Flow cytometric immunobead assay for detection of BCR-ABL1 fusion proteins in chronic myleoid leukemia: Comparison with FISH and PCR techniques

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence

reserved36noAlthough the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rb1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rb1 chain when cocultured with activated T cells or CD40L+cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.mixedCutrona, Giovanna*; Tripodo, Claudio; Matis, Serena; Recchia, Anna Grazia; Massucco, Carlotta; Fabbi, Marina; Colombo, Monica; Emionite, Laura; Sangaletti, Sabina; Gulino, Alessandro; Reverberi, Daniele; Massara, Rosanna; Boccardo, Simona; De Totero, Daniela; Salvi, Sandra; Cilli, Michele; Pellicanò, Mariavaleria; Manzoni, Martina; Fabris, Sonia; Airoldi, Irma; Valdora, Francesca; Ferrini, Silvano; Gentile, Massimo; Vigna, Ernesto; Bossio, Sabrina; De Stefano, Laura; Palummo, Angela; Iaquinta, Giovanni; Cardillo, Martina; Zupo, Simonetta; Cerruti, Giannamaria; Ibatici, Adalberto; Neri, Antonino; Fais, Franco; Ferrarini, Manlio; Morabito, FortunatoCutrona, Giovanna; Tripodo, Claudio; Matis, Serena; Recchia, Anna Grazia; Massucco, Carlotta; Fabbi, Marina; Colombo, Monica; Emionite, Laura; Sangaletti, Sabina; Gulino, Alessandro; Reverberi, Daniele; Massara, Rosanna; Boccardo, Simona; De Totero, Daniela; Salvi, Sandra; Cilli, Michele; Pellicanò, Mariavaleria; Manzoni, Martina; Fabris, Sonia; Airoldi, Irma; Valdora, Francesca; Ferrini, Silvano; Gentile, Massimo; Vigna, Ernesto; Bossio, Sabrina; De Stefano, Laura; Palummo, Angela; Iaquinta, Giovanni; Cardillo, Martina; Zupo, Simonetta; Cerruti, Giannamaria; Ibatici, Adalberto; Neri, Antonino; Fais, Franco; Ferrarini, Manlio; Morabito, Fortunat

Determination of a BCR-ABL^IS cut-off level.

<p>Receiver operator curve (ROC) analysis was used in 238 samples to determine the best BCR-ABL1^IS cut-off level [1.68% (AUC = 0.97, <i>P</i><0.001)] able to discriminate cases with a positive from negative FCBA result.</p

Comparison of Flow-Cytometric Bead Assay (FCBA) vs. RQ-PCR for Detection of BCR-ABL1 Expression in CML Follow-Up.

<p>N = 150 serial samples, belonging to 54 CML in follow-up* (*note that 1 CML patient with p230 transcripts was not included in this analysis).</p><p>Abbreviations: FCBA, Flow-cytometric Bead Assay, CHR = complete hematologic remission, CCyR = complete cytogenetic remission, MMR = major molecular response; (RQ-PCR) = quantitative real-time PCR; ELN = European Leukemia Net [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130360#pone.0130360.ref008" target="_blank">8</a>]</p><p>Comparison of Flow-Cytometric Bead Assay (FCBA) vs. RQ-PCR for Detection of BCR-ABL1 Expression in CML Follow-Up.</p

Comparison of FISH analysis and BCR-ABL1 Flow-cytometry Bead Assay on CML samples (N = 105 serial samples in 37 patients).

<p>* FCBA = Flow-cytometry Bead Assay; rMFI% range, 0.968–8.89; %BCR-ABL1^IS range 0.313–10.258, with 5/6 patients with %BCR-ABL1^IS ≥1%.</p><p>Comparison of FISH analysis and BCR-ABL1 Flow-cytometry Bead Assay on CML samples (N = 105 serial samples in 37 patients).</p

Detection limit using FCBA BCR-ABL1 protein serial dilution curve.

<p>The CML cell line K562 was diluted in the BCR-ABL-negative cell line, HL60. Negative cut-off median PE value for this series was 105. Assay sensitivity was compared with real-time quantitative PCR (RQ-PCR) of BCR-ABL1 transcripts for each dilution and standardized according to the International scale (IS). The protein assay sensitivity indicated a loss of sensitivity of the FCBA assay at a dilution between 0.1 and 0.01% K562 which corresponds to a %BCR-ABL1/ABL^IS mRNA ratio between 0.054 and 0.60%.</p

Screening clinical samples for BCR-ABL1 protein.

<p>A total of 278 cellular samples were screened for the presence of BCRABL1 protein using the FCBA assay; results are stratified by the Relative MFI% BCR-ABL. The cut-off MFI value of the Negative Control group was calculated as the mean MFI plus two standard deviations, this value was re-calculated for each test kit utilized. Relative %MFI (rMFI%) was then calculated as [(MFI_Sample−MFI_Normal cut-off) / MFI_Normal cut-off] × 100 in order to compare data across kits. A relative MFI%>1 indicates a positive test. Twenty-three samples from other hematological disorders (including 4 AML), 14 suspected ALL and 88 samples from patients with neutrophilia and/or thrombocytosis suspected of CML diagnosis; and 155 CML serial follow-up samples were studied. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130360#pone.0130360.s001" target="_blank">S1 Table</a> for patient details.</p