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.
•CALR mutations are prevalent in the general population but are much less frequent compared with the estimated JAK2 V617F prevalence.•JAK2 V617F and CALR mutations in the general population are linked to a distinct blood count profile, also in the absence of MPN diagnosis.
Display omitted
Circulating cell-free DNA (cfDNA) in plasma has shown potential as biomarker in various cancers and could become an importance source for tumour mutation detection. The objectives of our study were ...to establish a normal range of cfDNA in a cohort of healthy individuals and to compare this with four cohorts of metastatic colorectal cancer (mCRC) patients. We also investigated the prognostic value of cfDNA and analysed the tumour-specific KRAS mutations in the plasma.
The study was a prospective biomarker evaluation in four consecutive Phase II trials, including 229 patients with chemotherapy refractory mCRC and 100 healthy individuals. Plasma was obtained from an EDTA blood-sample, and the total number of DNA alleles and KRAS mutated alleles were assessed using an in-house ARMS-qPCR as previously described.
Median cfDNA levels were higher in mCRC compared to controls (p < 0.0001). ROC analysis revealed an AUC of 0.9486 (p<0.00001). Data showed impaired OS with increasing levels of baseline cfDNA both when categorising patients by quartiles of cfDNA and into low or high cfDNA groups based on the upper normal range of the control group (Median OS 10.2 (8.3-11.7) and 5.2 (4.6-5.9) months, respectively, HR 1.78, p = 0.0006). Multivariate analysis confirmed an independent prognostic value of cfDNA (HR 1.5 (95% CI 1.3-1.7) for each increase in the cfDNA quartile). The overall concordance of KRAS mutations in plasma and tissue was high (85%).
These data confirm the prognostic value of cfDNA measurement in plasma and utility for mutation detection with the method presented.
KRAS and BRAF mutations are responsible for primary resistance to epidermal growth factor receptor (EGFR) MoAbs in metastatic colorectal cancer (mCRC), but it is unknown what causes wildtype (wt) ...patients to develop resistance during treatment. We measured circulating free DNA (cfDNA), KRAS and BRAF in plasma and report the changes during third line treatment with cetuximab and irinotecan. One‐hundred‐and‐eight patients received irinotecan 350 mg/m2 q3w and weekly cetuximab (250 mg/m2) until progression (RECIST) or unacceptable toxicity. cfDNA and number of mutated KRAS/BRAF alleles in plasma at baseline and before each cycle was analyzed by an in‐house qPCR. cfDNA and pKRAS levels decreased from baseline to cycle three and increased at time of progression (p = 0.008). The decrease was larger in responding patients than in non‐responding (p < 0.05). Two patients with primary mutant disease had different types of mutations detected in the plasma, including synchronous KRAS and BRAF. Twelve patients had a primary KRAS mutant tumor, but wild‐type disease according to baseline plasma analysis, eight of these obtained stabilization of disease. In five patients with primary wt disease a mutation appeared in plasma before radiological evidence of progression. Loss of mutations may explain observed benefit of treatment in primary mutant disease, whereas appearance of mutations during therapy may be responsible for acquired resistance in primary wt disease. Benefit from EGFR MoAbs may be influenced by the quantitative level of mutational alleles rather than by mutational status alone, and plasma levels of cfDNA, KRAS and BRAF could be used to monitor patients during treatment.
What's new?
Mutations in the genes KRAS and BRAF contribute to chemotherapy resistance, but people whose primary tumors lack those mutations also develop resistance. Could there be a way to determine when resistance sets in, to avoid continuing an ineffective treatment? In this study, the authors sampled circulating free DNA, looking for mutations before and during treatment. They detected KRAS mutations popping up in patients who had wild‐type tumors, and these mutations heralded a turn for the worse in disease progression. Similarly, some patients had tumors that contained the mutation, but wild‐type KRAS in their plasma, and these patients responded better to treatment. Thus, circulating DNA might provide a useful indicator for how well the treatment is working.
Philadelphia-negative myeloproliferative neoplasms (MPNs) are a diverse group of diseases whose common feature is the presence of V617F mutation of the
JAK2
gene. In the era of novel therapeutic ...strategies in MPNs, such as JAK-inhibitor therapy, there is a growing need for establishing high sensitive quantitative methods, which can be useful not only at diagnosis but also for monitoring therapeutic outcomes, such as minimal residual disease (MRD). In this study, we compared the qPCR and ddPCR methods and their clinical utility for diagnosis, prognostication, and treatment monitoring of MPNs with
JAK2
V617F mutation in 63 MPN patients of which 6 were subjected to ruxolitinib treatment. We show a high conformance between the two methods (correlation coefficient
r
= 0.998 (
p
< 0.0001)). Our experiments revealed high analytical sensitivity for both tests, suggesting that they are capable of detecting the
JAK2
V617F mutation at diagnosis of MPN with a limit of detection (LoD) of 0.12% for qPCR and 0.01% for ddPCR. The alterations of
JAK2
V617F allele burden in patients treated with ruxolitinib were measured by both methods with equal accuracy. The results suggest an advantage of ddPCR in monitoring MRD because of allele burdens below the LoD of qPCR. Overall, the clinical utility of qPCR and ddPCR is very high, and both methods could be recommended for the routine detection of the V617F mutation at diagnosis, though ddPCR will probably supersede qPCR in the future due to cost-effectiveness.
We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included ...32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission was 0.85 and 0.75 for patients with polycythemia vera and myelofibrosis, respectively. The Myeloproliferative Neoplasm Symptom Assessment Form total symptom score decreased from 22 95% confidence interval (95% CI):, 16-29 at baseline to 15 (95% CI: 10-22) after 2 years. The median JAK2 V617F allele burden decreased from 47% (95% CI: 33-61%) to 12% (95% CI: 6-22%), and 41% of patients achieved a molecular response. The drop-out rate was 6% among patients with polycythemia vera and 32% among those with myelofibrosis. Of 36 patients previously intolerant of PEG-IFNα2, 31 (86%) completed the study, and 24 (67%) of these received PEG-IFNα2 throughout the study. In conclusion, combination treatment improved cell counts, reduced bone marrow cellularity and fibrosis, decreased JAK2 V617F burden, and reduced symptom burden with acceptable toxicity in several patients with polycythemia vera or myelofibrosis. #EudraCT2013-003295-12.
The purpose was to investigate total cell‐free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second‐line chemotherapy and in healthy controls and patients with different ...comorbidities. Patient treated with second‐line irinotecan for metastatic CRC (n = 100), a cohort of healthy controls with and without comorbidity (n = 70 and 100, respectively) were included. cfDNA was quantified by an in‐house developed quantitative polymerase chain reaction from plasma samples drawn prior to the first cycle of chemotherapy and at time of progression. cfDNA levels were significantly higher in CRC compared to controls, with a clear capability for discriminating between the groups (receiver operation curve analysis; area under the curve 0.82, p < 0.0001). Patients with high levels had a shorter survival from irinotecan compared to those with lover levels. The cohort independent upper normal limit divided patients into high and low risk groups. The progression‐free survival (PFS) was 2.1 months 95% confidence interval (CI) 2.0–3.4 and 6.5 (95% CI 4.2–7.2) months hazard ratio (HR) 2.53; 95% CI 1.57–4.06, p < 0.0001 and overall survival (OS) 7.4 months (95% CI 4.3–8.7) and 13.8 months (95% CI 11.9–18.9; HR 2.52; 95% CI 1.54–4.13, p < 0.0000), respectively. Cox regression multivariate analysis showed a PFS HR of 1.4 (95% CI 1.1–1.7) for each increase in cfDNA quartile, p = 0.03 and 1.6 (1.3–2.0) for OS, p < 0.0001, respectively. A combined marker analysis with plasma KRAS mutations added further prognostic impact, which was consistent when performed on the samples drawn at time of progression. In conclusion, cfDNA measurement holds important clinical information and could become a useful tool for prediction of outcome from chemotherapy in mCRC.
What's New?
Circulating nucleic acids have emerged as important diagnostic and prognostic markers in cancer biology. However, many researchers have focused on identifying tumor‐derived circulating DNA to study mutations. Here, the authors show that levels of total cell‐free DNA (cfDNA) can predict survival in patients with metastatic colorectal cancer. The levels of cfDNA were markedly higher in cancer patients compared to healthy controls and patients with significant comorbidities. Patients with high levels had a shorter survival when treated with irinotecan, a second‐line chemotherapeutic drug, supporting the notion that cfDNA may become a useful tool for prediction of outcome from chemotherapy in patients with metastatic colorectal cancer.
The chronic Philadelphia-negative myeloproliferative neoplasms (MPNs) are acquired stem cell neoplasms which ultimately may transform to acute myelogenous leukemia. Most recently, chronic ...inflammation has been described as an important factor for the development and progression of MPNs in the biological continuum from early cancer stage to the advanced myelofibrosis stage, the MPNs being described as "A Human Inflammation Model for Cancer Development". This novel concept has been built upon clinical, experimental, genomic, immunological and not least epidemiological studies. Only a few studies have described the development of MPNs by mathematical models, and none have addressed the role of inflammation for clonal evolution and disease progression. Herein, we aim at using mathematical modelling to substantiate the concept of chronic inflammation as an important trigger and driver of MPNs.The basics of the model describe the proliferation from stem cells to mature cells including mutations of healthy stem cells to become malignant stem cells. We include a simple inflammatory coupling coping with cell death and affecting the basic model beneath. First, we describe the system without feedbacks or regulatory interactions. Next, we introduce inflammatory feedback into the system. Finally, we include other feedbacks and regulatory interactions forming the inflammatory-MPN model. Using mathematical modeling, we add further proof to the concept that chronic inflammation may be both a trigger of clonal evolution and an important driving force for MPN disease progression. Our findings support intervention at the earliest stage of cancer development to target the malignant clone and dampen concomitant inflammation.
Liquid biopsies focusing on the analysis of cell‐free circulating tumor DNA (ctDNA) may have important clinical implications for personalized medicine, including early detection of cancer, ...therapeutic guidance, and monitoring of recurrence. Mutations in the oncogene, PIK3CA, are frequently observed in breast cancer and have been suggested as a predictive biomarker for PI3K‐selective inhibitor treatment. In this study, we analyzed the presence of PIK3CA mutations in formalin‐fixed, paraffin‐embedded, metastatic tissue and corresponding ctDNA from serum of patients with advanced breast cancer using a highly sensitive, optimized droplet digital PCR (ddPCR) assay. We found 83% of patients with PIK3CA mutation in the metastatic tumor tissue also had detectable PIK3CA mutations in serum ctDNA. Patients lacking the PIK3CA mutation in corresponding serum ctDNA all had nonvisceral metastatic disease. Four patients with detectable PIK3CA‐mutated ctDNA were followed with an additional serum sample during oncological treatment. In all cases, changes in PIK3CA ctDNA level correlated with treatment response. Our results showed high concordance between detection of PIK3CA mutations in tumor tissue and in corresponding serum ctDNA and suggest that serum samples from patients with advanced breast cancer and ddPCR may be used for PIK3CA mutation status assessment to complement imaging techniques as an early marker of treatment response.
PIK3CA mutations are frequent in breast cancer and proposed as a predictive biomarker for PI3K‐selective inhibitor treatment. Here, we show that PIK3CA mutations can be identified in ctDNA of serum samples from patients with PIK3CA‐mutated metastatic breast cancer and that the circulating PIK3CA mutation level can be used to monitor treatment response in these patients.
There is mounting evidence that the immune system can spontaneously clear malignant lesions before they manifest as overt cancer, albeit this activity has been difficult to demonstrate in humans. The ...calreticulin (
CALR
) exon 9 mutations are driver mutations in patients with chronic myeloproliferative neoplasms (MPN), which are chronic blood cancers. The
CALR
mutations generate a neo-antigen that is recognized by patient T cells, and T cells isolated from a patient with a
CALR
-mutation can recognize and kill autologous
CALR
-mutant cells. Surprisingly, healthy individuals display frequent and strong T cell responses to the CALR neo-antigens too. Furthermore, healthy individuals display immune responses to all parts of the mutant CALR epitope, and the CALR neo-epitope specific responses are memory T cell responses. These data suggest that although healthy individuals might acquire a
CALR
mutation, the mutant cells can be eliminated by the immune system. Additionally, a small fraction of healthy individuals harbor a
CALR
exon 9 mutation. Four healthy individuals carrying
CALR
mutations underwent a full medical examination including a bone marrow biopsy after a median follow up of 6.2 years. None of these patients displayed any signs of
CALR
-mutant MPN. Additionally, all healthy individuals displayed strong CALR neo-epitope specific T cell responses suggesting that these healthy individuals retained their
CALR
-mutant cells in the editing stage for several years. Thus, we suggest that
CALR
-mutant MPN could be a disease model of cancer immuno-editing, as we have demonstrated that
CALR
-mutant MPN displays all three stages described in the theory of cancer immuno-editing.
Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value ...in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.
PDF
