• Sowing Seeds of Discontent (Cover Story)
• Novel Proteasome Inhibitor Therapy
• A New Option for Relapsed Myeloma
• Another Piece in the Antiphospholipid Antibody Syndrome Puzzle
• Priming the Program for Cell Death in Cancer Cells
• Mutations Forming Subgroups in AML: Just Like Cells, They Are Constantly Dividing and Interacting
• SNP-Chip-Based Genome-Wide Analysis of Genetic Alterations in Hematologic Disorders: The Way Forward?

Novel Proteasome Inhibitor Therapy

Kenneth Anderson, MD

Dr. Anderson has done research work on proteasome inhibitors for multiple, different pharmaceutical companies.

Demo SD, Kirk CJ, Aujay MA, et al. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res. 2007;67:6383-91.

A new treatment paradigm targeting the tumor cell and its BM microenvironment to overcome drug resistance and improve patient outcome has now been developed in multiple myeloma (MM).¹ The boronic acid proteasome inhibitor bortezomib targets the tumor cell in its microenvironment in both laboratory² and animal models,³ and has rapidly been translated from the bench to the bedside and ultimately FDA approval first for treatment of relapsed refractory MM,⁴ and subsequently of relapsed MM.⁵ Bortezomib was then combined with dexamethasone⁶ or with melphalan and prednisone⁷ as initial therapy for newly diagnosed patients who were stem cell transplant candidates or elderly non-transplant candidates, respectively, and achieved high extent and frequency of response. However, although bortezomib is a major advance, not all patients respond, and those that do eventually acquire resistance.

Two strategies are under development to sensitize or overcome resistance to bortezomib. First, preclinical studies suggest that bortezomib combined with DNA damaging agents,⁸ heat shock protein 90 inhibitors,⁹ Akt inhibitors,¹⁰ and histone deacetylase inhibitors^11,12 mediates synergistic MM cell cytotoxicity; already the combination of bortezomib and pegylated doxorubicin has been FDA-approved for treatment of relapsed MM, as it has achieved significantly increased response rate, extent, and progression-free and overall survival compared to bortezomib alone.¹³ The second strategy is the development of next-generation proteasome inhibitors, NPI-0052 and PR-171. NPI-0052 inhibits caspase-like, trypsin-like, and chymotrypsin-like proteasome activities; overcomes bortezomib resistance and is well tolerated in preclinical models;¹⁴ and is now in a clinical trial in MM.

In the current report, Demo and colleagues report on PR-171, a novel epoxyketone-based irreversible proteasome inhibitor which exhibits equal potency but greater selectivity for the chymotrypsin-like activity of the proteasome.¹⁵ In preclinical studies at two-day or five-day consecutive schedules, it can inhibit proteasome activity by 80 percent and overcome bortezomib resistance. Additional in vitro data show that it inhibits chymotrypsin-like activity, not only of the proteasome, but also of the immunoproteasome, and induces apoptosis via both intrinsic and extrinsic apoptotic signaling.¹⁶ This promising preclinical data has rapidly translated to clinical trials of PR-171, which have achieved responses in relapsed refractory MM when given daily on either two-day or five-day schedules, and two phase II trials are soon to begin in relapsed and in relapsed refractory MM.

These exciting data both confirm the activity of proteasome inhibitors in MM and suggest that more potent chymotrypsin-like inhibition may increase MM cell cytotoxicity. In future clinical trials, gene and protein profiling of patients responsive versus resistant to proteasome inhibitors,¹⁷ coupled with the ability to utilize fluorescent probes to assess patterns of qualitative and quantitative proteasome and immunoproteasome inhibition in MM cells associated with response,¹⁸ will allow for the optimal use of this exciting class of novel therapeutics.

1. Hideshima T, Mitsiades C, Tonon G, et al. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7:585-98.
   
   
2. Hideshima T, Richardson P, Chauhan D, et al. The proteosome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001;61:3071-6.
   
   
3. LeBlanc R, Catley LP, Hideshima T, et al. Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model. Cancer Res. 2002;62:4996-5000.
   
   
4. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003;348:2609-17.
   
   
5. Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352:2487-98.
   
   
6. Jagannath S, Durie BG, Wolf J, et al. Bortezomib therapy alone and in combination with dexamethasone for previously untreated symptomatic multiple myeloma. Br J Haematol. 2005;129:776-83.
   
   
7. Mateos MV, Hernández JM, Hernández MT, et al. Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: results of a multicenter phase 1/2 study. Blood. 2006;108:2165-72.
   
   
8. Mitsiades N, Mitsiades CS, Richardson PG, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood. 2003;101:2377-80.
   
   
9. Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Antimyeloma activity of heat shock protein-90 inhibition. Blood. 2006;107:1092-100.
   
   
10. Hideshima T, Catley L, Yasui H, et al. Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells. Blood. 2006;107:4053-62.
    
    
11. Hideshima T, Bradner JE, Wong J, et al. Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc Natl Acad Sci USA. 2005;102:8567-72.
    
    
12. Catley L, Weisberg E, Kiziltepe T, et al. Aggresome induction by proteasome inhibitor bortezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells. Blood. 2006;108:3441-9.
    
    
13. Orlowski RZ, Nagler A, Sonneveld P, et al. Randomized phase III study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression. J Clin Oncol. 2007;25:3892-901.
    
    
14. Chauhan D, Catley L, Li G, et al. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib. Cancer Cell. 2005;8:407-19.
    
    
15. Demo SD, Kirk CJ, Aujay MA, et al. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res. 2007;67:6383-91.
    
    
16. Kuhn DJ, Chen Q, Voorhees PM, et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against pre-clinical models of multiple myeloma. Blood. 2007;110:3281-3290.
    
    
17. Mulligan G, Mitsiades C, Bryant B, et al. Gene expression profiling and correlation with outcome in clinical trials of the proteasome inhibitor bortezomib. Blood. 2007;109:3177-88.
    
    
18. Berkers CR, Verdoes M, Lichtman E, et al. Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib. Nature Methods. 2005;2:357-362.

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A New Option for Relapsed Myeloma

Michael E. Williams, MD

Orlowski RZ, Nagler A, Sonneveld P, et al. Randomized phase III study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression. J Clin Oncol. 2007;25:3892-901.

The proteasome inhibitor bortezomib (B) is an effective therapy for patients with relapsed multiple myeloma (MM), improving survival and time to progression as compared with single-agent, high-dose dexamethasone.¹ B also showed a high response rate and acceptable safety profile in combination with pegylated liposomal doxorubicin (PLD) in a phase I study.² In order to further assess the safety and efficacy of this combination, Orlowski and colleagues conducted a phase III clinical trial of B versus B+PLD for myeloma patients progressing after prior therapy and for those with primary refractory disease. This international, open-label trial enrolled 646 patients at 123 centers. Two-thirds of patients in each group had received prior anthracycline-containing regimens and over half had prior stem cell transplantation; none had received prior B treatment. Patients were randomized to receive either B 1.3 mg/m2 on days 1, 4, 8, and 11 of each 21-day cycle, or B in the same dose and schedule in combination with PLD 30 mg/m2 on day 4 following infusion of B. Eight cycles of treatment were planned, although responding patients could continue the assigned treatment if tolerated. There was no crossover from BB+PLD, but patients in the latter arm could continue protocol therapy with B alone if PLD was discontinued due to toxicity. Overall response rates did not differ between the two arms: B 41 percent and B+PLD 44 percent. Assessment at a planned interim analysis of intent-to-treat patients revealed a significant benefit in time to progression (TTP, the primary study endpoint) for B+PLD versus B (9.3 months versus 6.5 months, p=0.000004). Responses were observed in patients with prior anthracycline, stem cell transplantation, and immunomodulatory therapies. An updated survival analysis also showed a significant benefit for survival at 15 months of 76 percent for the combination versus 65 percent for B alone (p=0.03). Toxicity was increased in the combination arm, primarily myelosuppression, nausea, vomiting, or diarrhea, but with no increase in peripheral neuropathy. Hand-foot syndrome occurred in 16 percent of patients treated with PLD; 5 percent were grade 3 and PLD was discontinued in these patients. Only three patients in each group had a thromboembolic event; 7 percent of B and 9 percent of B+PLD patients had a decrease in left ventricular ejection fraction, although only three in each group developed congestive heart failure. Treatment-related death was recorded for three B-treated and four B+PLD-treated patients.

With the availability and efficacy of newer agents for MM treatment, including the immunomodulatory drugs and bortezomib, the use of traditional alkylator and anthracycline-containing cytotoxic regimens has markedly decreased. This clinical trial reintroduces doxorubicin in a potentially less toxic formulation and demonstrates an improved TTP as well as improved 15-month survival when given in combination with B for patients with relapsed and refractory MM, despite the absence of an increase in overall response rate versus B alone. Given the modest prolongation in TTP, it will be necessary for clinicians and patients to carefully consider the additional toxicities and cost of this combination regimen. As noted by the authors, the combination does provide an important option for patients intolerant or resistant to corticosteroid therapy and for those at high risk for thromboembolic disease. Further clinical research will be necessary to assess response rates in less heavily pretreated patients and with the addition of agents such as dexamethasone to B+PLD. The incorporation of correlative biomarker analyses in such trials will be an important means to identify molecular or phenotypic subsets of patients with MM most likely to benefit.

1. Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005; 352:2487-98.
   
   
2. Orlowski RZ, Voorhees PM, Garcia RA, et al. Phase 1 trial of the proteasome inhibitor bortezomib and pegylated liposomal doxorubicin in patients with advanced hematologic malignancies. Blood. 2005;105:3058-65.

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Another Piece in the Antiphospholipid Antibody Syndrome Puzzle

Roy L. Silverstein, MD

Hulstein JJJ, Lenting PJ, de Laat B, et al. β₂-Glycoprotein I inhibits von Willebrand factor-dependent platelet adhesion and aggregation. Blood. 2007;110:1483-1491.

Most patients with the pro-thrombotic condition known as antiphospholipid antibody (APLA) syndrome have circulating autoantibodies reactive with the phospholipid binding plasma protein β₂-glycoprotein I (β₂-GPI). Although β₂-GPI circulates at very high concentrations (3μM) and has been studied for many years, its biologic function remains obscure. In this manuscript, a team from the Netherlands has uncovered a potential role for β₂ -GPI in regulating primary hemostasis. Using two well-established ex vivo assays for platelet glycoprotein Ib complex-dependent binding to von Willebrand Factor (vWF) (ristocetin-induced platelet aggregation and shear stress induced adhesion of resting platelets to immobilized vWF), they found that adding increasing concentrations of β₂-GPI inhibited binding to a moderate, but statistically significant, degree. They then used in vitro binding assays to demonstrate that purified β₂-GPI binds directly to vWF with affinity well within the range of typical plasma levels. Importantly, β₂-GPI did not bind to "native" vWF, but only to the form rendered competent to bind GPIb induced when vWF is subjected to shear stress or incubated with ristocetin. Recombinant vWF proteins lacking the GPIB-binding A1 domain did not bind β₂-GPI while a mutant A1 domain found in patients with type IIb von Willebrand Disease (vWD) that is constitutively competent to bind GPIb bound β₂-GPI even in the absence of shear or ristocetin. To relate their findings to the APLA syndrome, the authors showed that anti-β₂-GPI antibodies, either monoclonal or isolated from patients’ sera, blocked binding of β₂-GPI to "active" vWF and blocked the inhibitory effect of β₂-GPI on platelet function. Furthermore, antibodies to β₂-GPI actually enhanced ristocetin-induced aggregation of platelet-rich plasma. Finally, the authors detected the "active" form of vWF in the circulation of patients with APLA syndrome with levels modestly, but significantly, higher in patients with history of thrombosis.

The APLA syndrome is a common and important acquired pro-thrombotic state unusual among the thrombophilias in that it is associated with both arterial and venous thrombosis and with spontaneous pregnancy loss. The syndrome is misnamed in that the autoantibodies circulating in patients do not react with phospholipids per se, but rather with various phospholipid-binding proteins, including β₂-GPI and prothrombin. By far the most commonly found reactivity is with β₂-GPI. Studies with animal models and human clinical studies linking antibody titer with thrombosis risk show that the pathophysiology of APLA syndrome is mediated by the circulating autoantibodies, but the precise molecular mechanisms by which these antibodies induce thrombosis is not clear. In fact, multiple potential mechanisms have been described. Some antibodies may have a direct prothrombotic effect by binding to and activating endothelial cells, leukocytes, and/or trophoblast cells. Some may have an indirect effect by preferentially inhibiting phospholipid-dependent anticoagulant pathways (for example, protein C) or by activating complement. In this new work, the authors propose a novel indirect effect related to the ability of β₂-GPI to act as a circulating antagonist of the GPIb complex, the platelet vWF receptor, and thereby modulate platelet adhesion to exposed subendothelial matrix at the site of a vascular injury. APLA antibodies, by blocking this natural "brake" on the system, create a prothrombotic state, perhaps akin to type IIB vWD or even TTP. (The unprocessed ultralarge vWF multimers seen in TTP also bind GPIb in the absence of ristocetin or shear.)

APLA syndrome is clearly a group of related conditions, not a well-defined single disease. Clinical presentations (and ultimately therapeutic approaches) are likely determined by a host of factors, including antibody titer, antibody class and isotype, and antigen specificity. Antibodies with lupus anticoagulant activity seem to carry the highest risk, but risk and type of presentation are also probably related to the ability of the antibodies to influence platelet, endothelial cell, placental, and plasma pro- and anti-coagulant functions. Although APLA syndrome does not share typical features of type IIb vWD or TTP, the mechanisms described in this manuscript could certainly contribute to the tendency of some of these patients to develop arterial thrombosis. These studies also suggest that anti-platelet therapy may have a place in the treatment of some patients.

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Priming the Program for Cell Death in Cancer Cells

Lilli Petruzzelli, MD

Deng J, Carlson N, Takeyama K, et al. BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents. Cancer Cell. 2007;12:171-85.

Cancer cells exhibit molecular changes that potentially could result in apoptosis; however, cancer cells often select for a block in apoptotic signaling that results in survival. BCL-2 family members function and control apoptotic signals in the mitochondria, and the authors probe the mechanism by which BCL-2 family members can be affected to overcome the proapoptotic signals. The work outlined in this manuscript takes a sweeping analysis of the BH3-containing family members and their expression in different lymphoma cell lines and correlates the mechanism of apoptotic block with sensitivity to chemotherapeutic agents.

The investigators draw from an array of knowledge about proteins in the BCL-2 family and their specific function in programmed cell death and apply this knowledge to classifying apoptotic block in lymphoma cell lines. Diffuse large B-cell lymphomas that represent the same class of disease but have heterogeneous mechanisms for overcoming apoptotic signals were used as a model in their studies. BH3 peptides were used to distinguish or profile three types of apoptotic block: a) inhibition of upstream activation of BH3-only proteins, b) loss of BAX and BAK so that the effector of apoptosis is lost, and c) expression of antiapoptotic proteins that prevent activation of BAX and BAK ("primed cancer cells"). Different classes of BH3 peptides were used to probe the models and predict the block in lymphoma cell lines that contained t(14;18) or did not. The different blocks were confirmed by analysis of BCL-2 proteins that were predicted by the BH3 peptide. Interestingly, their profiling predicted sensitivity to BCL-2 antagonism by the BH3 antagonist ABT-737. High abundance of BCL-2:BIM complex predicted sensitivity to ABT-737, and further characterization revealed that BCL-2 levels correlated with sensitivity. The investigators took this analysis beyond ABT-737 and treated the lymphoma cell lines with agents that have previously been shown to induce apoptosis through the mitochondrial pathway and confirmed that this model was predictive of relative chemotherapeutic sensitivity beyond inhibitors of BH3 domains.

Predicting sensitivity and understanding resistance to chemotherapeutic agents in diseases that are biologically similar but molecularly heterogeneous is of critical importance. The investigators focused on diffuse large-cell lymphoma and were able to classify cell lines based upon their sensitivity to BH3 peptides into groups that had differences in apoptotic block. Although this work was done in cell lines, the authors’ findings not only confirmed their ability to profile at the molecular level but also put forth an explanation of why high BCL-2 levels may not uniformly provide protection against chemotherapeutic agents. BCL-2, from their model, would be proposed to be occupied in contrast to cell lines where BCL-2 was over-expressed and therefore largely unoccupied. Because BCL-2 is largely occupied, the cancer cell is felt to be primed for high sensitivity to chemotherapeutic agents. The data using conventional chemotherapeutic agents that use intrinsic apoptotic pathways support the idea that cells primed for cell death are more likely to respond whereas those with other types of blocks are not as likely to respond. The work presented within this manuscript suggests that profiling is possible and that sensitivity to ABT-737 can be predicted by BH3 profiling. More detailed analysis may allow us to learn whether classification will enable us to distinguish sensitivity to different chemotherapeutic agents.

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Mutations Forming Subgroups in AML: Just Like Cells, They Are Constantly Dividing and Interacting

Peter Emanuel, MD

Mead AJ, Linch DC, Hills RK, et al. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood. 2007;110:1262-1270.

Dicker F, Haferlach C, Kern W, et al. Trisomy 13 is strongly associated with AML1/RUNX1 mutations and increased FLT3 expression in acute myeloid leukemia. Blood. 2007;110:1308-1316.

Bullinger L, Rücker FG, Kurz S, et al. Gene-expression profiling identifies distinct subclasses of core binding factor acute myeloid leukemia. Blood. 2007;110:1291-300.

As we move beyond a morphologic classification system of acute myeloid leukemia (AML) and into the era of genetic classifications for subgroups of AML, two of the major subgroups that have thus far emerged are those AML patients with disruptions of the core binding factor (CBF) complex and those patients with disruptions of the FLT3 gene.

The FMS-like tyrosine kinase 3 (FLT3) gene is a membrane-bound receptor tyrosine kinase. Many hematopoietic cells produce FLT3 ligand, which promotes dimerization and activation of the receptor tyrosine kinase, FLT3. Similar to many cytokine-signaling pathways, upon activation FLT3 exerts positive effects on a multitude of downstream pathways including RAS and phosphatidylinositol 3-kinase (PI 3-K). There are two major classes of disruptions of the FLT3 gene in AML. Internal tandem duplications (ITDs) in the juxtamembrane domain involve head-to-tail duplication of 3-400 base pairs in exons 14 or 15 (Figure 1). FLT3 ITDs occur in 15 to 35 percent of patients with AML. The other major class is missense point mutations in exon 20 in the intracellular domain at D835. These missense point mutations occur in 5 to 10 percent of patients with AML.^1,2

The CBF complex is a transcription factor complex critical for regulation of hematopoiesis and normal myeloid development. Disruptions of the CBF complex, t(8;21)(q22;q22) or inv(16)(p13q22)/ t(16,16)(p13;q22), constitute AML subgroups with favorable prognosis. The problem exists that in both the FLT3 and CBF groups of patients with AML considerable clinical heterogeneity exists that implies there must be additional biologic or genetic heterogeneity. Three manuscripts, all in the August 15, 2007, issue of Blood, shed new light on these subgroups and also highlight some possible interactions between genetic lesions.

Mead and colleagues screened 1,107 young adult patients with AML for FLT3 ITDs and for FLT3 point mutations (utilizing dHPLC analysis). They found a 23 percent incidence for FLT3 ITDs alone, 9 percent incidence for FLT3 point mutations alone, and, interestingly, 2 percent of patients that had both kinds of FLT3 aberrancies. There was a highly significant difference between patients with FLT3 ITD and patients with an FLT3 point mutation, both in terms of cumulative incidence of relapse and overall survival, with FLT3 ITD patients having a poorer prognosis in all analyses.

Bullinger and colleagues utilized gene-expression profiling to examine 93 AML patients with disruptions in the CBF complex, 55 with inv(16), and 38 with t(8;21). By unsupervised hierarchical clustering, they identified two subgroups of CBF AML patients based on distinct patterns of gene expression. The "unfavorable" subgroup was associated with elevated white blood cell counts and FLT3 ITDs. The gene-expression signatures associated with this "unfavorable" group included proliferative-type genes such as JUN, FOS, and others in the MAPK pathway as well as high-level expression of genes involved in the response to DNA damage and in DNA repair. Conversely, the "favorable" subgroup of the CBF AML patient samples was characterized by prominent gene-expression features of antiapoptotic pathways.

Finally, Dicker and colleagues investigated AML1/RUNX1 gene mutations as a part of disruption of the CBF complex. In different patient cohorts they noted a recurring theme of RUNX1 mutations being associated with trisomy 13 independent of the FAB subgroup. Since the FLT3 gene is localized on chromosome 13, they hypothesized that RUNX1 mutations might cooperate with trisomy 13 by increasing FLT3 transcript levels. These results pointed to a potential third type of involvement in AML by FLT3 gene abnormalities, namely, in the absence of ITDs or point mutations, FLT3 gene overexpression could be a third route for FLT3 activation, which could potentially cooperate in leukemic transformation together with RUNX1 (CBF) mutations.

Overall, these three papers teach us several things. First, that the FLT3 tyrosine kinase can be involved in leukemogenesis by a number of different genetic mechanisms. Secondly, it teaches us that even if it’s the same gene being activated, the different mechanisms for activation can have significantly different phenotypic behaviors. And finally, that the two major subgroups of genetic disruptions in AML (CBF complex and FLT3) can demonstrate interaction in initiating and maintaining the leukemic clone.

1. Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer. 2003;3:650-665.
   
   
2. Krause DS, Van Etten RA. Tyrosine kinases as targets for cancer therapy. N Engl J Med. 2005;353:172-187.

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SNP-Chip-Based Genome-Wide Analysis of Genetic Alterations in Hematologic Disorders: The Way Forward?

Yan-Tat Liu, DPhil, and Josef T. Prchal, MD

Mullighan CG, Goorha S, Radtke I, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;446:758-64.

Mohamedali A, Gaken J, Twine NA, et al. Prevalence and prognostic significance of allelic imbalance by single nucleotide polymorphism analysis in low risk myelodysplastic syndromes. Blood. Prepublished online July 18, 2007.

Gondek LP, Tiu R, Haddad AS, et al. Single nucleotide polymorphism arrays complement metaphase cytogenetics in detection of new chromosomal lesions in MDS. Leukemia. 2007;21:2058-61.

With the completion of the Human Genome Project and its sister HapMap Project that mapped single nucleotide polymorphisms (SNPs), the last few years have seen a revolution in how information about DNA sequences and genotypic variation are utilized in research. Soon, all these will be transferred to the clinical arena. The development of genotyping platforms, such as SNP chips, has allowed simultaneous assessment of hundreds of thousands of SNPs, as well as copy-number variations and the determination of the nature of loss-of-heterozygosity including uniparental disomy (UPD) across the whole human genome. High-resolution, genome-wide SNP chips, following the example of other high-tech devices, are including an ever-increasing number of SNPs at a rapidly declining cost. They are being increasingly used in association studies of inherited predispositions for both rare and common human diseases.¹ However, three recent studies also highlight their use for the identification of somatic point mutations and cryptic chromosomal lesions in blood disorders when applied to large cohorts of patient samples.

In an important study, Mullighan and colleagues describe an unprecedented genome-wide SNP analysis of 242 pediatric patients with acute lymphoblastic leukemia (ALL) using high-resolution SNP chips. This newer generation of chips features detections of more than 350,000 loci and permits analysis of copy-number changes at an average resolution of <5kb across the entire human genome. The study identified 54 recurrent somatic regions of deletion, of which 24 contained only a single gene. Further analysis of selected genes from these regions revealed that 40 percent of patients had deletions or mutations in genes that control B lymphocyte development and differentiation, with the PAX5 gene being the most frequent target of somatic mutation (in 31.7 percent of patients). The study also found deletions in additional genes important in B-cell differentiation including EBF1 and IKZF1 (IKAROS), suggesting a contribution of these genes to the pathophysiology of B-progenitor ALL.

Though examining a different clonal hematologic malignancy, myelodysplastic syndrome (MDS), two other papers published this year explored the usefulness of SNP chips in studies of acquired genetic imbalances. In MDS, a considerable percentage of patients do not exhibit cytogenetic abnormalities using conventional metaphase chromosome analysis. High-resolution SNP chips, however, offer improved levels of genomic resolution, facilitating detection of both cryptic defects as well as their copy number. In independent studies, Mohamedali et al. and Gondek et al. carried out genome-wide SNP analysis in 119 and 66 cases of MDS, respectively. Surprisingly, both groups identify a high frequency of segmental loss-of-heterozygosity due to UPD, which is not detectable by traditional cytogenetic analysis. UPD results from duplication of one of the parental alleles during mitotic recombination but is not detectable by cytogenetic analysis. UPD was previously found to be common in polycythemia vera² and other myeloproliferative disorders, but had not been appreciated in other hematologic malignancies. These three studies confirm the greater power of SNP chips over the cumbersome metaphase-dependent cytogenetics, bypassing the need for laborious low-resolution and costly metaphase examination. It remains to be established whether SNP chips could avoid the need for bone marrow tissue since easily accessible clonal circulating granulocytes may provide the same information as analysis of hematopoietic progenitors.

Step 1: Individual digestion of genomic DNA (500ng) with Nsp I and Sty I restriction enzymes (RE). Step 2: Ligation of digested products to corresponding adaptors. Step 3: Preferential amplification of adaptor-ligated RE digested DNA fragments with generic primer to generate products of sizes from 200 to 1100 bp. Step 4: Combination of PCR products from each RE digest. Step 5: Purification of products using polystyrene beads.

Taken together, these three pioneering manuscripts show the amazing ability of SNP chips to detect somatic mutations associated with acquired clonal hematologic disorders and demonstrate UPD as one of the most common genetic mechanisms found. These elegant studies highlight the increasing speed, specificity, and power of genome-wide approaches in the systematic search for pathogenic lesions. As the cost of SNP chips has recently dropped to within reach of most laboratories ($250 per sample), and as genome-wide chips are being produced with even higher densities (the current-generation chip contains 1 million SNPs), the way we investigate, diagnose, and stratify therapy for both germline and somatic mutations will likely be transformed. With these rapidly evolving technologies, the possibility of having the entire 3-billion-base-pairs human genome sequence hybridized to a single chip could actually come a lot sooner than we thought.

1. Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007;447:661-78.
   
   
2. Kralovics R, Guan Y, and Prchal JT. Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera. Exp Hematol. 2002;30:229-236.

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