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Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

We report a case of a pancreatic ductal adenocarcinoma (PDAC) presenting synchronously with a paraganglioma (PGL) in a Whipple reaction specimen. The patient was a 72-year-old female with a history of breast and vulvar cancer. The simultaneous occurrence of two synchronous tumours in the pancreas was striking. Due to the presence of PGL and multiple meta- and synchronous tumours, the patient was referred to genetic counselling. Tumour tissue from the vulvar carcinoma, the PDAC and the PGL was analysed by targeted next-generation sequencing (NGS) of 161 cancer-related genes and by whole exome sequencing (WES). Peripheral blood was also examined by NGS and WES. These genetic analyses revealed germline polymorphisms in AXIN2 (NM_004655.4:c 0.2272 G>A; p.Ala758Thr), BRCA2 (NM_000059.3:c.9976 A>T; p.Lys3326Ter), NCOR1 (NM_006311.4:c 0.6544 G>A; p.Ala2182Thr) and SPTA1 (NM_003126.3:c 0.373 G>A; p.Ala125Thr) and somatic mutations of KRAS (NM_033360.3;c 0.35 G>A; p.Gly12Asp) and TP53 (NM_000546.5; c.602delT; p.Leu201CysfsTer46) in the PDAC and of TP53 (NM_000546.5; c 0.733 G>A; p.Gly245Ser) and TERT (NM_198253.2; c.-124 C>T; promotor region) in the vulvar carcinoma. Breast carcinoma tissue was not available for genetic analysis. The results of the genetic analyses did not explain the presence of multiple tumours in this patient, despite a slightly increased risk of breast cancer associated with the identified BRCA2 polymorphism. To our knowledge, this is the first report of the synchronous occurrence of PDAC and PGL. This case emphasizes the importance of thorough macroscopic examination of pancreatic resection specimens, as coexisting neoplasms may otherwise be missed.

Rosette-forming glioneuronal tumor (RGNT) is a rare brain neoplasm that primarily affects young adults. Although alterations affecting the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathway have been associated with this low-grade entity, comprehensive molecular investigations of RGNT in larger series have not been performed to date, and an integrated view of their genetic and epigenetic profiles is still lacking. Here we describe a genome-wide DNA methylation and targeted sequencing-based characterization of a molecularly distinct class of tumors (n = 30), initially identified through genome-wide DNA methylation screening among a cohort of > 30,000 tumors, of which most were diagnosed histologically as RGNT. FGFR1 hotspot mutations were observed in all tumors analyzed, with co-occurrence of PIK3CA mutations in about two-thirds of the cases (63%). Additional loss-of-function mutations in the tumor suppressor gene NF1 were detected in a subset of cases (33%). Notably, in contrast to most other low-grade gliomas, these tumors often displayed co-occurrence of two or even all three of these mutations. Our data highlight that molecularly defined RGNTs are characterized by highly recurrent combined genetic alterations affecting both MAPK and PI3K signaling pathways. Thus, these two pathways appear to synergistically interact in the formation of RGNT, and offer potential therapeutic targets for this disease.

Activating mutations in the epidermal growth factor receptor gene occur as early cancer-driving clonal events in a subset of patients with non-small cell lung cancer (NSCLC) and result in increased sensitivity to EGFR-tyrosine-kinase-inhibitors (EGFR-TKIs). Despite very frequent and often prolonged clinical response to EGFR-TKIs, virtually all advanced EGFR-mutated (EGFRM+) NSCLCs inevitably acquire resistance mechanisms and progress at some point during treatment. Additionally, 20−30% of patients do not respond or respond for a very short time (EGFRM+ NSCLC at baseline has illustrated the co-existence of multiple genetic, phenotypic, and functional mechanisms that may contribute to tumor progression and cause intrinsic TKI-resistance. Several of these mechanisms have been further corroborated by preclinical experiments. Intrinsic resistance can be caused by mechanisms inherent in EGFR or by EGFR-independent processes, including genetic, phenotypic or functional tumor changes. This comprehensive review describes the identified mechanisms connected with intrinsic EGFR-TKI-resistance and differences and similarities with acquired resistance and among clinically implemented EGFR-TKIs of different generations. Additionally, the review highlights the need for extensive pre-treatment molecular profiling of advanced NSCLC for identifying inherently TKI-resistant cases and designing potential combinatorial targeted strategies to treat them.

Activating mutations in the epidermal growth factor receptor gene occur as early cancer-driving clonal events in a subset of patients with non-small cell lung cancer (NSCLC) and result in increased sensitivity to EGFR-tyrosine-kinase-inhibitors (EGFR-TKIs). Despite very frequent and often prolonged clinical response to EGFR-TKIs, virtually all advanced EGFR-mutated (EGFRM+) NSCLCs inevitably acquire resistance mechanisms and progress at some point during treatment. Additionally, 20–30% of patients do not respond or respond for a very short time (

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

Background Recombinant Interferon Alpha-2a (r-IFNα) is a potent immunomodulating agent, which has been used off-label for the treatment of polycythemia vera (PV) for more than three decades and has been demonstrated to induce high rates of clinical, hematological and molecular responses. Only few studies have compared efficacy and safety of r-IFNα vs. hydroxyurea (HU), which is considered first line therapy for PV patients > 60 years in most parts of the world. However, recent studies have provided encouraging results for the treatment of PV with r-IFNα compared to HU irrespective of age (R. Hoffmann 2016; H. Gisslinger 2018). Aims To examine the difference in efficacy and safety of low-dose r-IFNα in PV patients ≤ 60 or > 60 years of age compared to HU > 60 years of age. Methods Ninety newly diagnosed or previously phlebotomized PV patients only (WHO 2008) were enrolled in the DALIAH trial (NCT01387763). All patients provided written informed consent. Patients ≤ 60 years were randomized (I:I) to r-IFNα-2a (Pegasys®) or to r-IFNα-2b (PegIntron®) whereas patients > 60 years were randomized (I:I:I) to either r-IFNα-2a, r-IFNα-2b or to HU. The starting dose of r-IFNα-2a and r-IFNα-2b was 45 or 35 µg/week, respectively. The HU dose was 500 to 2000 mg/day. Patients randomized to r-IFNα who presented with major thrombosis or platelets > 1500 109/L received HU from inclusion and until normalization of the platelet count. Efficacy assessment consisted of the clinicohematological and the molecular response rates by intention to treat analysis (ITT) using the European Leukemia Net (ELN) 2009 criteria. JAK2V617F analysis was performed by qPCR. Groups were compared by Fisher's Exact Test. Results Three-year analysis was available in all but five patients (n=85) at time of abstract submission (Table 1). The analysis was performed after a median of 36 months (range: 33-39 months). The median treatment dose was 684 mg/day (IQR: 131 - 942) for HU, 51 μg/week (IQR: 30-90 μg/week) and 54 μg/week (IQR: 30-66 μg/week) for r-IFNα-2a age ≤ 60 and > 60, respectively, and 41 μg/week (IQR: 29-45 μg/week) and 36 μg/week (IQR: 31-37 μg/week) for r-IFNα-2b age ≤ 60 and > 60. The overall clinicohematological response rate (ORR) was 68% (13/19) for HU, 42% (14/33) for r-IFNα ≤ 60 years and 39% (13/33) for r-IFNα > 60 years. The partial clinicohematological response rate (PHR) and the complete clinicohematological response rate (CHR) was 53% (10/19) and 16% (3/19) for HU, 9% (3/33) and 33% (11/33) for r-IFNα ≤ 60 years and 9% (3/33) and 30% (10/33) for r-IFNα > 60 years. Neither the ORR, CHR nor the PHR was significantly different between the three groups. Maintenance of CHR from first occurrence to data analysis after 36 months was 11% (2/19) for HU, 21% (7/33) for r-IFNα ≤ 60 years and 18% (6/33) for r-IFNα > 60 years. Forty-seven JAK2V617F positive patients were available for molecular response analysis after 36 months of therapy. A partial molecular response (PMR) was detected in 21% (4/19) of HU treated patients and in 24% (7/29) of r-IFNα treated patients ≤ 60 years and 18% (6/33) of r-IFNα > 60 years. Notably, 7% (2/29) of the r-IFNα treated patients ≤ 60 years obtained a complete molecular response (CMR). The median JAK2V617F reduction from baseline was 38% (IQR: 31-63%) for HU, 79% (IQR: 59-92%) for r-IFNα ≤ 60 years and 73% (IQR: 49-97%) for r-IFNα > 60 years. There was no statistically significant difference in the PMR between groups. Discontinuation of treatment for any reason after 36 months of therapy was 21% (4/19) for HU, 52% (17/33) for r-IFNα ≤ 60 years and 45% (15/33) for r-IFNα > 60 years. Toxicity related discontinuation was 5% (1/19) for HU and 30% (10/33) for both r-IFNα ≤ 60 and > 60 years. Grade 3-4 AEs occurred in 32% (6/19) of HU treated patients, 27% (9/33) in r-IFNα treated patients ≤ 60 years and in 42% (14/33) r-IFNα treated patients > 60 years. SAEs were reported in 21% (4/19) for HU, 9% (3/33) for r-IFNα ≤ 60 years and 24% (8/33) for r-IFNα > 60 years. The numbers of grade 3-4 AEs as well as SAEs were comparable between groups. Conclusion After 36 months of therapy CHR was non-significantly higher in PV patients treated with r-IFNα compared to HU by ITT, irrespective of age. Also, maintenance of CHR was longer for r-IFNα. However, ORR was non-significantly higher for HU. PMR was almost similar between the three groups but the median JAK2V617F reduction was greater for r-IFNα. Toxicity related discontinuation from study therapy was higher for r-IFNα compared to HU. Disclosures Stentoft: Bristol-Myers Squibb: Research Funding; Merck Sharp&Dohme: Research Funding. Hasselbalch:Novartis: Research Funding.

Until recently, diagnostics of brain tumors were almost solely based on morphology and immunohistochemical stainings for relatively unspecific lineage markers. Although certain molecular markers have been known for longer than a decade (combined loss of chromosome 1p and 19q in oligodendrogliomas), molecular biomarkers were not included in the WHO scheme until 2016. Now, the classification of diffuse gliomas rests on an integration of morphology and molecular results. Also, for many other central nervous system tumor entities, specific diagnostic, prognostic and predictive biomarkers have been detected and continue to emerge. Previously, we considered brain tumors with similar histology to represent a single disease entity. We now realize that histologically identical tumors might show alterations in different molecular pathways, and often represent separate diseases with different natural history and response to treatment. Hence, knowledge about specific biomarkers is of great importance for individualized treatment and follow-up. In this paper we review the biomarkers that we currently use in the diagnostic work-up of brain tumors.

Background: To investigate the role of genomics in determining response and resistance to front line treatment in MPN, we performed somatic mutational profiling of the DALIAH trial, a randomized controlled phase III trial of interferon versus hydroxyurea in newly diagnosed MPN patients. Methods: We performed genomic analyses on 202 pre-treatment primary MPN samples obtained from patients enrolled on the DALIAH trial (NCT01387763) and 135 samples obtained after 24 months of treatment. Genomic profiling comprised targeted next generation sequencing (NGS) of 100 genes, selected on the basis of their known or suspected involvement in the pathogenesis of myeloid malignancies, and 1609 informative single nucleotide polymorphisms (SNPs) on chromosome 9p. Clinicohematological response was determined by central review using ELN 2009 (ET, PV and pre-MF) and EUMNET 2005 (PMF) criteria. We evaluated the association of somatic mutations with clinical parameters and with attainment of clinicohematological complete response (CR) at 24 months. Results: Prior to treatment, 191 of 202 (95%) patients had somatic mutations, including 93% with canonical MPN phenotypic drivers: JAK2 (74%), CALR (14%), and MPL (5%). Among those with JAK2 mutations 37% had JAK2 copy-neutral loss of heterozygosity (JAK2 CN-LOH). Patients with PV were more likely to have JAK2 CN-LOH (p = 0.0001) as compared with patients with other MPN subtypes. At baseline, patients with JAK2 CN-LOH had significantly higher hemoglobin (p = 0.0001), higher white blood cell count (WBC, p = 0.002) and lower platelet count (p=0.0001) than patients without JAK2 CN-LOH. Mutations in TET2 (24%), DNMT3A (16%), and ASXL1 (10%) were the most frequent co-occurring non-MPN phenotypic driver mutations and they occurred across all MPN subtypes. In addition, 5% of patients had spliceosome gene mutations, and 6% had mutations in genes involved in RAS/MAPK signaling. Patients with TET2, DNMT3A or ASXL1 mutations were significantly older than patients without these mutations (p= 0.0001) and there was a significant association between the presence of a TET2, DNMT3A or ASXL1 mutation and prior stroke (p = 0.004). There were no other significant associations between somatic mutation status and baseline clinical characteristics. The probability of attaining clinicohematological CR at 24 months was independent of baseline somatic mutations. Among patients with JAK2 mutations who remained on interferon treatment at 24 months, those with CR had a greater reduction in mean variant allele fraction (VAF) (28% to 8%, p Among patients who remained on treatment for 2 years, 44 mutations in 35 patients were newly detected or expanded on serial sampling. DNMT3A mutations were the most frequently acquired, accounting for 41% of new mutations. ASXL1, TET2, PPM1D, TP53, IDH2, and CBL mutations were also recurrently acquired. 97% of patients who acquired new mutations were JAK2-mutant. Among those with acquired DNMT3A mutations, 85% were treated with interferon, and 23% had CR at 24 months. Among those that acquired non-DNMT3A mutations, 38% were treated with interferon and 47% had CR at 24 months. The VAF of newly detected mutations was low (median 1.5%), and half of the patients with newly acquired mutations had at least 50% reduction in JAK2V617F VAF, suggesting that new mutations could either have arisen independently or be subclonal to the dominant JAK2-mutant clone. Conclusions: Using sequential genomic analyses of a phase III clinical trial of interferon versus hydroxyurea in MPN patients, we found mutation-specific and treatment-specific patterns of response. We uncovered distinct patterns of response to interferon in JAK2-mutant MPN as compared with CALR-mutant MPN. We found that DNMT3A mutations were the most frequent acquired mutations at 24 months and that these were enriched in patients treated with interferon. In aggregate, these results provide insights into molecular response and resistance to interferon and inform the clinical use of interferon in MPN patients. Disclosures Hansen: Alexion: Research Funding. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Hasselbalch:Novartis: Research Funding; AOP Orphan Pharmaceuticals: Other: Data monitoring board. Lindsley:Jazz Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding. Mullally:Janssen: Research Funding.

We present a multiplex analysis for genes known to have prognostic value in an attempt to design a clinically useful classification model in patients with diffuse large B-cell lymphoma (DLBCL). Real-time polymerase chain reaction was used to measure transcript levels of 28 relevant genes in 194 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Including International Prognostic Index (IPI) as a variable in a penalized Cox regression, we investigated the association with disease progression for single genes or gene combinations in four models. The best model was validated in data from an online available R-CHOP treated cohort. With progression-free survival (PFS) as primary endpoint, the best performing IPI independent model incorporated the LMO2 and HLADQA1 as well as gene interactions for GCSAMxMIB1, GCSAMxCTGF and FOXP1xPDE4B. This model assigned 33% of patients (n = 60) to poor outcome with an estimated 3-year PFS of 40% vs. 87% for low risk (n = 61) and intermediate (n = 60) risk groups (P < 0·001). However, a simpler, IPI independent model incorporated LMO2 and BCL2 and assigned 33% of the patients with a 3-year PFS of 35% vs. 82% for low risk group (P < 0·001). We have documented the impact of a few single genes added to IPI for assignment in new drug trials.

Essential thrombocythemia (ET) and polycythemia vera (PV) are Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) characterized by the JAK2 V617F mutation, which can be found in more than 98% of PV patients and in ∼ 50% of ET patients. Assessment of the JAK2 V617F allele burden by a highly sensitive quantitative PCR (qPCR) assay appears to be a useful tool for monitoring minimal residual disease (MRD) and evaluating treatment efficacy. This report expands and substantiates existing data, showing that IFN-alpha2 is a highly potent immunomodulating agent capable of inducing MRD with low-burden JAK2 V617F, major molecular response (MMR), complete hematological remission (CHR) and complete histomorphological normalization of the bone marrow in a sub-set of patients with ET and PV after long-term treatment (≥ 3.5 years). Furthermore, long-lasting hematological, molecular and histomorphological remissions are sustained after discontinuation of IFN-alpha2 for up to ∼ 5-6 years.

Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients. We investigated the response to IFN in a cohort of 21 CALR mutated MPN patients including ET, prefibrotic primary myelofibrosis (pre-PMF), and primary myelofibrosis (PMF) with a median follow-up of 31 months. For evaluation of a molecular response, we developed highly sensitive quantitative PCR (qPCR) assays for monitoring the mutant allele burden of the two most prevalent CALR mutations (type 1 and type 2). Thirteen patients (62%) experienced a decrease in the mutant allele burden with a median decline of 29% from baseline. However, only four patients, including patients with ET, pre-PMF, and PMF diagnosis, achieved molecular responder (MR) status with >50% reduction in mutant allele burden according to European LeukemiaNet (ELN) guidelines. MR patients displayed significant differences in the dynamics of the CALR mutant load with regard to time to response and dynamics in mutant allele burden after discontinuation of IFN treatment. Furthermore, we highlight the prognostic value of the CALR mutant allele burden by showing a close association with leucocyte- and platelet counts, hemoglobin concentration, in addition to plasma lactate dehydrogenase (LDH) irrespective of molecular response and treatment status.

Determining the presence of MYC gene rearrangements is becoming an increasingly important part of the diagnostic workup in aggressive lymphoma. Cytogenetic MYC alterations aid in differentiating diffuse large B-cell lymphoma (DLBCL) from Burkitt lymphoma. In addition, MYC aberrations are associated with poor prognosis in DLBCL. Fluorescence in situ hybridization and karyotyping are standard tests for detecting MYC aberrations, but these techniques are laborious and expensive. Here, we studied MYC status of 219 DLBCLs and Burkitt lymphomas using fluorescence in situ hybridization, immunohistochemistry, and quantitative real-time polymerase chain reaction (QRT-PCR). Overall, 15% of the cases had an MYC break. QRT-PCR analysis of MYC expression showed that 72% of DLBCLs with an MYC break had aberrantly high or low levels of MYC transcript. Excluding the cases with aberrantly low MYC expression, we found a significant positive correlation between levels of MYC transcripts and MYC tumor cells; however, QRT-PCR is not readily applicable as a screening tool. Immunohistochemically, all tumors showed a nuclear staining pattern that was simple to evaluate. The percentage of MYC lymphoma cells correlated closely with MYC rearrangement status. In all, 93% of cases with an MYC break had ≥80% MYC cells, in contrast to 3% of nonrearranged cases (P

BACKGROUND: Until recently there has been no proven second-line therapy for patients with advanced gastro-esophageal cancer (GEC). Since 2004, Denmark has had a national health program where non-proven therapy can be offered to patients with advanced cancer, after approval by an expert panel appointed by the National Board of Health. This program has accelerated the introduction and implementation of new therapies in Denmark. Inspired by therapy in metastatic colorectal cancer, a combination of cetuximab and irinotecan (Cetiri) was chosen for second-line therapy in GEC patients. We report our experience with Cetiri as second-line therapy in patients with GEC. METHODS: All patients had histologically confirmed GEC and all patients had progressive disease during or after first-line platinum-containing chemotherapy. The patients received cetuximab 500 mg/m(2) on day 1 and irinotecan 180 mg/m(2) on day 1 every 2nd week until progression or unacceptable toxicity. Toxicity was prospectively evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0. RESULTS: From December 2007 to February 2009, 50 consecutive patients received Cetiri as second-line therapy. Median performance status (PS) was 1. The median number of courses was seven. Seven patients (14%) had a partial response. Median progression-free survival (PFS) was 3.3 months and overall survival (OS) was 5.5 months; two patients are still alive without progressive disease. Major toxicities were: diarrhea (8%), fatigue (10%), neutropenia (16%), and febrile neutropenia (2%). CONCLUSION: Cetiri every two weeks is a convenient and well-tolerated second-line regimen in GEC patients. A promising effect was seen in patients with PS 0-1 and in patients who developed a rash.

High-grade gliomas have an aggressive clinical course and new clinical biomarkers and therapeutic targets are highly needed. WEE1 is a regulator of the G2 checkpoint in glioblastoma (GBM) cells. Inhibition of this kinase has, in experimental glioma studies, been suggested to enhance sensitivity to irradiation and temozolomide. However, expression level and prognostic potential of WEE1 protein in gliomas remain uninvestigated. In this study, glioma samples from 235 patients across all four WHO grades were analyzed by immunohistochemistry. Using image analysis, we calculated the area fraction of WEE1 positive nuclei. We found that WEE1 protein was localized in tumor cell nuclei and expressed in all glioma types and grades. Although WEE1 protein levels are higher in GBMs (mean 24.5 %) relative to grade III (mean 14,0 %, p

The chaperone and calcium storing protein calreticulin is coded by CALR, and newly identified mutations in CALR are found in respectively 49-70% and 56-88% of JAK2- and MPL-negative patients with essential thrombocytaemia (ET) and primary myelofibrosis (PMF). A total of 41 mutations have been identified, all located to exon 9 which codes the protein's C-terminal. CALR mutations are present only in myeloid malignancies and confer a more indolent disease than JAK2-mutated ET and PMF. CALR mutations as a diagnostic and prognostic tool are promising and the mutations are potential targets for immune therapy.

Background Treatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs. Patients and Methods Newly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0. Results Ninety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET). The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range Conclusions Development of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. Disclosures Off Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.

Within recent years data has accumulated demonstrating the efficacy of recombinant interferon alpha2 (rIFN-alpha2) in the treatment of chronic myeloproliferative neoplasms (MPNs). We report on clinical and molecular data in the largest cohort of JAK2 V617F mutant MPN Danish patients (n=102) being treated long-term with rIFN-alpha2 (rIFN-alpha2a and rIFN-alpha2b in a non-clinical trial setting. The median follow-up was 42 months. We substantiate the capacity of rIFN-alpha2 to induce complete hematologic remissions (ET 95%, PV 68%) and molecular response. In total 76 patients (74.5%) had a decline in JAK2 V617F allele burden with a median reduction from baseline of 59% (95% c.i. 50-73%, range 3-99%). A decline in JAK2 V617F allele burden was recorded in both ET (median 24-10% (95% c.i.: 8-16%), and PV (median 59-35% (95% c.i.: 17-33%). Patients with the lowest pre-treatment JAK2 V617F allele burdens tend to achieve the most favourable responses on long term treatment with rIFN-alpha2. Eleven patients (10%) had deep molecular remissions with ≤ 2% JAK2 V617F mutant DNA. Finally, long term treatment with rIFN-alpha2 was associated with a very low thrombosis rate. Our observations are supportive of the concept of early up-front treatment with rIFN-alpha2.

Contains fulltext : 110952.pdf (Publisher’s version ) (Closed access)

Contains fulltext : 108255.pdf (Publisher’s version ) (Closed access) In recent years, the mutational status of the KRAS oncogene has become incorporated into standard medical care as a predictive marker for therapeutic decisions related to patients with metastasized colorectal cancer. This is necessary, because these patients benefit from epidermal growth factor receptor (EGFR)-targeted therapy with increased progression-free survival only if the tumor does not carry a mutation in KRAS. Many different analytical platforms, both those commercially available and those developed in house, have been used within pathology laboratories to assess KRAS mutational status. For a testing laboratory to become accredited to perform such tests, it is essential that they perform reliability testing, but it has not previously been possible to perform this kind of testing on the complete workflow on a large scale without compromising reproducibility or the mimicry of the control sample. We assessed a novel synthetic control for formalin-fixed, paraffin-embedded (FFPE) tumor samples in a blind study conducted within nine laboratories across Europe. We show that FFPE material can, at least in part, mimic clinical samples and we demonstrate this control to be a valuable tool in the assessment of platforms used in testing for KRAS mutational status.

Learning Objectives After completing this course, the reader will be able to: Identify the most frequent errors made in KRAS testing in this study and the possible consequences for a patient.Describe factors that could increase the chance of an error during KRAS testing. This article is available for continuing medical education credit at CME.TheOncologist.com The use of epidermal growth factor receptor–targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false-positive and false-negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization.

Udgivelsesdato: 2009-Dec Polycythemia vera (PV) is characterized by the presence of the JAK2V617F mutation in virtually all patients. Several studies have shown that the JAK2V617F mutational load decreases during treatment with alpha-interferon 2. We report on molecular and histomorphological bone marrow responses in seven PV patients with profound molecular responses during and after long-term treatment with alpha-interferon 2b. All patients obtained a major molecular response (MMR). Subsequently all patients discontinued alpha-interferon and sustained complete hematological remission with a follow-up period of median 10 months (range 4-30 months). Complete normalization of the bone marrow was seen in three of five patients. Long term treatment with IFN2b is able to induce 'minimal residual disease' with very low JAK2 V617F allele burden and may induce profound, and in some patients total, regression of histomorphological bone marrow features of PV. Finally, hematological remissions and major molecular responses can be sustained after discontinuation of long-term treatment with IFN2b.

Udgivelsesdato: 2009-Dec Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P

Introduction: The JAK2 V617F mutation has become a key molecular marker in the diagnostic setting in Philadelphia chromosome negative chronic myeloproliferative (CMPDs) disorders, virtually all patients with polycythemia vera (PV) and approximately half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF) being JAK2 V617F positive. Apart from being a clonal marker of CMPD, a quantification of the JAK2 V617F allele burden may allow a stratification of phenotype in regard to degree of myeloproliferation and - although still controversial - risk of thrombosis and transformation to a myelofibrotic disease state. The treatment of choice in elderly patients is hydroxyurea (HU). Preliminary data on the efficacy of HU in lowering the JAK2 V617F allele burden have been conflicting (1–3). In the present study we show that long-term treatment with HU for a median of two years does not induce significant reductions in the JAK2 V617F allele burden in patients with CMPDs. Methods: 24 patients (newly diagnosed: PV=9, ET=3, PMF=2; non-newly diagnosed: PV=9, ET=1) were included. Quantitative PCR was performed on un-fractionized peripheral blood leukocytes every 3-6 months. Comparison of changes in median JAK2 V617F allele burdens was performed by linear regression analysis, calculating the slope of the regression line between medians at the various time points. Results: A median of 4.7 samples (range 3–7) were collected from each patient. Median follow-up was 24.2 months (range 11–36). Seventeen patients were treated with HU and four patients with anagrelide (ANA). Three patients not receiving cytoreductive therapy were also included. The median JAK2 V617F allele burden at baseline in HU treated patients including both newly diagnosed patients and pre-baseline HU treated patients was 33% (95% c.i.: 11–36%) and 24 % (95% c.i.: 13–48%) at the end of follow-up (median 24.2 months, range 11–36). Although indicating a small decline in JAK2 V617F allele burden, the slope of the regression line between medians were 6.7 (p=0.32 (95% c.i.: −7.1–20.7)) and accordingly not significant. Looking solely on newly diagnosed patients no significant reduction was recorded in median JAK2 V617F allele burden at baseline 34% (9–47%) as compared to 21% (6–55%) at end of study, (slope -0.31 (p=0.96) (95%c.i.: −14.6–13.9))). Some patients on HU had an initial decline in JAK2 V617F allele burden during the first 3–6 months of therapy (pt. no. 1, 4, 8, 18, 19 and 24). However, the overall change in JAK2 V617F allele burden during the first 3–6 months of therapy in newly diagnosed patients was not significant with a slope of the regression line between medians (baseline and 3–6 months) of 1.7, (p= 0.75 (95%, c.i.: −13.3– 9.8)). In patients treated with anagrelide there was a trend towards increasing JAK2 V617F allele burden (p=0.16), whereas in patients followed without myelosuppresive therapy the JAK2 V617F allele burden significantly increased during follow up (p=0.04). Conclusions: The present study of the dynamics of the JAK2 V617F allele burden during cytoreductive therapy with HU has clearly shown that HU has no or only modest effect on the JAK2 V617F allele burden. HU may have a capacity to prohibit clonal expansion to some extend. However, HU is cytostatic and only active against cycling precursors and has no selective clonal advantages. Our results are in accordance with the clinical experience of rapid increase in leukocyte and platelet counts when HU is discontinued contrasting the long lasting effects of immune therapy with alpha-interferon, which in a subgroup of patients has been shown to induce major and sustained molecular responses even after cessation of long-term treatment. Monitoring of molecular responses during conventional and novel targeted therapies is an important objective to be implemented in future clinical trials.

Objectives: Our aim is to investigate the expression of kit protein (KIT) and epidermal growth factor receptor (EGFR) in parotid carcinomas in order to correlate the expression to histology and prognosis. Further we want to perform mutation analysis of KIT-positive adenoid cystic carcinomas. Patients and Methods: Formalin-fixed paraffin-embedded sections from 73 patients with parotid gland carcinomas were used for the study. The sections were stained with both KIT and EGFR polyclonal antibodies. Twelve KIT-positive adenoid cystic carcinomas were examined for c-kit mutation in codon 816. Results: Of all carcinomas 25% were KIT-positive and 79% were EGFR-positive. Ninety-two percentage of the adenoid cystic carcinomas were KIT-positive. None of the adenoid cystic carcinomas had mutations in codon 816 of the c-kit gene. Conclusion: Neither KIT- nor EGFR-expression seem to harbour significant prognostic information. Adenoid cystic carcinomas express KIT, but no mutations in codon 816 of the c-kit gene were identified.

An epidemic of foot-and-mouth disease (FMD) causing a total of 23 cases in 1982–1983, primarily on the island of Funen, Denmark, was subjected to molecular epidemiological investigations. In an attempt to exploit the quasi-species nature of foot-and-mouth disease virus strains for molecular high-resolution strain identification in order to analyse the dynamics of this epidemic, full-length VP1 coding regions were sequenced for 17 isolates collected at different farms during the epidemic. The sequence information together with epidemiological information gathered during the epidemic suggests that the epidemic was caused by at least three introductions across Danish borders and one case of airborne transmission between two islands in Denmark over a distance of 70 km. The assortment of nucleotide markers among the three strains is indicative of common recombination events in their evolutionary history, and the prerequisite of co- or superinfection of animals with variant strains in turn implies that they have a common source or epidemiologically related sources originating from an area with endemic FMD.

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

Genetic variation of the glutamate-rich protein (GLURP) of Plasmodium falciparum was analysed in 29 field isolates and 15 laboratory lines of diverse geographical origin, by DNA sequencing of the non-repetitive 5′-region (R0) of the glurp gene. Polymorphism with respect to the length of the GLURP R2 repeat region was also analysed by a polymerase chain reaction method. As reference, the nucleotide sequence of the highly polymorphic 3′-region of the circumsporozoite protein gene was determined in the same isolates. It was found that even in the presence of variations in the GLURP R2 repeat region, immunodominant parts of the GLURP R0 region are surprisingly well conserved and the conservation is most pronounced in isolates from locations with very high malaria transmission. This might indicate that the R0 structure plays an important role in the parasite.

Background Treatment with pegylated interferon-alpha2 (IFN) is associated with complete hematologic remissions (CHR) in a large majority of patients with essential thrombocythemia (ET) and PV. In a subset of patients, IFN can induce major molecular remissions that can be sustained for up to 4 years after discontinuation of therapy. In hypercellular myelofibrosis (MF), IFN may also induce regression of bone marrow fibrosis. To this end, long-term IFN treatment with normalization of elevated blood counts is accompanied by a reduced risk of thrombosis and hemorrhage. When initiated at the early disease stage, IFN may have the greatest chance to induce “minimal residual disease” with a low JAK2 V617F allele burden. For these reasons, IFN is foreseen to be a cornerstone in the treatment of these neoplasms. Ruxolitinib (RUX) is a JAK1-2 inhibitor that is highly efficacious in alleviating hypermetabolic symptoms and enlarged spleens in patients with MF. Although the earliest reports suggested that RUX did not significantly lower JAK2 V617F mutational load, newer findings clearly show that in a subset of patients RUX may indeed profoundly decrease JAK2 V617F allele burden; these reductions could be even larger if treatment is instituted in early stages of MF or PV. Preliminary data show RUX to be highly efficacious in PV as well. With both drugs having potent antiproliferative and anti-inflammatory effects, combination therapy with IFN and RUX may potentially promote tumor regression in MPNs, as assessed by rapid induction of CHR, reduction in spleen size, and enhanced reduction of JAK2 V617F allele burden. Case report A 55-year-old woman with PV diagnosed in the precursor stage of ET 12 years ago was referred for a second opinion due to intolerance of hydroxyurea (HU; monosymptomatic fever) and pegylated interferon-alpha2b (PEG-Intron; exanthema). The patient had suffered 2 episodes of transitory ischemic attack (TIA) since 2009, and therefore, permanent treatment with clopidogrel had been instituted. Several CT scans were normal. At the time of referral, the patient was receiving anagrelide and clopidogrel. The patient's hemoglobin was 11.9 g/dL, her leukocyte count was 21.9 x 109/L, and platelets were elevated at 526 x 109/L. CRP levels were normal. An abdominal ultrasound revealed a spleen length of 20 cm, causing intermittent pain and abdominal discomfort. Because of hypermetabolic symptoms, pronounced abdominal discomfort, and intolerance of PEG-Intron and HU, treatment with RUX was initiated at a dose of 20 mg twice daily. Within the first 3 days of RUX therapy, the patient experienced remarkable clinical improvement with resolution of severe fatigue, night sweats, abdominal pain, and pruritus, which had negatively impacted the patient's quality of life during the past 2 to 3 years. Four days after starting RUX, the patient experienced acute TIA-like symptoms with transient decrease of strength in the left arm and abnormal sensations in the left half of the tongue and neck. The symptoms were similar to those that the patient experienced during previous TIAs. The blood values disclosed a slight decrease in the leukocyte count (17.6 x 109/L) and an increase in the platelet count (573 x 109/L). A CT scan was normal. Consequently, RUX therapy was combined with PEG-IFN-alpha2a (Pegasys) at a very low dose of 45 µg every second week. After 1 month, a CHR was achieved (Figure 1), and after 2.5 months, an abdominal ultrasound showed a spleen length reduction from 20 to 13.8 cm. After 6 months, the spleen was no longer palpable. The patient had no need of phlebotomies during treatment with RUX and Pegasys. The combination therapy was exceedingly well tolerated without any side effects to either drug. Remarkably, despite an advanced PV stage, JAK2 V617F allele burden was rapidly lowered from 90% mutated alleles at referral to 59% at 6 months and 20% within 1 year (Figure 2). Conclusion This case report demonstrates for the first time that combination therapy with RUX and “low–low-dose” Pegasys is highly efficacious in rapidly reducing JAK2 V617F allele burden in concert with achieving CHR and resolution of splenomegaly. This observation warrants prospective trials of this combinatorial approach in patients with PV and hyperproliferative MF to assess whether combination therapy induces a more rapid decline in the JAK2 V617F allele burden than monotherapy with either drug. Disclosures: Hasselbalch: Novartis: Consultancy, Honoraria, Speakers Bureau. Off Label Use: Interferon-alpha2a is used routinely in Denmark for treatment of essential thrombocythemia polycythemia vera and in some myelofrbrosis patients , as well. Ruxolitinib is being used routinely in myelofibrosis patients on strict indications including hypermetabolic symptoms and abdominaldiscomfort due to large splenomegaly ln rare cases Ruxolitinib is being used in the transitional stage of PV towards myelofibrosis when other options are not available due to intolerance to eg. interferonlor hydroryurea.

Background : Polycythemia vera (PV) is a clonal myeloproliferative disorder characterized by the presence of the JAK2V617 mutation in virtually all patients. Recently several studies have shown that the JAK2V617F mutational load decreases during treatment with alpha-interferon2 (1–6). Aim: To report on molecular and histomorphological bone marrow responses in seven PV patients with complete molecular remissions during and after long-term treatment with alpha-interferon 2b. Patients: Seven patients treated with alpha-interferon2b for a median of 84 months (range 31–120) are reported. In four of the patients alpha-interferon2b was started at the time of diagnosis and in three patients 9, 36 and 42 months from the time of diagnosis, respectively. Methods: The mutation was determined by allele specific PCR (n=2 only) (7) and quantitative PCR (qPCR) (n=5) (8). In three out of these patients qPCR JAK2V617F was performed on archived bone marrow from diagnosis (2 patients) and on peripheral blood (one patient) prior to treatment with alpha-interferon2b. A complete molecular remission (CMoR) was defined by less than 2 % JAK2 V617F mutated alleles (7). Results: Molecular Responses. All patients obtained a CMoR after a median of 84 months (29–120 months) of treatment with alpha-interferon2b. Subsequently all patients have discontinued alpha-interferon with a follow-up period of median 10 months (range 4–30 months) and sustained complete hematological remission. Furthermore, in three patients molecular responses have recently been updated – April and May 2008 - showing CMoRs in all (1,2 %, 0,9 % 0,1 % mutated alleles, respectively). Bone Marrow Responses. Follow-up bone marrow biopsies were available in five patients. Complete normalization of the bone marrow was seen in three patients after treatment with alpha-interferon2b for 84, 132 and 132 months, respectively. In the bone marrow from the patient being treated with alpha-interferon for 132 months a qPCR JAK2V617 analysis was performed detecting the mutation at a very low level (0,5 % mutated alleles). In two other patients, being treated with alpha-interferon2b for 24 and 120 months, respectively, and having obtained a CmoR in peripheral blood the bone marrow histomorphology showed marked regression of PV-features but in both patients still with focal areas displaying an increased number of morphologically abnormal megakaryocytes. Updated histomorphological and molecular response patterns will be presented. Discussion and Conclusion : Previous studies on the molecular response during alpha-interferon2a treatment have shown that a substantial proportion of patients achieve a significant molecular response after 12 months with a continuous decrease in the JAK2V617F mutation load at 24 and 36 months (1,5,6). This report confirms and extends preliminary data, showing that long-term treatment with alpha-interferon 2b in a subgroup of PV-patients is able to induce complete molecular remissions with normalization of the bone marrow morphology, which may even be sustained after discontinuation of alpha-interferon2b for up to 20 months (5). Prolonged treatment for several years seems necessary to induce such sustained responses, since treatment for only a few months has been reported to be followed by rapid recurrence of clonal hematopoiesis (9). In conclusion, a state of “minimal residual disease” may be achieved in PV by long-term immune therapy using alpha-interferon 2. Our observations call for large prospective clinical studies in which treatment with alpha-interferon is initiated up-front in patients with JAK2-positive PV and allied diseases. These studies should also aim at exploring the minimal dose of alpha-interferon needed to elicit complete molecular responses in order to minimize side effects of the drug and accordingly diminish the high drop-out rates reported in most previous studies.

The chaperone and calcium storing protein calreticulin is coded by CALR, and newly identified mutations in CALR are found in respectively 49-70% and 56-88% of JAK2- and MPL-negative patients with essential thrombocytaemia (ET) and primary myelofibrosis (PMF). A total of 41 mutations have been identified, all located to exon 9 which codes the protein's C-terminal. CALR mutations are present only in myeloid malignancies and confer a more indolent disease than JAK2-mutated ET and PMF. CALR mutations as a diagnostic and prognostic tool are promising and the mutations are potential targets for immune therapy.