Rosette-forming glioneuronal tumors share a distinct DNA methylation profile and mutations in FGFR1, with recurrent co-mutation of PIK3CA and NF1.

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.

Papillary glioneuronal tumor (PGNT) exhibits a characteristic methylation profile and fusions involving PRKCA.

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.

Multiplex polymerase chain reaction-based prognostic models in diffuse large B-cell lymphoma patients treated with R-CHOP.

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.

Expression and prognostic value of the WEE1 kinase in gliomas.

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 < 0.05) and grade II (mean 6.8%, p < 0.001) gliomas, high WEE1 protein was associated with better survival in GBMs (p = 0.002). This was confirmed in multivariate analysis (HR 0.60, p = 0.003) even when adjusted for MGMT status (HR 0.60, p = 0.005). In conclusion, we report a nuclear expression of WEE1 protein in all glioma grades and types. The WEE1 positive nuclear area was correlated with malignancy grade but it was inversely associated with prognosis in GBM. Although WEE1 is a frequently occurring protein and has been proposed as a novel target in GBM, the role of WEE1 in glioma patient survival appears to be connected to the MGMT status and is more complex than previously anticipated.

High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of >850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %-100 % and 0-59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96-8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.

Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses.

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.

External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds.

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.

Biweekly cetuximab and irinotecan as second-line therapy in patients with gastro-esophageal cancer previously treated with platinum.

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.

Synchronous detection of pancreatic adenocarcinoma and paraganglioma in a Whipple resection specimen.

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.

Biomarkers in tumors of the central nervous system - a review.

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.

Differential Dynamics of CALR Mutant Allele Burden in Myeloproliferative Neoplasms during Interferon Alfa Treatment.

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.

Minimal residual disease after long-term interferon-alpha2 treatment: a report on hematological, molecular and histomorphological response patterns in 10 patients with essential thrombocythemia and polycythemia vera.

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.

[New disease markers within the chronic myeloproliferative neoplasms]. / Nye sygdomsmarkører ved de kroniske myeloproliferative neoplasier.

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.

Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA.

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.

Long term molecular responses in a cohort of Danish patients with essential thrombocythemia, polycythemia vera and myelofibrosis treated with recombinant interferon alpha.

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.

High levels of nuclear MYC protein predict the presence of MYC rearrangement in diffuse large B-cell lymphoma.

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<0.0001). Receiver operating characteristic curve analysis showed ≥70% MYC tumor cells to be the optimal cutoff (sensitivity=100%, specificity=93%). Area under the receiver operating characteristic curve was 0.992, indicating that immunostaining for Myc protein is an excellent screening test to predict whether an MYC rearrangement is present.

Implementation of formalin-fixed, paraffin-embedded cell line pellets as high-quality process controls in quality assessment programs for KRAS mutation analysis.

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.

Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue.

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<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

Limited efficacy of hydroxyurea in lowering of the JAK2 V617F allele burden.

Besides being an invaluable marker of clonal disease in chronic myeloproliferative disorders (CMPDs), the JAK2 V617F mutation and the mutated allele burden have an impact on disease phenotype and may provide information on prognosis. Recently, hydroxyurea (HU) has been shown to induce a rapid decline in the JAK2 V617F allele burden. The aim of the present study was to assess the dynamics of the JAK2 V617F allele burden during long-term treatment with HU in a series of patients with CMPDs. The JAK2 V617F allele burden was determined by quantitative PCR in 24 patients of whom 17 received HU, four received anagrelide and three were followed without any cytoreductive therapy. During a median follow-up of 24.2 months, no significant reductions in the JAK2 V617F allele burden were seen in patients treated with HU. We conclude that HU has only a limited effect on the JAK2 V617F allele burden in CMPD.