Øystein Fluge

Molecular portraits of human breast tumours

Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cells identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.

Benefit from B-Lymphocyte Depletion Using the Anti-CD20 Antibody Rituximab in Chronic Fatigue Syndrome. A Double-Blind and Placebo-Controlled Study

Background Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients. Methods and Findings In this double-blind, placebo-controlled phase II study (NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m2 or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients. The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responders to Rituximab was 25 weeks (range 8–44). Four Rituximab patients had clinical response durations past the study period. General linear models for repeated measures of Fatigue scores during follow-up showed a significant interaction between time and intervention group (p = 0.018 for self-reported, and p = 0.024 for physician-assessed), with differences between the Rituximab and Placebo groups between 6–10 months after intervention. The primary end-point, defined as effect on self-reported Fatigue score 3 months after intervention, was negative. There were no serious adverse events. Two patients in the Rituximab group with pre-existing psoriasis experienced moderate psoriasis worsening. Conclusion The delayed responses starting from 2–7 months after Rituximab treatment, in spite of rapid B-cell depletion, suggests that CFS is an autoimmune disease and may be consistent with the gradual elimination of autoantibodies preceding clinical responses. The present findings will impact future research efforts in CFS. Trial registration ClinicalTrials.gov NCT00848692

Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis

Background:Enhancer of zeste homologue 2 (EZH2) is a member of the Polycomb group of genes that is involved in epigenetic silencing and cell cycle regulation.Methods:We studied EZH2 expression in 409 patients with colorectal cancer stages II and III. The patients were included in a randomised study, and treated with surgery alone or surgery followed by adjuvant chemotherapy.Results:EZH2 expression was significantly related to increased tumour cell proliferation, as assessed by Ki-67 expression. In colon cancer, strong EZH2 expression (P=0.041) and high proliferation (⩾40%; P=0.001) were both associated with better relapse-free survival (RFS). In contrast, no such associations were found among rectal cancers. High Ki-67 staining was associated with improved RFS in colon cancer patients who received adjuvant chemotherapy (P=0.001), but not among those who were treated by surgery alone (P=0.087). In colon cancers stage III, a significant association between RFS and randomisation group was found in patients with high proliferation (P=0.046), but not in patients with low proliferation (P=0.26). Multivariate analyses of colon cancers showed that stage III (hazard ratio (HR) 4.00) and high histological grade (HR 1.80) were independent predictors of reduced RFS, whereas high proliferation indicated improved RFS (HR 0.55).Conclusion:Strong EZH2 expression and high proliferation are associated features and both indicate improved RFS in colon cancer, but not so in rectal cancer.

Dose-densified chemoimmunotherapy followed by systemic central nervous system prophylaxis for younger high-risk diffuse large B-cell/follicular grade 3 lymphoma patients : results of a phase II Nordic Lymphoma Group study

BACKGROUND Many patients with aggressive B-cell lymphomas and high clinical risk score still die of lymphoma after conventional R-CHOP chemoimmunotherapy. We hypothesized that intensified chemoimmunotherapy including systemic central nervous system (CNS) prophylaxis improves outcome and reduces the incidence of CNS-related events. PATIENTS AND METHODS Inclusion criteria were age 18-65 years, primary diffuse large B-cell lymphoma or grade III follicular lymphoma without clinical signs of CNS disease and negative cerebrospinal fluid cytology, age-adjusted International Prognostic Index 2-3 and WHO performance score 0-3. Treatment consisted of six courses of R-CHOEP-14 followed by a course of high-dose cytarabine and a course of high-dose methotrexate. Primary end point was failure-free survival (FFS) at 3 years. RESULTS A total of 156 eligible patients with a median age of 54 years (range 20-64) were included. Three toxic deaths were observed. Three-year overall survival (OS) and FFS rates (median observation time 52 months for survivors) were 81% and 65%, respectively. Seven patients experienced CNS relapse, all within 6 months. CONCLUSIONS The results are promising with favorable 3-year OS and FFS rates, a low toxic death rate and a lower than expected number of CNS events. CNS progression might be further reduced by earlier CNS prophylaxis. CinicalTrials.gov. identifier NCT01502982.

NATH, a novel gene overexpressed in papillary thyroid carcinomas.

In this study a replica cDNA screening (RCS) approach to identify genes differentially expressed in papillary thyroid carcinomas (PTC) was used, as compared to non-neoplastic thyroid tissues. RCS is based on hybridization of radioactively labeled cDNA probes made from the biopsies to replica membranes with 15 000 clones from a PTC cDNA library. Among the genes overexpressed in PTC, and especially in clinically aggressive tumors with histologic evidence of poorly differentiated or undifferentiated areas, a novel gene named NATH was found. NATH has two mRNA species, 4.6 and 5.8 kb, both harboring the same open reading frame encoding a putative protein of 866 amino acids. The NATH protein is homologous to yeast N-acetyltransferase (NAT)1 and to mouse NARG1 (mNAT1) and contains four tetratricopeptide repeat (TPR) domains, suggesting that NATH may be part of a multiprotein complex. Overlapping RT–PCR fragments from several PTC biopsies confirmed the NATH mRNA sequence. Northern blots, semiquantitative RT–PCR experiments, TaqMan real-time RT–PCR experiments, and in situ hybridization verified the overexpression of NATH mRNA localized to tumor cells in PTC biopsies. NATH was expressed at a low level in most human adult tissues, including the normal thyroid gland. Increased NATH expression was seen especially in a Burkitt lymphoma cell line and in adult human testis. Recombinant in vitro expression showed that NATH protein was located mainly in the cytoplasm, and was present as a single protein band of the expected 105 kDa molecular weight. Heterologous expression of NATH in the papillary carcinoma cell line (NPA) and 293 cells did not alter the cellular proliferation rate. The biological function of NATH remains to be elucidated, but the overexpression in classic PTC and especially in poorly differentiated or undifferentiated components may indicate a function in the progression of papillary thyroid carcinomas.

Antibodies to β adrenergic and muscarinic cholinergic receptors in patients with Chronic Fatigue Syndrome.

Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in CFS point to an autoimmune disease directed against neurotransmitter receptors. Autoantibodies against G-protein coupled receptors were shown to play a pathogenic role in several autoimmune diseases. Here, serum samples from a patient cohort from Berlin (n=268) and from Bergen with pre- and post-treatment samples from 25 patients treated within the KTS-2 rituximab trial were analysed for IgG against human α and β adrenergic, muscarinic (M) 1-5 acetylcholine, dopamine, serotonin, angiotensin, and endothelin receptors by ELISA and compared to a healthy control cohort (n=108). Antibodies against β2, M3 and M4 receptors were significantly elevated in CFS patients compared to controls. In contrast, levels of antibodies against α adrenergic, dopamine, serotonin, angiotensin, and endothelin receptors were not different between patients and controls. A high correlation was found between levels of autoantibodies and elevated IgG1-3 subclasses, but not with IgG4. Further patients with high β2 antibodies had significantly more frequently activated HLA-DR+ T cells and more frequently thyreoperoxidase and anti-nuclear antibodies. In patients receiving rituximab maintenance treatment achieving prolonged B-cell depletion, elevated β2 and M4 receptor autoantibodies significantly declined in clinical responder, but not in non-responder. We provide evidence that 29.5% of patients with CFS had elevated antibodies against one or more M acetylcholine and β adrenergic receptors which are potential biomarkers for response to B-cell depleting therapy. The association of autoantibodies with immune markers suggests that they activate B and T cells expressing β adrenergic and M acetylcholine receptors. Dysregulation of acetylcholine and adrenergic signalling could also explain various clinical symptoms of CFS.

High dose chemotherapy with autologous stem cell support for patients with histologically transformed B-cell non-Hodgkin lymphomas. A Norwegian multi centre phase II study

We present a prospective phase II study of patients with relapse after chemotherapy showing transformation of follicular lymphoma to diffuse large B‐cell lymphoma, performed before rituximab was included in standard treatment. Patients in complete (CR) or partial remission (PR) after salvage chemotherapy were eligible for high‐dose chemotherapy with autologous stem cell support (HDT). Forty‐seven patients from five Norwegian centres were included, of whom 30 (63%) received HDT. Eighteen (60%) achieved CR, seven (23%) PR and five (10%) had progressive disease following HDT. Median follow‐up for the surviving patients was 75 months; median progression‐free (PFS) and overall survival (OS) were 26 and 47 months, respectively. Median OS for all patients was 43 months, compared to only 10 months for patients not eligible for HDT. Patients receiving CD34+ enriched/B‐cell depleted grafts had inferior PFS and a trend for inferior OS compared to patients receiving non‐purged grafts (Log Rank 0·025 and 0·151, respectively). In conclusion, two thirds of patients with transformation of follicular lymphoma were eligible for HDT. The majority of patients achieved CR and a considerable number had prolonged OS. The use of in vitro purged grafts did not result in a survival benefit compared to that of non‐purged grafts.

Final results of a randomised phase III study on adjuvant chemotherapy with 5 FU and levamisol in colon and rectum cancer stage II and III by the Norwegian Gastrointestinal Cancer Group

Background. The recommendation of adjuvant chemotherapy for colon cancer with lymph node metastases, based on two studies from USA, was reluctantly accepted by Norwegian medical doctors. It was therefore decided to assess the role of adjuvant therapy with 5fluorouracil (5-FU) combined with levamisole (Lev) in a confirmatory randomised study. Material and methods. Four hundred and twenty five patients with operable colon and rectum cancer, Stage II and III (Dukes’ stage B and C), were from January 1993 to October 1996, included in a randomised multicentre trial in Norway. The age limits were 18–75 years. Therapy started with a loading course of bolus i.v. 5-FU (450 mg/m2) daily for 5 days and p.o. doses of Lev (50 mg x 3) for 3 days. From day 28 a weekly i.v. 5-FU dose (450 mg/m2) were administered for 48 weeks. From day 28 also p.o. doses of Lev (50 mg x 3) for 3 days were given every 14 days. In total 214 patients were randomised to 5FU/Lev and 211 were included in the control group with surgery alone. Some did not comply with the inclusion and exclusion criteria, thus leaving 206 evaluable patients in each group. Results. There was no significant survival difference between the two groups at 5 years: Disease-free survival (DFS) was 73% after chemotherapy, 68% (p=0.24) in the control group, and corresponding cancer specific survival (CSS) 75% and 71%, respectively (p=0.69). There was no difference between the two groups when analysed for colon and rectum separately. However, the subgroup of colon cancer with stage III exhibited a statistically significant difference both for DFS, 58% vs. 37% (p=0.012) and CSS, 65% vs. 47% (p=0.032) in favour of adjuvant chemotherapy. The benefit was further statistically significant for women but not for men. Toxicity was generally mild and acceptable with no drug related fatalities. Conclusions. Colon cancer patients with lymph node metastases benefit from adjuvant chemotherapy with 5-FU/Lev with acceptable toxicity. In a subgroup analysis females did better than males. Rectal cancer does not benefit from this regimen.

Clinical impact of B-cell depletion with the anti-CD20 antibody rituximab in chronic fatigue syndrome: a preliminary case series

BackgroundChronic fatigue syndrome (CFS) is a disease of unknown aetiology. A patient with CFS had unexpected, marked recovery of CFS symptoms lasting for five months during and after cytotoxic chemotherapy for Hodgkins disease. We reasoned that the transient CFS recovery was related to methotrexate treatment, which induces immunomodulation in part through B-cell depletion.MethodsIn a case series, this patient and two additional CFS patients were B-cell depleted by infusion of the monoclonal anti-CD20 antibody rituximab.ResultsAll three had improvement of all CFS symptoms. Patients 1 and 2 had major amelioration from 6 weeks after intervention, patient 3 slight improvement from the same time, but then improved markedly from 26 weeks after intervention. The symptomatic effect lasted until weeks 16, 18 and 44, respectively. At relapse, all were retreated with a single (patient 1) or double rituximab infusion (patients 2 and 3). Again, all three had marked symptom improvement, mimicking their first response. After new symptom recurrence, patients 1 and 2 were given weekly oral methotrexate, patient 1 having effect also from this agent. Patients 1 and 2 were again treated for a third rituximab infusion after new relapse, again with a marked clinical benefit. No unexpected toxicity was seen.ConclusionThese observations suggest that B-lymphocytes are involved in CFS pathogenesis for a subset of patients. Benefit for all CFS symptoms, the delayed symptom relief following B-cell depletion, the kinetics of relapses, and the effect also from methotrexate treatment, provide suggestive evidence that B-cells play a significant role in the ongoing clinical features, and that CFS may be amenable to therapeutic interventions aimed at modifying B-cell number and function. More systematic investigations of this therapeutic strategy, and of its biological basis, are now needed.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT–PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cystein-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.