Molecular monitoring of the BCR‐ABL1 transcript for patients with chronic phase chronic myeloid leukemia (CML) has become increasingly demanding. Real‐time quantitative PCR (qPCR) is the routinely ...used method, but has limitations in quantification accuracy due to its inherent technical variation. Treatment recommendations rely on specific BCR‐ABL1 values set at timed response milestones, making precise measurement of BCR‐ABL1 a requisite. Furthermore, the sensitivity of qPCR may be insufficient to reliably quantify low levels of residual BCR‐ABL1 in patients in deep molecular response (DMR) who could qualify for an attempt to discontinue Tyrosine Kinase Inhibitor (TKI) therapy. We reviewed the current use of digital PCR (dPCR) as a promising alternative for response monitoring in CML. dPCR offers an absolute BCR‐ABL1 quantification at various disease levels with remarkable precision and a clinical sensitivity reaching down to at least MR5.0. Moreover, dPCR has been validated in multiple studies as prognostic marker for successful TKI treatment discontinuation, while this could not be achieved using classical qPCR. dPCR may thus prospectively be the preferred method to reliably identify patients achieving treatment milestones after initiation of TKI therapy as well as for the selection and timing for TKI discontinuation.
Treatment with PEGylated interferon‐alpha2 (IFN) of patients with essential thrombocythemia and polycythemia vera induces major molecular remissions with a reduction in the JAK2V617F allele burden to ...undetectable levels in a subset of patients. A favorable response to IFN has been argued to depend upon the tumor burden, implying that institution of treatment with IFN should be as early as possible after the diagnosis. However, evidence for this statement is not available. We present a thorough analysis of unique serial JAK2V617F measurements in 66 IFN‐treated patients and in 6 untreated patients. Without IFN treatment, the JAK2V617F allele burden increased exponentially with a period of doubling of 1.4 year. During monotherapy with IFN, the JAK2V617F allele burden decreased mono‐ or bi‐exponentially for 33 responders of which 28 patients satisfied both descriptions. Bi‐exponential description improved the fits in 19 cases being associated with late JAK2V617F responses. The decay of the JAK2V617F allele burden during IFN treatment was estimated to have half‐lives of 1.6 year for the monoexponential response and 1.0 year in the long term for the bi‐exponential response. In conclusion, through data‐driven analysis of the JAK2V617F allele burden, we provide novel information regarding the JAK2V617F kinetics during IFN‐treatment, arguing for early intervention.
Data‐driven mathematics of the kinetics of the JAK2V617F allele burden during treatment with IFN is presented. The half‐life of the JAK2V617F allele burden was predicted as 1.6 year, and 1.0 year in the long term, supporting early treatment with IFN.
As supplement to KRAS mutational analysis, BRAF and PIK3CA mutations as well as expression of PTEN may account for additional non-responders to anti-EGFR-MoAbs treatment. The aim of the present study ...was to investigate the utility as biomarkers of these mutations in a uniform cohort of patients with metastatic colorectal cancer treated with third-line cetuximab/irinotecan.
One-hundred-and-seven patients were prospectively included in the study. Mutational analyses of KRAS, BRAF and PIK3CA were performed on DNA from confirmed malignant tissue using commercially available kits. Loss of PTEN and EGFR was assessed by immunohistochemistry.
DNA was available in 94 patients. The frequency of KRAS, BRAF and PIK3CA mutations were 44%, 3% and 14%, respectively. All were non-responders. EGF receptor status by IHC and loss of PTEN failed to show any clinical importance. KRAS and BRAF were mutually exclusive. Supplementing KRAS analysis with BRAF and PIK3CA identified additional 11% of non-responders. Patient with any mutation had a high risk of early progression, whereas triple-negative status implied a response rate (RR) of 41% (p<0.001), a disease control (DC) rate of 73% (p<001), and a significantly higher PFS of 7.7(5.1-8.6 95%CI) versus 2.3 months (2.1-3.695%CI) (p<0.000).
Triple-negative status implied a clear benefit from treatment, and we suggest that patient selection for third-line combination therapy with cetuximab/irinotecan could be based on triple mutational testing.
Purpose
Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment ...monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in cfDNA during neoadjuvant chemotherapy.
Patients and methods
The study included 50 patients from a prospective cohort analyzed during neoadjuvant chemotherapy. Fifty healthy women with no history of cancer served as control group and 15 patients with metastatic breast cancer were used to validate the assay. Total cfDNA and HER2 gene amplification were measured by quantitative real-time polymerase chain reaction.
Results
Plasma HER2 gene copy number (
p
= 0.794), HER2 gene amplification (
p
= 0.127) and total cfDNA (
p
= 0.440) did not differ significantly from the levels in the control group. Eighteen patients (36 %) obtained pathological complete response (pCR). HER2 gene copy number before the operation was significantly higher than the baseline level (
p
< 0.0001), but there was no difference between patients with and without pCR (
p
= 0.569). Likewise, there was no difference in plasma HER2 gene amplification between tissue HER2-positive and -negative patients (
p
= 0.754).
Conclusions
The results indicate that neither total cfDNA nor HER2 gene copy number is elevated in primary breast cancer patients compared to healthy controls. The level of both parameters increased during neoadjuvant chemotherapy, but without any relation to treatment effect. There was no indication of plasma HER2 gene amplification in the HER2-positive patients in the neoadjuvant setting.
We have developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) reaction, which enables us to detect 29 translocations/chromosomal aberrations in patients with acute lymphoid ...leukemia (ALL) and acute myeloid leukemia (AML). Through the construction and optimization of specific primers for each translocation, we have been able to reduce the set-up to 8 parallel multiplex PCR reactions, thus greatly decreasing the amount of work and reagents. We show the value of our set-up in a retrospective analysis on cryopreserved material from 102 AML and 62 ALL patients. The multiplex RT-PCR detected a hybrid mRNA resulting from a structural chromosomal aberration in 45 of 102 (44%) of the AML and in 28 of 62 (45%) of the pediatric ALL cases. Importantly, in 33% of AML and in 47% of the ALL cases with cytogenetic data, submicroscopic chromosomal aberrations or masked translocations were shown that were not detected in the cytogenetic analysis either for structural reasons or because of an insufficient number of metaphases obtained. This multiplex RT-PCR system, which can handle up to 10 patients with a response time of 2 working days, is thus an important tool that complements cytogenetic analysis in the up-front screening of acute leukemia patients and should provide a rapid and efficient characterization of leukemia cells, even in situations with sparse patient material.
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.
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.
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 = 0.85, P < 10(-4)). The disease control rate was 77% in patients with low cfDNA (<25% quartile) and 30% in patients with high cfDNA >75% quartile (P = 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 = 0.048). Cox analysis confirmed the prognostic importance of both cfDNA and pmKRAS. High levels were clear indicators of a poor outcome.
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.
To develop an affordable and robust pipeline for selection of patient-specific somatic structural variants (SSVs) being informative about radicality of the primary resection, response to adjuvant ...therapy, incipient recurrence and response to treatment performed in relation to diagnosis of recurrence.
We have established efficient procedures for identification of SSVs by next-generation sequencing and subsequent quantification of 3-6 SSVs in plasma. The consequence of intratumour heterogeneity on our approach was assessed. The level of circulating tumour DNA (ctDNA) was quantified in 151 serial plasma samples from six relapsing and five non-relapsing colorectal cancer (CRC) patients by droplet digital PCR, and correlated to clinical findings.
Up to six personalised assays were designed for each patient. Our approach enabled efficient temporal assessment of disease status, response to surgical and oncological intervention, and early detection of incipient recurrence. Our approach provided 2-15 (mean 10) months' lead time on detection of metastatic recurrence compared to conventional follow-up. The sensitivity and specificity of the SSVs in terms of detecting postsurgery relapse were 100%.
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.
Objectives and background:
Clinical studies are currently initiated where treatment of CML patients with tyrosine kinase inhibitors is stopped. One of the main criteria for patients to be eligible ...for these studies is that they must have achieved a molecular response of MR4.0. However, in order to obtain a reliable molecular measurement at the MR4.0 sensitivity level a higher sensitivity is needed in the analysis. We have tested a multiplex droplet digital PCR (ddPCR) approach in order to obtain reliable measurements at the MR5.0 level. When using a standard laboratory protocol with three reference genes and singleplex qPCR, only ¼ of the sample is tested for BCR-ABL1 transcripts. By using multiplex PCR the entire sample can be tested. The advantage of ddPCR is that an absolute concentration is measured and there is no need for calibration or standardisation of the analysis.
Methods:
Blood samples stabilized in Paxgene tubes from 36 CML patients were used for the study. Samples were analysed according to the standard laboratory protocol with qPCR using B2M, BCR and GUSB as reference genes. For the study presented here RNA was purified from 5 million cells using the QiaSymphony RNA kit (Qiagen). 2.5 ug of RNA was used for cDNA synthesis using SuperScript VILO cDNA synthesis kit (LifeTechnologies). Samples were analysed with multiplex dd PCR on the QX100 system (Biorad). The WHO reference genes BCR and GUSB were used as reference. Eight wells were analysed for BCR-ABL1 (FAM labelled probe). Four of the wells were also analysed for BCR and four were analysed for GUSB (HEX labelled probes).
Results:
The median number of GUSB transcripts in the samples was 605,000 copies (range 195,520-934,400). According to the EUTOS “Working definitions of Molecular Response in CML” (ref), MR5.0 sensitivity is obtained when analysing 240,000 GUSB transcripts. All samples except one were above this level. MR5.5 is obtained when analysing 759,000 GUSB transcripts and 20% of the samples (7/36) reached this level. The ddPCR results were compared to the qPCR results and a high concordance was seen in the positive samples (r2=0.94). 21 samples were positive by qPCR, and an additional 4 samples were positive by ddPCR.
Conclusions:
Using the multiplex ddPCR protocol developed 97% of the analysed samples had a sensitivity of MR5.0 or better. Compared to the labs routine qPCR protocol 20% more samples were found positive with ddPCR. Due to the multiplex set-up of the ddPCR the entire cDNA sample can be tested for BCR-ABL1 transcripts. Contrary to qPCR there is no need for calibration of the ddPCR analysis and the method has potential for routine use when a high sensitivity is needed.
References:
“Working definitions of Molecular Response in CML”, EUTOS Molecular Monitoring Steering Group, Nick Cross, Martin Müller, Fabrizio Pane, Andreas Hochhaus, November 2013.
No relevant conflicts of interest to declare.
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