Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqManbased real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n ¼ 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic Correspondence: Professor J

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 ¼ 126) and diagnostic leukemic (n ¼ 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-PCRbased 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.

We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

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.

Purpose: The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma.Experimental Design: Inclusion criteria were histopathologically verified chemotherapy-resistant mCRC, adequate Eastern Cooperative Oncology Group performance status, and organ function. Treatment consisted of irinotecan being administered at 350 mg/m 2 for 3 weeks and weekly administration of 250 mg/m 2 cetuximab until progression or unacceptable toxicity. A quantitative PCR method was developed to assess the number of cfDNA alleles and KRAS and BRAF mutation alleles in plasma at baseline. Results: The study included 108 patients. Only three patients were positive for BRAF mutations. The majority of KRAS mutations detected in tumors were also found in the plasma [32 of 41 (78%)]. Plasma cfDNA and plasma mutant KRAS levels (pmKRAS) were strongly correlated (r ¼ 0.85, P < 10 À4). The disease control rate was 77% in patients with low cfDNA (<25% quartile) and 30% in patients with high cfDNA [>75% quartile (P ¼ 0.009)]. Patients with pmKRAS levels higher than 75% had a disease control rate of 0% compared with 42% in patients with lower pmKRAS (P ¼ 0.048). Cox analysis confirmed the prognostic importance of both cfDNA and pmKRAS. High levels were clear indicators of a poor outcome.Conclusions: KRAS analysis in plasma is a viable alternative to tissue analysis. Quantitative levels of cfDNA and pmKRAS are strongly correlated and hold promise of clinical application.

The JAK2 V617F and calreticulin mutations (CALR) are frequent within myeloproliferative neoplasms (MPNs). JAK2 V617F has been detected in the general population, but no studies have previously investigated the CALR prevalence. Thus, we aimed to determine the CALR and JAK2 V617F population prevalence and assess the biochemical profile and lifestyle factors in mutation-positive individuals with and without MPN. 19 958 eligible participants, enrolled from 2010-2013, from the Danish General Suburban Population Study were screened for JAK2 V617F and CALR by droplet digital polymerase chain reaction with (3.2%) mutation positives of which 16 (2.5%) had MPN at baseline. Of 645 participants, 613 were JAK2 V617F positive, and 32 were CALR positive, corresponding to a population prevalence of 3.1% (confidence interval [CI], 2.8-3.3) and 0.16% (CI, 0.11-0.23), respectively. Increasing age, smoking, and alcohol were risk factors for the mutations. JAK2 V617F positives with and without MPN presented elevated odds for prevalent venous thromboembolism. The odds ratio for a diagnosis of MPN per percentage allele burden was 1.14 (95% CI, 1.09-1.18; P = 1.6 × 10−10). Mutation positives displayed higher blood cell counts than nonmutated participants, and 42% of mutation positives without MPN presented elevation of ≥1 blood cell counts; 80 (13%) even presented blood cell counts in accordance with current MPN diagnostic criteria. In conclusion, we present a novel population prevalence of CALR and a JAK2 V617F prevalence that is 3 to 30 times higher compared with less sensitive methods. Mutation-positive non-MPNs with elevated blood cell counts raise concerns of MPN underdiagnosis in the population.

Summary The JAK2 V617F mutation is a frequent genetic event in the three classical Philadelphia‐chromosome negative chronic myeloproliferative disorders (Phneg.‐CMPD), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Its occurrence varies in frequency in regards to phenotype. The mutation is found in the majority of patients with PV and about half of the patients with ET and IMF. These diseases are clonal stem cell disorders arising in an early stem cell progenitor. The level in the stem cell hierarchy on which the initiating genetic events and the JAK2 V617F mutation occurs is not known. The mutation has so far been detected in all cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In this study, we detected the JAK2 V617F mutation by real‐time quantitative PCR (qPCR) in both B‐lymphocytes and T‐lymphocytes in a subgroup of patients with Phneg.‐CMPDs. These results demonstrate the origin of the JAK2 V617F positive disorders in an early stem cell with both lymphoid and myeloid differentiation potential.

The present study support the concept of the JAK2 V617F positive chronic myeloproliferative disorders as a biological continuum with phenotypic presentation in part influenced by JAK2 V617F mutational load.