Zbigniew Rudzki

Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials

An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo

The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

BCR-ABL messenger RNA levels continue to decline in patients with chronic phase chronic myeloid leukemia treated with imatinib for more than 5 years and approximately half of all first-line treated patients have stable undetectable BCR-ABL using strict sensitivity criteria.

Purpose: In the first years of imatinib treatment, BCR-ABL remained detectable in all but a small minority of patients with chronic myeloid leukemia. We determined whether BCR-ABL continues to decline with longer imatinib exposure and the incidence and consequence of undetectable BCR-ABL. Experimental Design: BCR-ABL levels were measured in a subset of 53 imatinib-treated IRIS trial patients for up to 7 years (29 first-line, 24 second-line). Levels were deemed undetectable using strict PCR sensitivity criteria. Results: By 18 months, the majority achieved a 3-log reduction [major molecular response (MMR)]. BCR-ABL continued to decline but at a slower rate (median time to 4-log reduction and undetectable BCR-ABL of 45 and 66 months for first-line). The probability of undetectable BCR-ABL increased considerably from 36 to 81 months of first-line imatinib {7% [95% confidence interval (95% CI), 0-17%] versus 52% (95% CI, 32-72%)}. Undetectable BCR-ABL was achieved in 18 of 53 patients and none of these 18 lost MMR after a median follow-up of 33 months. Conversely, MMR was lost in 6 of 22 (27%) patients with sustained detectable BCR-ABL and was associated with BCR-ABL mutations in 3 of 6. Loss of MMR was recently defined as suboptimal imatinib response. There was no difference in the probability of achieving molecular responses between first- and second-line patients but first-line had a significantly higher probability of maintaining MMR [P = 0.03; 96% (95% CI, 88-100%) versus 71% (95% CI, 48-93%)]. Conclusions: With prolonged therapy, BCR-ABL continued to decline in most patients and undetectable BCR-ABL was no longer a rare event. Loss of MMR was only observed in patients with sustained detectable BCR-ABL.

A novel BCR-ABL transcript (e8a2) with the insertion of an inverted sequence of ABL intron 1b in a patient with Philadelphia-positive chronic myeloid leukaemia.

An atypical BCR–ABL transcript in a patient with Philadelphia (Ph)‐positive chronic myeloid leukaemia (CML) was detected using a long polymerase chain reaction technique. Sequence analysis revealed a joining of BCR exon e8 and ABL exon a2, with a 55‐base pair (bp) insert of an inverted sequence of ABL intron 1b between them, giving rise to an in frame e8a2 BCR–ABL transcript. This is the first report of a BCR–ABL transcript with the insertion of an inverted sequence. The long PCR technique is a useful screen for atypical BCR–ABL transcripts not detected by standard techniques.

Enzyme replacement therapy reduces Gaucher cell burden but may accelerate osteopenia in patients with type I disease – a histological study

Enzyme replacement treatment (ERT) is effective in controlling the clinical and biochemical manifestation of type I Gaucher disease. Little is known on the evolution of bone marrow histology caused by ERT. We compared the pretreatment trephine bone marrow biopsies in five patients (F32, F41, F50, M55, and M46) with the control biopsies performed after 26–32 months of imiglucerase treatment. The planimetric estimates revealed significant decrease in Gaucher cell burden in all the patients. The post‐ERT values ranged from 24% to 65% of the initial total volumes occupied by the Gaucher cells. This resulted mainly from disappearance of Gaucher cells, and to a lesser extent from their shrinkage. Normal hemopoiesis was markedly increased in four of five patients, fat tissue in all the five patients. Surprisingly, the estimated volumes of trabecular bone in the control biopsies were significantly smaller than in the pre‐ERT trephines (0.61%, 0.64%, 0.64%, 0.97%, and 0.22% of the initial volumes, P = 0.043). The bone loss correlated rather with the degree of reconstitution of normal hemopoiesis than with the decrease in the Gaucher cell burden. The histological response did not correlate strictly with initial clinical parameters and the genotype, with different reactions to ERT in two sibs (cases 3 and 4). Particularly, ERT alleviated bone manifestations in all four patients with previous bone pains or fractures. ERT may accelerate progress of osteopenia in men and premenopausal women. The clinical significance of this phenomenon remains to be established. Its mechanism may be linked to increased osteolytic activity exerted directly or indirectly by regenerating hemopoietic cells.

ABL-kinase domain point mutation as a cause of imatinib (STI571) resistance in CML patient who progress to myeloid blast crisis

Imatinib mesylate (STI571) is a major therapeutic advance for the management of chronic myeloid leukaemia (CML), however, a proportion of patients are refractory to it, particularly those in more advanced phases of CML. Different mechanisms of resistance to imatinib are suggested, including point mutations within ABL-kinase domains. A point mutation leading to substitution at the ATP binding site of ABL-kinase and insensitivity to imatinib was detected in our patient treated with imatinib, who progressed to blast crisis. Additionally, clonal evolution could lead to BCR-ABL-independent proliferation. Early detection of ABL-kinase mutation could predict the progression of CML treated with imatinib.

Dual transcription of b2a2 and b3a2 BCR-ABL transcripts in chronic myeloid leukaemia is confined to patients with a linked polymorphism within the BCR gene.

Summary.  We propose a mechanism for dual expression of b2a2 and b3a2 BCR–ABL in chronic myeloid leukaemia (CML). We have identified a BCR allele, present in approximately 29% of the population, which includes an adenine to guanine polymorphism in the putative branchpoint of BCR intron 13. CML patients who expressed both b2a2 and b3a2 transcripts had the allele, which was also associated with alternative transcription of the normal BCR allele. We conclude that the BCR intronic polymorphism is associated with activation of a cryptic branchpoint resulting in reduced efficiency of RNA splicing and exon 14 (b3) skipping in BCR and BCR–ABL.

Familial occurrence of warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome

Introduction:Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder with an autosomal-dominant pattern of inheritance and low fatality rate but significant lifelong morbidity.Materials and Methods:A 27-year-old mother of two children has been suffering from severe neutropenia and recurrent infections with the diagnosis of sporadic WHIM syndrome established by sequencing the CXCR4 gene and the finding of a heterozygous 1000 C→T nonsense mutation in the second CXCR4 exon. The first child was an apparently healthy boy delivered at full term. Umbilical cord blood cells were obtained for genetic analysis. Peripheral blood cells were also analyzed at 8 months of life. Both analyses revealed the same mutation as that of his mother. The child was in a good condition, manifesting neutropenia without infections until 11 months of life. He subsequently developed pneumonia requiring a more aggressive treatment. After that, the regular substitution of immunoglobulins (IVIGs) and G-CSF has been preventing serious infections. Six months ago the second boy was delivered who also demonstrated neutropenia without severe infections. Genetic studies using cord blood and also peripheral blood cells in the fourth month showed an identical mutation of the CXCR4 gene as in his mother. Moreover, the mother and her first son demonstrated monocytopenia.Results:The results indicate that genetic defects connected with WHIM syndrome may influence not only the granulocyte, but also the monocytic lineage. Moreover, a perinatal diagnosis of WHIM syndrome made by sequencing the CXCR4 gene should be performed in cases where either parent is known to be affected with this disease.Conclusions:This would facilitate an earlier detection of the deficiency in children, thereby allowing a more comprehensive follow-up and administration of appropriate therapy.

NK cell function is markedly impaired in patients with chronic lymphocytic leukaemia but is preserved in patients with small lymphocytic lymphoma.

Chronic lymphocytic leukemia (B-CLL) and small lymphocytic lymphoma (SLL) are part of the same disease classification but are defined by differential distribution of tumor cells. B-CLL is characterized by significant immune suppression and dysregulation but this is not typical of patients with SLL. Natural killer cells (NK) are important mediators of immune function but have been poorly studied in patients with B-CLL/SLL. Here we report for the first time the NK cell phenotype and function in patients with B-CLL and SLL alongside their transcriptional profile. We show for the first time impaired B-CLL NK cell function in a xenograft model with reduced activating receptor expression including NKG2D, DNAM-1 and NCRs in-vitro. Importantly, we show these functional differences are associated with transcriptional downregulation of cytotoxic pathway genes, including activating receptors, adhesion molecules, cytotoxic molecules and intracellular signalling molecules, which remain intact in patients with SLL. In conclusion, NK cell function is markedly influenced by the anatomical site of the tumor in patients with B-CLL/SLL and lymphocytosis leads to marked impairment of NK cell activity. These observations have implications for treatment protocols which seek to preserve immune function by limiting the exposure of NK cells to tumor cells within the peripheral circulation.

Reduced Intensity Allogeneic Stem Cell Transplant for Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm is a rare, aggressive tumor characterized by skin and/or marrow infiltration by CD4+ CD56+ cells. Historically, the tumor was variably thought to arise from either monocytes, T cells or NK cells giving rise to terms such as CD4+/CD56+ acute monoblastic leukemia, primary cutaneous CD4+/CD56+ hematodermic tumor and blastic NK-cell lymphoma. Whilst considerable progress has been made in understanding the histogenesis, the best modality of treatment remains to be defined. We are therefore reporting this case which was successfully treated with a T-deplete allogeneic transplant and the patient is currently alive and in remission 4 years post transplant.