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Simple Summary The introduction to clinical practice of a treatment-free remission approach in chronic myeloid leukemia patients with a stable deep molecular response highlighted how crucial it is to monitor the molecular levels of BCR–ABL1 as accurately and precisely as possible. In this context, the droplet digital PCR (ddPCR) presents an alternative methodology for such quantification. To hypothesize the introduction of this technology in routine practice, we performed a multicentric study that compares ddPCR with the standard methodology currently used. Our results demonstrate that the use of ddPCR in clinical practice is feasible and could be beneficial. Abstract BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTM BCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.

Philadelphia positive acute lymphoblastic leukemia is well documented nowadays but very little is known about Philadelphia positive lymphoblastic lymphoma (LBL). Only two cases are available in literature and both of them died during treatment whereas the patient treated in our center is still alive 3 years after the initial diagnosis. A chemo-free regimen was used in induction with dasatinib plus steroids with local radiotherapy on the mass, and then the patient underwent bone marrow transplant. Philadelphia positive lymphoblastic lymphoma is a difficult diagnosis to make and the management of this extremely rare disease is very challenging.

More than 15 years ago, imatinib entered into the clinical practice as a “magic bullet”; from that point on, the prognosis of patients affected by chronic myeloid leukemia (CML) became comparable to that of aged-matched healthy subjects. The aims of treatment with tyrosine kinase inhibitors (TKIs) are for complete hematological response after 3 months of treatment, complete cytogenetic response after 6 months, and a reduction of the molecular disease of at least 3 logs after 12 months. Patients who do not reach their goal can switch to another TKI. Thus, the molecular monitoring of response is the main consideration of management of CML patients. Moreover, cases in deep and persistent molecular response can tempt the physician to interrupt treatment, and this “dream” is possible due to the quantitative PCR. After great international effort, today the BCR-ABL1 expression obtained in each laboratory is standardized and expressed as “international scale.” This aim has been reached after the establishment of the EUTOS program (in Europe) and the LabNet network (in Italy), the platforms where biologists meet clinicians. In the field of quantitative PCR, the digital PCR is now a new and promising, sensitive and accurate tool. Some authors reported that digital PCR is able to better classify patients in precise “molecular classes,” which could lead to a better identification of those cases that will benefit from the interruption of therapy. In addition, digital PCR can be used to identify a point mutation in the ABL1 domain, mutations that are often responsible for the TKI resistance. In the field of resistance, a prominent role is played by the NGS that enables identification of any mutation in ABL1 domain, even at sub-clonal levels. This manuscript reviews how the molecular tools can lead the management of CML patients, focusing on the more recent technical advances.

Background: Monitoring of BCR-ABL1 molecular levels is essential for the management of Chronic Myeloid Leukemia (CML) patients treated with Tyrosine Kinases Inhibitors (TKIs). Real Time Quantitative PCR (RT-qPCR) is currently the standard method for assessing molecular remission (MR) in patients with CML. Recently, droplet digital PCR (ddPCR) has emerged to provide a more accurate detection of minimal residual disease (MRD). In order to hypothesize the use of this new technology in the clinical practice, in the era of TKI discontinuation, we designed a multi-centric study to evaluate the potential value of ddPCR in diagnostic routine. Aims of the study were:1) the evaluation of the agreement between the measures obtained by ddPCR and RT-qPCR and 2) the assessment of the repeatability of the two methods. Methods: Total RNA was extracted from 37 CML patients using Maxwell 16 LEV simplyRNA Blood kit (Promega), following the manufacturer's instructions. Samples were divided in 5 groups based on molecular response (MR) as follow: group 1, MR Statistical analysis: Bland-Altman analysis was performed. For the measurement of the agreement we reported the bias, which is the mean of the difference between the methods, the 95% limits of agreement and the coefficient of variation. Residual variance and coefficients of repeatability (i.e. the upper limits of a prediction interval for the absolute difference between two measurements by the same method on the same item) were computed to achieve the second endpoint. An analysis of sensitivity on the labs was also carried out. All analyses were stratified by the level of disease. Results: A total of 370 measures were included in the analysis, 185 for ddPCR and 185 for RT-qPCR divided as follow: 50 for group 1 and for group 2, 90 for group 3, 4 and 5. In Table 1 we reported the median and interquartile range (IQR) for all levels of disease. Results of the Bland-Altman analysis are shown in Table 2. The coefficients of variation, which expresses the standard deviation as a percentage of the mean, were 2.35, 2.31, 1.10, 1.34, 39.12 in group 1, 2, 3, 4 and 5 respectively. The repeatability coefficients of ddPCR were smaller than qRT-PCR across all the levels of disease, showing a slightly better precision of ddPCR (Table 2). Conclusions: Higher coefficients of variation in group 1 and 2 were probably due to a greater heterogeneity of the patients. In fact, BCR-ABL1/ABL1 levels by RT-qPCR ranged between 1.43 and 6.94 and between 0.10 and 0.25 in group 1 and in group 2 respectively (Table 1). Sensitivity analysis showed that the high coefficient of variation in group 5 can be explained almost all by the variability observed in Lab B. Coefficient of repeatability of ddPCR was always smaller than RT-qPCR for all level of disease showing a slightly better precision. Our results showed that ddPCR has a good agreement with RT-qPCR and it is more precise to quantify BCR-ABL1 transcript levels, particularly for MR 4 and MR 4.5. Thus, ddPCR may be valuable in diagnostic routine. Disclosures Fava: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Martino:Bio-Rad: Employment. Saglio:Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy. Martinelli:Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Real-time quantitative PCR (RT-qPCR) is commonly used for follow-up of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors, but its current sensitivity does not allow detection of very low BCR-ABL levels. Therefore RT-qPCR negativity is not synonymous with complete molecular response. Replicate RT-qPCR had shown increased sensitivity in tyrosine kinase inhibitor–treated patients and was, therefore, used here to evaluate whether RT-qPCR–negative post–allogeneic stem cell transplantation (SCT) patients harbor detectable disease. Samples from 12 patients were tested at 2 time points using 82 replicates of BCR-ABL RT-qPCR. One patient (38 months after SCT) had detectable transcripts at baseline and none at the follow-up test, done at a median of 107 months after SCT. This suggests cure from CML in the majority of allogeneic SCT patients who have no transcripts detectable by replicate RT-qPCR for BCR-ABL.

An early molecular response has a strong predictive value in chronic myeloid leukemia (CML). Recently, the halving time (velocity of early BCR-ABL1 transcript elimination) has been shown to represent an additional prognostic index. Our objective was the evaluation of the prognostic significance of the 3-month point in our population. We retrospectively collected BCR-ABL1 transcript data at different time points, events, and survival data of patients with CML treated at the Division of Hematology, San Luigi Hospital, University of Turin, Turin, Italy. Of 71 patients diagnosed from January 2005 to March 2015 in our center and treated with front-line tyrosine kinase inhibitors (imatinib, nilotinib and dasatinib), we selected those who had undergone a molecular evaluation at 3 months. The event-free survival (EFS) by the median follow-up time was the primary endpoint. The data from 50 patients with CML chronic phase were analyzed. Overall, 34 of the 50 patients (68%) had a transcript ≤ 10% at 3 months. Of those in the10% group, 63% had experienced an event compared with 12% in the ≤ 10% group by the median follow-up point (P .001). The halving time threshold for discriminating between EFS was 17 days. None of the patients with a transcript10% at 3 months had a halving time of ≤ 17 days. Patients with BCR-ABL1 ≤ 10% and a halving time of ≤ 17 days had significantly better EFS than that of patients with BCR-ABL1 ≤ 10% and a halving time17 days and of patients with BCR-ABL110% (96% group 1 vs. 60% group 2 vs. 27% group 3; P .001). Irrespective of the tyrosine kinase inhibitor used, the prognosis was significantly superior for patients with BCR-ABL1 ≤ 10% and halving time of ≤ 17 days. Our data revealed that the use of ABL1 as a control gene is reliable for the determination of the halving time in the clinical setting and highlight the importance of measuring the BCR-ABL1 transcript at CML diagnosis.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR 1 -MR 4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR 4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR 4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Effective management of tyrosine kinase inhibitor (TKI) therapy for patients with CML requires frequent patient testing to monitor patient disease status. The gold standard for this testing is a PCR measurement of the BCR-ABL/ABL transcript ratio. Hence, standardized, accurate and reproducible molecular analyses are fundamental for clinicians in order to correctly address therapeutic decision and to achieve a better patient management. Based on these clinical needs, it is widely recognized that inter-laboratory reproducibility is a crucial issue that requires standardization and strict alignment of BCR-ABL values on the international scale (IS), as established by the International Randomized Study of Interferon and STI571 (IRIS) laboratories. In addition to this, another important aspect in terms of patient management is the availability of a rapid response, improving patient quality of life by reducing anxiety linked to delay in results delivery. Xpert® BCR-ABL Monitor, developed and manufactured by Cepheid (Sunnyvale, CA), is a cartridge-based automated assay for quantification of BCR-ABL1 mRNA, reporting results in International Scale (IS). Alignment to the IS, based upon the quality control standards derived from the WHO BCR-ABL Standards, is performed lot to lot automatically by the software of the Cepheid GeneXpert® DX Instrument. Xpert® BCR-ABL Monitor automates and integrates sample processing, nucleic acids extraction and amplification and target sequence detection in peripheral blood specimens collected either in EDTA or PAXgene tubes using real-time RT-PCR. The cartridge includes reagents to detect BCR-ABL fusion genes resulting from two major breakpoints, translocation e13a2 (b2a2) and e14a2 (b3a2) and the ABL transcript as an endogenous control. In the following study, Xpert® BCR-ABL Monitor was independently evaluated in the three Italian reference centers for BCR-ABL mRNA monitoring of the LabNet project. A total of 150 peripheral blood samples from CML patients, equally divided between centers, were analyzed both with the standard laboratory method and with Xpert® BCR-ABL Monitor Assay, in order to compare the results expressed in International Scale. BCR-ABL mRNA of the specimens covered a broad IS range, allowing to compare data also at low levels of disease (MR 4.5, 0.00316% BCR-ABL/ABL). Overall, linear regression demonstrated that there was a good correlation between Xpert® BCR-ABL Monitor and reference centers data (R2= 0.92). Correlation slightly increased when the data produced with Xpert® BCR-ABL Monitor were refined using a correction tool not automatically adopted by the current version of the assay software (R2= 0.93). Moreover, using assay comparison criteria proposed by Müller et al. (Leukemia 2009), Xpert® BCR-ABL Monitor was considered comparable to the standard laboratory methods of the reference centers, considering that all the three acceptance criteria were satisfied (58% of the samples between 0.5 and 2-fold variation, 78% of the samples between 0.33 and 3-fold variation, 91% of the samples between 0.2 and 5-fold variation). In conclusion, Xpert® BCR-ABL Monitor demonstrated to be a rapid, reliable and accurate test for monitoring of BCR-ABL mRNA transcript levels. Results were available within approximately 2 hours, potentially allowing clinicians to report results to patient the same day of the visit. From the technical point of view, cartridge-based automated system significantly reduced operator hands-on time from several hours to approximately 15 minutes. Concerning the assay comparative performance, Xpert® BCR-ABL showed to have a good inter-laboratory reproducibility, with no significant differences between the three reference centers standard methods. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Saglio: NOVARTIS: Consultancy. Pane:NOVARTIS: Consultancy, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Current diagnostic criteria for Philadelphia-negative myeloproliferative neoplasia (MPN) have been redefined by the discovery of Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL) and calreticulin (CALR) genetic alterations. Only few cases of coexistence of CALR-mutated MPN and Philadelphia-positive chronic myeloid leukemia (CML) have been described so far. Here we report the case of a patient with CML diagnosed in 2001, treated with imatinib and pegylated interferon (IFN) frontline. She reached complete molecular remission (CMR) and discontinued imatinib, maintaining treatment free remission. Due to persistent thrombocytosis, we repeated bone marrow (BM) analysis and diagnosed CARL-mutated essential thrombocythemia (ET). A CALR-positive clone was found to be present since 2001, and was unaffected by imatinib treatment, possibly representing a molecular abnormality arising at stem cell level.

Allogeneic stem cell transplant (SCT) is thought to be the only curative therapy of Phþ acute lymphoblastic leukemia (ALL). Recent results showed that Phþ ALL with minimal residual disease (MRD) after SCT benefitted from the use of imatinib. Three patients with Phþ ALL fromour institution showed different degrees of MRD increase after transplant and were treated with second-generation tyrosine kinase inhibitors, obtaining a complete molecular remission. These cases suggest a posttransplant application of TKIs to traet or prevent the relapse of Phþ ALL.

Juvenile or type 2 hemochromatosis is a rare autosomal recessive disorder which leads to severe iron overload early in life. As in the classic adult form of the disease iron toxicity causes liver cirrhosis, cardiomyopathy, and endocrine complications, but the onset of the disease is anticipated in the second to third decades of life. Experience of this disease in children is limited. Molecular diagnosis is unfeasible because the type 2 hemochromatosis gene is still unknown, although it is known that the disease locus maps to chromosome 1q. Combining linkage analysis with markers encompassing chromosome 1 locus and a non-invasive method for liver iron quantitation we diagnosed juvenile hemochromatosis in a presymptomatic stage in an 11-year-old Italian child. A regular phlebotomy protocol reduced iron overload preventing all the disease complications. Conclusion: Juvenile hemochromatosis patients have severe iron overload within the first years of life, strengthening the greater iron absorption that occurs in this as compared to other types of hemochromatosis. Early detection is essential, because treatment in presymptomatic stages prevents organ damage.

Background Consensus has been achieved that standardized molecular quantitative analysis (RQ-PCR) on peripheral blood (PB) is a suitable method for monitoring residual disease in chronic myeloid leukemia (CML). However, BM is still obtained at specific timepoints, and in a number of cases, only bone marrow (BM) sample collected for cytogenetic analysis is available. Being one of the laboratory involved in the standardization process of molecular monitoring for CML patients, we decided to perform a comparative analysis of BM and PB samples in order to evaluate the consistency of the results. Methods Between March 2009 and January 2013, 230 consecutive RQ-PCR tests to assess BCR-ABL transcript levels from simultaneously collected PB and BM samples were performed (for a total of 460 analysis) on 77 patients affected by Ph+ CML in chronic phase treated in our center. All samples were analyzed in the same laboratory following international guidelines (Cross N, Leukemia 2012) and results were expressed according to the International Scale; ABL1 was used as control gene. Time from blood-drawn to processing was within 3-4 hours. Results Among the 230 pairs, 3 were considered as not evaluable because of inadequate material; for the purpose of this study, the remaining 227 pairs were considered as “evaluable”. 204 pairs were classified as “fit” when both BM and PB ABL amplification resulted in more than 10.000 copies; 23 pairs were considered unfit for ABL1 Conclusions In a single center experience of molecular analysis, BCR/ABL ratio was highly consistent in BM and PB samples. In less than 10% of the cases a single test did not reach the required sensitivity of 10.000 ABL copies and the double testing allowed to obtain a valid result. This may be especially valuable in evaluating an early response (i.e. at 3 months), when the amount of disease has prognostic relevance. The analysis will be expanded to include samples coming from different centers to evaluate a possible role of timing and transport on data consistency. Disclosures: Saglio: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Abstract 1690 RQ-PCR has become the major method for the follow up of CML pts in the tyrosine kinase inhibitor (TKI) era. In CML pts undergoing allogeneic SCT (Allo-SCT) it has been shown that successive increase in quantitative BCR ABL transcript levels is in correlation with the clinical outcome and may serve as an early sign of disease relapse and an indication for a therapeutic intervention such as the addition of a TKI or DLI. However, the sensitivity of current RQ-PCR methods which is limited to approximately 10−4 in the majority of routine clinical laboratories, do not allow for the detection of very low BCR ABL transcript levels, and therefore RQ-PCR negativity cannot be regarded as CMR in the majority of pts. Studies of replicate RQ-PCR (rRQ-PCR) have shown that the number of measurement repetitions is relevant in the detection of rare transcripts and rRQ-PCR was found to be more sensitive than conventional RQ-PCR in TKI treated CML pts. We evaluated the role of rRQ-PCR in the detection of MRD in a cohort of long term post Allo-SCT CML patients. Samples from 12 CML pts (M=7, F=5), median age 43 y, at a median time of 85 months after SCT (range 28–136) were tested. SCT was myeloablative (n=6) or with RIC (n=6), from a matched sibling (n=7) or a MUD (n=5), in first chronic phase (CP1) (n=8) or at a later phase (n=4). rRQ-PCR was done in 82 replicates using the same conditions of conventional RQ-PCR. Results of rRQ-PCR are given in Table 1. 75% of the patients had negative RQ-PCR in all 82 wells, while 3 patients had positive results in 1 (1.2%), 2 (2.4%) and 6 (7.3%) wells, respectively, which was above the background determined in normal PB samples (6/901 positive wells, 0.66%). The sensitivity of rRQ-PCR was 10−5.5 or higher in all patients. Negative rRQ-PCR result was not related to the phase at SCT (CP1 vs. others), conditioning regimen (myeloablative vs. RIC), donor type (sibling vs. MUD) or the presence of GVHD. Median time from diagnosis to SCT was borderline longer in patients with positive rRQ-PCR results (61 vs. 25 months, p=0.08). Higher sensitivity RQ-PCR methods are able to further define subgroups of post BMT CML pts and allow for a more accurate follow up of MRD. We will present update clinical data at 1 year after rRQ-PCR determination. Table 1. Results of rRQ-PCR. Patient number Disease status at SCT Conditioning Time from diagnosis to SCT (months) Time from SCT to rRQ-PCR (months) rRQ-PCR result BCR ABL/ABL1 (IS) Total quantity of ABL transcripts Level of MR 1 AP RIC 97 57 0 575034.8 MR5.5 2 CP2 MA 6 30 0.0000862 705622.3 3 CP2 MA 273 48 0.0007393 563237.5 4 CP1 RIC 4 52 0 997521.8 MR6 5 CP1 MA 16 34 0 555768.9 MR5.5 6 CP1 MA 39 30 0 700630.1 MR5.5 7 CP1 MA 6 40 0 874247.9 MR5.5 8 CP1 RIC 61 68 0.0000484 1067353 9 CP1 RIC 33 46 0 938046.4 MR5.5 10 CP1 RIC 18 58 0 1467748 MR6 11 AP RIC 6 28 0 776271.9 MR5.5 12 CP2 MA 3 23 0 860350.6 MR5.5 RIC reduced intensity conditioning; MA myeloablative conditioning; MR molecular response. 1 Average result of positive wells normalized to the international standard. PCR positive in 12, 63 and 24 wells of 82 tested. Disclosures: No relevant conflicts of interest to declare.