3–7, 3–9) were exclusively unmutated in CS patients while they were mutated in non-CS patients. Several genes were exclusively used in either CS group (3–13, 3–15, 4–31, 4–39, 4–61, 5–51) or non-CS group (3–64, 3–65, 3–71, 3–74, 4–30, 7–4). They were mutated in most cases. Despite the drawbacks of our study (classification based on patients history and medical records), we provide unique and novel data on the frequency of chronic antigenic stimulation in an unselected cohort of newly diagnosed CLL patients. We noted considerable discrepancies between CS and non-CS patients regarding mutational status and VH gene usage. Our data warrant further evaluation of the role of CS in the development and course of B-CLL.

P036 Clinical data of CLL patients with unmutated IGHV1-69 gene expression

P036 Clinical data of CLL patients with unmutated IGHV1-69 gene expression

Polyreactive Monoclonal Antibodies Synthesized by Some B-CLL Cells Recognize Specific Antigens on Viable and Apoptotic T Cells.

B-cell chronic lymphocytic leukemia (CLL) is a disease characterized by uncontrolled clonal expansion of a CD5+/CD19+ B cell. Clones from different patients often express receptors/mAbs with remarkably similar VHDJH rearrangements, suggesting that the same antigen(s) or epitope(s) led to the initial clonal expansion. Although the specific antigens and epitopes remain to be identified, recent data indicate that B-CLL mAbs often bind autoantigens. In certain systemic autoimmune diseases, autoantibodies can react with cell surface molecules of viable lymphocytes (anti-lymphocyte mAbs) and with intracellular molecules of apoptotic cells that are transported to the cell membrane and function as autoantigens. Therefore, we expressed recombinant antibodies from B-CLL cells as IgG1 mAbs and tested them by flow cytometry for immunoreactivity with surface molecules on irradiated (apoptotic) and non-irradiated Jurkat cells, a human T-cell line. We chose to use T cells as our target for analysis to avoid difficulties that might arise from binding to FcgRIIb on B lymphocytes.Initial screening indicated that 5 of 25 mAbs showed intense binding. Antibodies from CLL Nos. 141 and DO8 bound to live but not apoptotic cells; in contrast, CLL Nos. 014, 114, and DO13 bound to apoptotic but not live cells. These five antibodies express unmutated VH genes. CLL 141 and DO8 express VH4-34. CLL 014 expresses VH1-69, CLL 114 VH4-39 and DO13 VH1-02. Other antibodies expressing VH1-69 in its unmutated form also bound Jurkat cells, albeit weaker than CLL 014, whereas mAbs expressing different unmutated genes from the VH1 family did not react. Among the mAbs expressing VH1-02 that were tested, only DO13 showed reactivity. This indicates that the VH1-02 gene was not solely responsible for the observed binding, and that DJ and/or VLJL segments were also involved. Remarkably, none of the tested mAbs belonging to VH3 family recognize antigens on Jurkat cells; all of these VH3 mAbs were mutated. Only CLL 114 belongs to a previously identified stereotypic set. Of interest, although CLL 014 and 141 are polyreactive, binding ssDNA, dsDNA, insulin, LPS, and HEp-2 cell lysates, their reactivities with surface antigens were specific in that CLL 141 bound only live cells and CLL 014 reacted solely with apoptotic cells.Our results indicate that, despite their inherent polyreactivity, certain B-CLL mAb bind specifically to antigenic epitopes on the surface of live as well as apoptotic Jurkat cells. The identity of these autoantigens is currently being investigated. These data highlight the importance of autoreactivity in the promotion and evolution of B-CLL.

Comparison of bone marrow and peripheral blood ZAP‐70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia

: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. : We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19(+) B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t-test. : A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. : These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. (c) 2006 International Society for Analytical Cytology.

Flow Cytometric Detection of ZAP-70 in Chronic Lymphocytic Leukemia: Addition of a Novel Second Monoclonal Antibody Improves Correlation with IgVH Mutation Status.

Background: Expression of ZAP-70 is associated with unmutated immunoglobulin heavy chain variable region genes and poor prognosis in B-cell CLL. Reliable clinical flow cytometric assays have been difficult to establish, as many anti-ZAP-70 antibodies show a suboptimal signal-to-noise ratio, and an imperfect correlation with IgVH mutation status.Methods: We studied fresh peripheral blood or bone marrow specimens from forty-three patients with CLL/SLL. Samples were stained for intracytoplasmic ZAP-70 using a standard monoclonal antibody, 1E7.2/PE (Caltag, Burlingame, CA), and the novel monoclonal antibody J13–1164/PE (kindly provided by BD Biosciences, San Diego, CA). Threshholds were set using isotype-matched negative control antibodies. Mutational status of the IgVH genes was assessed by RT-PCR, followed by standard Sanger DNA sequencing. Divergence from germline IgVH segments (IMGT database) was calculated using VBASE, with 2% or less changes over codons 1–94 of IgVH regarded as unmutated.Results: Staining with J13–1164 revealed a clear apparent cutpoint in levels of positivity, with 26/43 (60%) of the CLL cases showing >49% of cells above the isotype threshhold. Most of the remaining cases (15/43, 35%) showed <26% of cells positive. The 1E7.2/PE reagent yielded a similar number of positive cases, with 28/43 (65%) showing >20% positive cells (the cutpoint suggested by Rassenti et al., N Engl J Med 2004, 351:893). However, staining with 1E7.2 was more difficult to interpret, as many cases had levels of positivity that were close to the cutpoint, with 15/43 (35%) showing 10–30% of cells positive. Both antibodies showed similar degrees of correlation with IgVH mutation status, with 72–75% of unmutated cases showing the expected positivity for ZAP-70, and 57–64% of mutated cases negative for ZAP-70 (see Table 1). The two antibodies yielded concordant results in about half of cases (21/43), and the concordant cases showed a strong correlation with IgVH mutation status: 15/16 cases positive with both antibodies had unmutated genes, and 4/5 cases negative with both antibodies had mutated genes (see Table 2). The cases with discordant staining between the two antibodies were a mixture of cases with mutated or unmutated IgVH genes.Conclusions: The J13–1164 anti-ZAP-70 antibody provides more easily interpretable results in routine clinical use than the 1E7.2 clone, with brighter staining in many cases and a more distinct difference between positive and negative cases in our series. Compared to the 1E7.2 clone, the J13–1164 clone shows a similar degree of correlation with the IgVH mutation status. A combination of both antibodies provides the strongest correlation with IgVH mutation status, with 19/21 concordant cases showing the expected staining pattern.ZAP-70 staining vs IgVH mutation statusUnmutatedMutated1164 positive2151164 negative891E7.2 positive2261E7.2 negative78IgVH mutation status in concordant vs. discordant cases1E7.2 positive1E7.2 negative1164 positive15/16 unmutated6/10 unmutated1164 negative7/12 unmutated4/5 mutated