ctDNA-guided adjuvant treatment after radical-intent treatment of metastatic spread from colorectal cancer-the first interim results from the OPTIMISE study.

BACKGROUND: Patients with detectable ctDNA after radical-intent treatment of metastatic spread from colorectal cancer (mCRC) have a very high risk of recurrence, which may be prevented with intensified adjuvant chemotherapy (aCTh). In the OPTIMISE study, we investigate ctDNA-guided aCTh after radical-intent treatment of mCRC. Here we present results from the preplanned interim analysis. MATERIAL AND METHODS: The study is an open-label 1:1 randomized clinical trial comparing ctDNA-guided aCTh against standard of care (SOC), with a run-in phase investigating feasibility measures. Key inclusion criteria; radical-intent treatment for mCRC and clinically eligible for triple-agent chemotherapy. Patients underwent a PET-CT scan before randomization. ctDNA analyses of plasma samples were done by ddPCR, detecting CRC-specific mutations and methylation of the NPY gene. In the ctDNA-guided arm, ctDNA positivity led to an escalation strategy with triple-agent chemotherapy, and conversely ctDNA negativity led to a de-escalation strategy by shared-decision making. Patients randomized to the standard arm were treated according to SOC. Feasibility measures for the run-in phase were; the inclusion of 30 patients over 12 months in two Danish hospitals, compliance with randomization >80%, rate of PET-CT-positive findings <20%, and eligibility for triple-agent chemotherapy >80%. RESULTS: Thirty-two patients were included. The rate of PET-CT-positive cases was 22% (n = 7/32). Ninety-seven percent of the patients were randomized. Fourteen patients were randomly assigned to SOC and sixteen to ctDNA-guided adjuvant treatment and follow-up. All analyses of baseline plasma samples in the ctDNA-guided arm passed the quality control, and 19% were ctDNA positive. The median time to result was three working days. All ctDNA-positive patients were eligible for triple-agent chemotherapy. CONCLUSION: The study was proven to be feasible and continues in the planned large-scale phase II trial. Results from the OPTIMISE study will potentially optimize the adjuvant treatment of patients undergoing radical-intent treatment of mCRC, thereby improving survival and reducing chemotherapy-related toxicity.

The Importance of Feasibility Assessment in the Design of ctDNA Guided Trials - Results From the OPTIPAL II Study.

INTRODUCTION: Both quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response. MATERIALS AND METHODS: Patients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level. RESULTS: Forty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue (p < .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p= .02), and 4.6 months (HR = 11.4, p= .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p= .03), and 9.0 months (HR = 2.6, p= .03) in patients with ctDNA maximum response, partial response, and progression, respectively. CONCLUSION: Pretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value.

Liquid Biopsies with Circulating Plasma HPV-DNA Measurements-A Clinically Applicable Surveillance Tool for Patients with HPV-Positive Oropharyngeal Cancer.

PURPOSE: To evaluate the accuracy of cell-free human papillomavirus-DNA (cfHPV-DNA) measurements in liquid biopsies in predicting disease in patients with HPV-positive/p16-positive (HPV+/p16+) oropharyngeal squamous cell carcinoma (OPSCC). EXPERIMENTAL DESIGN: This was a prospective cohort study. Plasma samples were collected before treatment, serially after curative intended therapy at follow-up visits 2 weeks, and 6, 9, 12, 18, 24, and 30 months after treatment. A droplet digital PCR assay comprising eight HPV genotypes was used. HPV genotypes found in plasma and tumor tissue were compared. We correlated biopsy- or imaging-verified tumor progression to cfHPV-DNA in follow-up samples. RESULTS: We enrolled 72 patients with HPV+/p16+ OPSCC. Baseline sensitivity for cfHPV-DNA detection was 97.2% (95% confidence interval, 90.3%-99.6%). CfHPV-DNA copy number/milliliter plasma correlated with tumor stage. We found a 100% concordance between HPV genotype in tumor tissue and plasma. Fifty-four patients were followed with serial blood samples for a median of 19.7 months (interquartile range, 13.5-25.5 months). Forty-one patients had undetectable plasma cfHPV-DNA in all follow-up samples, and none developed recurrences. Thirteen patients were classified as cfHPV-DNA-positive in a follow-up plasma sample. Of these, five patients developed a recurrence, and three had residual cancer. It was possible to detect cfHPV-DNA in plasma 97 to 166 days prior to the proven recurrence. CONCLUSIONS: To our knowledge, to date, our study, comprising the largest study of patients with HPV+/p16+ OPSCC, using an ultrasensitive multiplex HPV gene panel, revealed a high sensitivity of cfHPV-DNA detection in the liquid biopsies. We recommend serial plasma HPV samples for clinical monitoring of patients with HPV+/p16+ OPSCC.

Endoscopic calcium electroporation for colorectal cancer: a phase I study.

Background and study aims Colorectal cancer is one of the most common malignancies, with approximately 20 % of patients having metastatic disease. Local symptoms from the tumor remain a common issue and affect quality of life. Electroporation is a method to permeabilize cell membranes with high-voltage pulses, allowing increased passage of otherwise poorly permeating substances such as calcium. The aim of this study was to determine the safety of calcium electroporation for advanced colorectal cancer. Patients and methods Six patients with inoperable rectal and sigmoid colon cancer were included, all presenting with local symptoms. Patients were offered endoscopic calcium electroporation and were followed up with endoscopy and computed tomography/magnetic resonance scans. Biopsies and blood samples were collected at baseline and at follow-up, 4, 8, and 12 weeks after treatment. Biopsies were examined for histological changes and immunohistochemically with CD3/CD8 and PD-L1. In addition, blood samples were examined for circulating cell-free DNA (cfDNA). Results A total of 10 procedures were performed and no serious adverse events occurred. Prior to inclusion, patients reported local symptoms, such as bleeding (N = 3), pain (N = 2), and stenosis (N = 5). Five of six patients reported symptom relief. In one patient, also receiving systemic chemotherapy, clinical complete response of primary tumor was seen. Immunohistochemistry found no significant changes in CD3 /CD8 levels or cfDNA levels after treatment. Conclusions This first study of calcium electroporation for colorectal tumors shows that calcium electroporation is a safe and feasible treatment modality for colorectal cancer. It can be performed as an outpatient treatment and may potentially be of great value for fragile patients with limited treatment options.

Circulating tumour DNA and its clinical utility in predicting treatment response or survival in patients with metastatic colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: We investigate the current knowledge on circulating tumour DNA (ctDNA) and its clinical utility in predicting outcomes in patients with metastatic colorectal cancer (mCRC). METHODS: PubMed, Embase, Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials were searched. Last search 16/12/2020. We included studies on patients with mCRC reporting the predictive or prognostic value of ctDNA. We performed separate random-effects meta-analyses to investigate if baseline ctDNA and early changes in ctDNA levels during treatment were associated with survival. The risk of bias was assessed according to the Quality in Prognosis Studies tool. RESULTS: Seventy-one studies were included with 6930 patients. Twenty-four studies were included in meta-analyses. High baseline ctDNA level was associated with short progression-free survival (PFS) (HR = 2.2; 95% CI 1.8-2.8; n = 509) and overall survival (OS) (HR = 2.4; 95% CI 1.9-3.1; n = 1336). A small or no early decrease in ctDNA levels during treatment was associated with short PFS (HR = 3.0; 95% CI 2.2-4.2; n = 479) and OS (HR = 2.8; 95% CI 2.1-3.9; n = 583). Results on clonal evolution and lead-time were inconsistent. A majority of included studies (n = 50/71) had high risk of bias in at least one domain. CONCLUSIONS: Plasma ctDNA is a strong prognostic biomarker in mCRC. However, true clinical utility is lacking.

Perioperative changes in cell-free DNA for patients undergoing surgery for colon cancer.

BACKGROUND: Various conditions with cellular decay are associated with elevated cell-free DNA (cfDNA). This study aimed to investigate if perioperatively measured cfDNA levels were associated with the surgical approach, complications, or recurrence. METHODS: Plasma was obtained from patients who underwent surgery for colon cancer at admission and at the time of discharge. Quantitative measurement of cfDNA was performed by amplifying two amplicons of 102 base pairs (bp) and 132 bp of Beta-2-Microglobulin (B2M) and Peptidyl-Prolyl cis-trans Isomerase A (PPIA), respectively. RESULTS: cfDNA was measured in 48 patients who underwent surgery for colonic cancer. Sixteen patients had recurrence during the follow-up period, fifteen developed a postoperative complication, and seventeen patients developed neither, acting as the control group. Postoperative cfDNA levels were significantly elevated from baseline samples, across all groups, with a median preoperatively B2M level of 48.3 alleles per mL and postoperatively of 220 alleles per mL and a median preoperatively level PPIA of 26.9 alleles per mL and postoperatively of 111.6 alleles per mL (p < 0.001 for B2M and p < 0.001 for PPIA). Postoperative levels of PPIA, but not B2M, were significantly higher in patients experiencing complications than in the control group (p = 0.036). However, a tendency towards an association between the surgical approach and the changes in cfDNA levels was found for PPIA (p = 0.058), and B2M (p = 0.087). CONCLUSIONS: Plasma cfDNA was increased after surgery in all patients with colon cancer. Postoperative PPIA levels were significantly higher in patients experiencing surgical complications but not in B2M levels.

Total cell-free DNA measurement in metastatic colorectal cancer with a fast and easy direct fluorescent assay.

Treatment for metastatic colorectal cancer (mCRC) is focused on prolonging survival and maintaining quality of life. It is important to establish prognostic and predictive markers to avoid extended, ineffective treatment. The aim of the present study was, by a novel approach, to analyze the association between cell-free (cf)DNA levels and outcome in patients receiving systemic therapy for incurable mCRC. The study was a prospective non-interventional biomarker study for patients receiving standard of systemic treatment for mCRC. Patients with mCRC, who, according to standard guidelines, were considered for treatment with EGFR inhibitors, were included. The cfDNA levels in consecutive plasma samples were measured by a direct fluorescence assay. The study included 47 patients. Blood samples were available at baseline (n=47); prior to the third treatment cycle (n=31); the first (n=33), second (n=22) and third response evaluation during treatment (n=17); and at progression (n=22). The disease control rate was 42 and 91% in patients with high (≥75th percentile of baseline cfDNA levels) and low cfDNA levels (<75th percentile of baseline cfDNA levels), respectively (P<0.001). Median progression-free survival (PFS) was 3.8 and 8.5 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.03, 95% CI 1.46-6.29, P<0.01). Median overall survival (OS) was 5.0 and 26.6 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.48, 95% CI 1.44-8.44, P<0.01). In the multivariate analysis, baseline cfDNA levels remained a significant predictor of PFS and OS. In conclusion, cfDNA is a promising prognostic tool in the personalized treatment of mCRC. cfDNA levels were estimated by a simple, rapid and inexpensive method (OPTIPAL II: ClinicalTrials.gov identifier no. NCT03750175; registered November 21, 2018).

Circulating Tumor DNA Monitoring Reveals Molecular Progression before Radiologic Progression in a Real-life Cohort of Patients with Advanced Non-small Cell Lung Cancer.

Purpose: The clinical potential of liquid biopsy in patients with advanced cancer is real-time monitoring for early detection of treatment failure. Our study aimed to investigate the clinical validity of circulating tumor DNA (ctDNA) treatment monitoring in a real-life cohort of patients with advanced non-small cell lung cancer (NSCLC). Experimental Design: Patients with advanced or noncurative locally advanced NSCLC were prospectively included in an exploratory study (NCT03512847). Selected cancer-specific mutations were measured in plasma by standard or uniquely designed droplet digital PCR assays before every treatment cycle during first-line treatment until progressive disease (PD). Correlation between an increase in ctDNA (= molecular progression) and radiologic PD was investigated, defined as lead time, and the corresponding numbers of likely futile treatment cycles were determined. Utility of ctDNA measurements in clarifying the results of nonconclusive radiologic evaluation scans was evaluated. Results: Cancer-specific mutations and longitudinal plasma sampling were present in 132 of 150 patients. ctDNA was detectable in 88 (67%) of 132 patients treated by respectively chemotherapy (n = 41), immunotherapy (n = 43), or combination treatment (n = 4). In 66 (90%) of 73 patients experiencing PD, a ctDNA increase was observed with a median lead time of 1.5 months before radiologic PD. Overall, 119 (33%) of 365 treatment cycles were administered after molecular progression. In addition, ctDNA measurements could clarify the results in 38 (79%) of 48 nonconclusive radiologic evaluations. Conclusions: ctDNA monitoring leads to earlier detection of treatment failure, and clarifies the majority of nonconclusive radiologic evaluations, giving the potential of sparing patients from likely futile treatments and needless adverse events. Significance: Treatment monitoring by ctDNA has the clinical potential to reveal PD before radiologic evaluation and consequently spare patients with advanced cancer from likely ineffective, costly cancer treatments and adverse events.

Assessment of droplet digital polymerase chain reaction for measuring BCR-ABL1 in chronic myeloid leukaemia in an international interlaboratory study.

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.

The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus.

BACKGROUND AND PURPOSE: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around 90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. MATERIAL AND METHODS: Patients treated at Aarhus University Hospital from 2016-2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. RESULTS: Samples from 88 patients were drawn PT (n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. CONCLUSION: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.

Clinicopathological factors associated with tumour-specific mutation detection in plasma of patients with RAS-mutated or BRAF-mutated metastatic colorectal cancer.

Detection of tumour-specific circulating cell-free DNA in plasma (ctDNA) fails in a significant number of cases depending on the clinical context. The primary aim was to investigate clinicopathological factors associated with detection of ctDNA in patients with RAS-/BRAF-mutated metastatic colorectal cancer (mCRC) prior to first-line therapy. A secondary aim was to evaluate the prognostic impact of ctDNA compared to other biomarkers. Patients were included from the NORDIC-VII study (N = 253). ctDNA was sampled prior to treatment and analysed for hotspot tissue mutations (KRAS, NRAS, and BRAF) using droplet digital PCR. Multivariable regression models were constructed to predict the probability of mutation detection and survival. Increasing radiological size of target lesions by increments of 1 cm (odds ratio [OR] = 1.18; 95% confidence interval [CI] 1.09-1.27; P < .001), intact primary tumour (OR = 3.17; 95% CI 1.22-8.22; P = .018) and more than one metastatic site (OR = 3.08; 95% CI 1.32-7.19; P = .009) were associated with mutation detection in plasma. Metastatic involvement of the lung was associated with non-detection (OR = 0.26; 95% CI 0.12-0.58; P = .001). Preanalytical and analytical factors modulated detection. High allele frequencies of ctDNA indicated poor prognosis independently of CEA and CA19-9 (hazard ratio [HR] = 2.38; 95% CI 1.74-3.26; P < .001; N = 206). Clinicopathological characteristics should be carefully considered when evaluating ctDNA results from mCRC patients, especially when confronted with a plasma negative result. ctDNA may prove to be a clinically useful marker in the evaluation of mCRC treatment.

Digital PCR for BCR-ABL1 Quantification in CML: Current Applications in Clinical Practice.

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.

Ruxolitinib and interferon-α2 combination therapy for patients with polycythemia vera or myelofibrosis: a phase II study.

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.

Circulating tumor DNA as a marker of minimal residual disease following local treatment of metastases from colorectal cancer.

BACKGROUND: Local treatment of liver and/or lung metastases from colorectal cancer (CRC) is increasingly used in daily practice and comprises resection, radiofrequency ablation (RFA) and stereotactic radiotherapy (SBRT). The need for prognostic markers for patients undergoing such treatment is currently unmet. We investigated post-treatment circulating tumor-specific DNA (ctDNA) analysis and address a possible prognostic value in a pilot study. MATERIALS: From July 2015 to September 2017, patients undergoing standard of care local treatment of liver and/or lung metastases were included in a prospective translational study. Blood samples were drawn 2 weeks after local treatment and during follow-up. CtDNA was detected by ddPCR and a mass spectrometry-based platform MassARRAY®. RESULTS: Post treatment blood samples were available for 35 patients including five with detectable ctDNA (KRAS mutation, n = 2; NRAS mutation, n = 2; BRAF mutation, n = 1) by ddPCR. 17 out of 35 patients (49%) developed recurrence within a median of 273 days (95%CI 95-NA) among patients positive for ctDNA, while the median time to recurrence was not reached for the group of patients negative for ctDNA (p = .03). CONCLUSION: The presence of ctDNA following local treatment of metastatic CRC is associated with an increased risk of recurrence and a short time to failure.

Data-driven analysis of JAK2V617F kinetics during interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms.

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.

Evidence of immune elimination, immuno-editing and immune escape in patients with hematological cancer.

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.

Prevalence and phenotypes of JAK2 V617F and calreticulin mutations in a Danish general population.

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.