Enrico Gottardi

Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – a Europe against cancer program

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study

PURPOSE Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. PATIENTS AND METHODS Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. RESULTS Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). CONCLUSION Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.

Impact of Baseline BCR-ABL Mutations on Response to Nilotinib in Patients With Chronic Myeloid Leukemia in Chronic Phase

PURPOSE Nilotinib is a second-generation tyrosine kinase inhibitor indicated for the treatment of patients with chronic myeloid leukemia (CML) in chronic phase (CP; CML-CP) and accelerated phase (AP; CML-AP) who are resistant to or intolerant of prior imatinib therapy. In this subanalysis of a phase II study of nilotinib in patients with imatinib-resistant or imatinib-intolerant CML-CP, the occurrence and impact of baseline and newly detectable BCR-ABL mutations were assessed. PATIENTS AND METHODS Baseline mutation data were assessed in 281 (88%) of 321 patients with CML-CP in the phase II nilotinib registration trial. RESULTS Among imatinib-resistant patients, the frequency of mutations at baseline was 55%. After 12 months of therapy, major cytogenetic response (MCyR) was achieved in 60%, complete cytogenetic response (CCyR) in 40%, and major molecular response (MMR) in 29% of patients without baseline mutations versus 49% (P = .145), 32% (P = .285), and 22% (P = .366), respectively, of patients with mutations. Responses in patients who harbored mutations with high in vitro sensitivity to nilotinib (50% inhibitory concentration [IC(50)] <or= 150 nM) or mutations with unknown nilotinib sensitivity were equivalent to those responses for patients without mutations (not significant). Patients with mutations that were less sensitive to nilotinib in vitro (IC(50) > 150 nM; Y253H, E255V/K, F359V/C) had less favorable responses, as 13%, 43%, and 9% of patients with each of these mutations, respectively, achieved MCyR; none achieved CCyR. CONCLUSION For most patients with imatinib resistance and with mutations, nilotinib offers a substantial probability of response. However, mutational status at baseline may influence response. Less sensitive mutations that occurred at three residues defined in this study, as well as the T315I mutation, may be associated with less favorable responses to nilotinib.

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580–5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients.

In order to verify if quantitative assessment of the WT1 transcript amount by the real time quantitative PCR (RQ-PCR) can be used as a marker for minimal residual disease detection, the WT1 transcript amount was determined in BM and PB samples of patients with myeloid and lymphoid acute leukemia, in normal controls, in regenerating bone marrow samples and in purified CD34-positive cells from normal subjects. In 10 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment, we performed a simultaneous analysis of the WT1 and of the fusion-gene transcript at sequential time intervals during follow-up. Sequential WT1 analysis was also performed in five AML patients lacking additional molecular markers. The data obtained show that normal and regenerating BM samples and purified CD34-positive cells consistently express minimal amounts of WT1 transcript and that this is extremely low and frequently undetectable in normal PB. By contrast, high levels of WT1 expression are present in the BM and PB samples of all acute leukemia (AL) cases at diagnosis. The WT1 levels during follow-up were found to follow the pattern of the other molecular markers (fusion gene transcripts) used for MRD monitoring and increased WT1expression in the BM and/or PB during follow-up of AL patients was always found to be predictive of an impending hematological relapse.

The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRα-positive hypereosinophilic syndrome. Results of a multicenter prospective study

Background and Objectives The hypereosinophilic syndrome (HES) may be associated with the fusion of the platelet derived growth factor receptor α (PDGFRα) gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated PDGFRα tyrosine kinase. These cases with FIP1L1-PDGFRα rearrangement have been reported to be very sensitive to the tyrosine kinase inhibitor imatinib mesylate. Design and Methods A prospective multicenter study of idiopathic or primary HES was established in 2001 (Study Protocol Registration no. NCT 0027 6929). One hundred and ninety-six patients were screened, of whom 72 where identified as having idiopathic or primary HES and 63 were treated with imatinib 100 to 400 mg daily. Results Twenty-seven male patients carried the FIP1L1-PDGFRα rearrangement. All 27 achieved a complete hematologic remission (CHR) and became negative for the fusion transcripts according to reverse transcriptase polymerase chain reaction (RT-PCR) analysis. With a median follow-up of 25 months (15–60 months) all 27 patients remain in CHR and RT-PCR negative, and continue treatment at a dose of 100 to 400 mg daily. In three patients imatinib treatment was discontinued for few months, the fusion transcript became rapidly detectable, and then again undetectable upon treatment reassumption. Thirty-six patients did not carry the rearrangement; of these, five (14%) achieved a CHR, which was lost in all cases after 1 to 15 months. Interpretation and Conclusions All patients meeting the criteria for idiopathic or primary HES should be screened for the FIP1L1-PDGFRα rearrangement. For all patients with this rearrangement, chronic imatinib treatment at doses as low as 100 mg daily ensures complete and durable responses.

Significant Correlation Between the Degree of WT1 Expression and the International Prognostic Scoring System Score in Patients With Myelodysplastic Syndromes

PURPOSE To determine whether pattern of WT1 gene expression is a useful marker for establishing prognosis and tracking disease progression in patients with myelodysplastic syndromes (MDS). PATIENTS AND METHODS We performed a quantitative assessment of the WT1 transcript amount by real-time quantitative polymerase chain reaction (RQ-PCR) in 173 samples (131 bone marrow samples and 42 peripheral-blood samples) from 131 patients with MDS (79 patients with refractory anemia [RA], 31 with RA with excess blasts [RAEB], 18 with secondary acute myeloid leukemia [s-AML] evolved from MDS, and three with deletion of 5q as the sole cytogenetic abnormality). Values obtained were correlated with the blast percentage and International Prognostic Scoring System (IPSS) score. RESULTS Sixty-five percent of BM and 78% of PB samples for RA and 100% of BM and PB samples of RAEB and s-AML expressed WT1 transcript amounts greater than the level observed in healthy volunteers. The degree of WT1 expression was highly correlated with the type of MDS, was much higher in RAEB and s-AML compared with RA, and increased during disease progression. Moreover, a significant correlation was found between WT1 expression levels, blast cell percentage, and the presence of cytogenetic abnormalities. Therefore, we found a significant correlation between the amount of WT1 transcripts and the IPSS score, which currently represents the most reliable risk index of disease progression available for MDS patients. CONCLUSION WT1 is a useful molecular marker for risk assessment in MDS patients.

Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

Assessment of minimal residual disease (MRD) in CBFbeta/MYH11 -positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts

The inv(16)(p13q22) chromosomal rearrangement associated with FAB M4Eo acute myeloid leukemia (AML) subtype is characterized by the presence of the CBFbeta/MYH11 fusion transcript that can be used to detect minimal residual disease (MRD). However, qualitative RT-PCR studies of MRD have so far produced conflicting results and seem of limited prognostic value. We have evaluated retrospectively MRD in a large series of CBFbeta/MYH11-positive patients employing both qualitative and quantitative (real-time PCR) approaches. 186 bone marrow samples from 36 patients were examined with a median follow-up of 27.5 months; 15 patients relapsed during follow-up. In qualitative studies, carried out by ‘nested’ RT-PCR assay, all patients in complete remission (CR) immediately after induction/consolidation therapy were found to be PCR positive. However, follow-up samples at later time points were persistently negative (except one case) in patients remaining in continuous CR (CCR) for more than 12 months. 16 patients were evaluated by quantitative real-time PCR assay: CBFbeta/MYH11 transcript copy number was normalized for expression of the housekeeping gene ABL, expressed as fusion gene copy number per 104 copies of ABL. A 2–3 log decline in leukemic transcript copy number was observed after induction/consolidation therapy. After achieving CR, the mean copy number was significantly higher in patients destined to relapse compared to patients remaining in CCR (151 vs 9, P < 0.0001 by Mann–Whitney test). Moreover, in CCR patients, the copy number dropped below the detection threshold after the treatment protocol was completed and remained undetectable in subsequent MRD analysis in accordance with results obtained by qualitative RT-PCR. On the contrary, in the seven patients who relapsed, the copy number in CR never declined below the detection threshold; thus a cut-off value discriminating these two groups of patients could be established. The findings of our study, if confirmed, might confer an important predictive value to quantitative real-time PCR determinations of MRD in patients with inv(16) leukemia.

Harmonization of BCR-ABL mRNA quantification using a uniform multifunctional control plasmid in 37 international laboratories

Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-β (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.