Wolfgang Eisterer

CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia

OBJECTIVE Expression of the catalytic subunit of the telomerase enzyme hTERT is essential for prolonging the replicative lifespan and is the rate-limiting step in cellular immortalization and carcinogenesis. Because hTERT expression is positively correlated with telomerase activity, its regulation is suggested as the major determinant of enzymatic activity. The hTERT promoter region contains two CpG islands, which are known to be target sites for de novo DNA methylation. To elucidate the impact of this epigenetic mechanism on telomerase activity, we analyzed the degree of hTERT promoter methylation in 30 patients with B-cell chronic lymphocytic leukemia. MATERIALS AND METHODS hTERT promoter methylation was assessed using a methylation-specific competitive polymerase chain reaction assay. The assay is based on digestion of genomic DNA with a methylation-sensitive restriction enzyme before amplification with an internal standard. RESULTS Patients exhibiting high telomerase activity showed significantly less methylation of the hTERT promoter core domain than patients with low enzyme activity. In addition, telomerase activity was significantly associated with telomere length and overall survival. CONCLUSIONS Our data show that the degree of CpG island methylation of the hTERT promoter exhibits an impact on telomerase activity in a subgroup of patients with B-cell chronic lymphocytic leukemia and therefore is assumed to play a role in regulating hTERT gene expression in these patients.

Preoperative treatment with capecitabine, bevacizumab and radiotherapy for primary locally advanced rectal cancer – A two stage phase II clinical trial

BACKGROUND AND PURPOSE The aim of this single-arm multicenter phase II clinical trial was to assess the feasibility and tolerability of preoperative radiotherapy and simultaneous capecitabine and bevacizumab. Secondary endpoints were downstaging-rate and induction of complete pathological response. MATERIAL AND METHODS Patients with cT3 rectal cancer were eligible. Capecitabine (825 mg/sqm twice daily on radiotherapy-days weeks 1-4) and bevacizumab (5 mg/kg on days 1, 15 and 29) were administered concurrently to pelvic radiotherapy (1.8 Gy daily up to 45 Gy in 5 weeks). Surgery followed 6-8 weeks later. A two-stage trial was designed with early termination at eight patients if more than three patients had experienced a common toxicity criteria ≥grade 3 according to the NCI CTC guidelines. RESULTS In the first stage eight patients were enrolled. Median age was 70 years (range 55-76) and ECOG PS 0/1 (%) was 87.5/12.5. Major side effects were mostly intestinal bleeding (grade 3, 25%), diarrhea (grade 3, 25%), perianal and abdominal pain (grades 3 and 4, 25%) followed by anemia (grade 3, 12.5%). Tumor downstaging was observed in 37.5% of patients with complete pathological response in two patients (25%). CONCLUSIONS After interim analysis of feasibility and tolerability, accrual was terminated according to protocol due to ≥grade 3 toxicities in 50% of patients. Complete pathological response was seen in 25% of patients but was accompanied by considerable toxicity. Further clinical trials are needed to clarify the role of bevacizumab in this setting.

Safety and efficacy of regorafenib in patients with advanced soft tissue sarcoma (REGOSARC): a randomised, double-blind, placebo-controlled, phase 2 trial

BACKGROUND Regorafenib is a multikinase inhibitor with proven activity in refractory gastrointestinal stromal tumours and chemotherapy-refractory advanced colorectal cancers. We assessed this agents efficacy and safety in patients with metastatic soft tissue sarcomas previously treated with anthracycline. METHODS In this randomised, double-blind, phase 2 trial undertaken in France and Austria, we enrolled patients aged 18 years and older with advanced soft tissue sarcomas who had received previous doxorubicin or other anthracycline treatment. These patients were randomly assigned (1:1) into one of the following four cohorts: liposarcoma, leiomyosarcoma, synovial sarcoma, and other sarcomas. Participants were treated with oral regorafenib (160 mg per day 3 weeks on and 1 week off) or matched placebo. Patients receiving placebo were offered optional crossover in case of centrally confirmed disease progression. The random allocation schedule was computer-generated with permuted blocks of four patients, with two stratification factors: country (France or Austria) and previous exposure to pazopanib (yes or no). Eligibility criteria included patients with histologically proven advanced and inoperable soft tissue sarcomas with intolerance or failure to doxorubicin or other anthracycline-based chemotherapy and at least one unidimensionally or bidimensionally measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1). The primary endpoint was RECIST-based progression-free survival after central radiological review in the intention-to-treat population. Patients, physicians, and radiologists of the panel were masked to treatment allocation. This study is still open for recruitment for an additional stratum (patients previously treated with pazopanib) and registered with ClinicalTrials.gov, NCT01900743. FINDINGS From Aug 5, 2013, to Nov 26, 2014, 182 patients were randomly assigned to one of four cohorts and included in the final analysis. At the cutoff date (Jan 7, 2016), the number of required events was reached for the four cohorts. In the liposarcoma cohort, progression-free survival was 1·1 months (95% CI 0·9-2·3) with regorafenib versus 1·7 months (0·9-1·8) with placebo (HR 0·89 [95% CI 0·48-1·64] p=0·70). In the leiomyosarcoma cohort, progression-free survival was 3·7 months (95% CI 2·5-5·0) with regorafenib versus 1·8 (1·0-2·8) months with placebo (HR 0·46 [95% CI 0·46-0·80] p=0·0045). In the synovial sarcoma cohort, progression-free survival was 5·6 months (95% CI 1·4-11·6) with regorafenib versus 1·0 (0·8-1·4) with placebo (HR 0·10 [95% CI 0·03-0·35] p<0·0001). In the other sarcoma cohort, progression-free survival was 2·9 months (95% CI 1·0-7·8) with regorafenib versus 1·0 (0·9-1·9) with placebo (HR 0·46 [95% CI 0·25-0·81] p=0·0061). Before crossover, the most common clinically significant grade 3 or higher adverse events were arterial hypertension (17 [19%] events in the 89 patients in the regorafenib group vs two [2%] events in the 92 patients in the placebo group), hand and foot skin reaction (14 [15%] vs no events) and asthenia (12 [13%] vs six [6%]). One treatment-related death occurred in the regorafenib group due to liver failure. INTERPRETATION Regorafenib has an important clinical antitumour effect in non-adipocytic soft tissue sarcomas, improving progression-free survival. Regorafenib should be further evaluated in this setting, and its therapeutic role has to be defined in the context of the growing therapeutic armamentarium, already including one approved multikinase inhibitor, pazopanib. FUNDING Bayer HealthCare.

Decreased potency of MDR‐modulators under serum conditions determined by a functional assay

Summary. A variety of agents are capable of overcoming P‐glycoprotein‐mediated multidrug resistance (MDR) in vitro. However, the clinical potential of these compounds is often limited due to high plasma protein binding. We compared the efficacy of several MDR‐reversing compounds in serum‐free culture medium and under serum conditions by means of a functional assay. Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rhl23) was measured from normal peripheral blood CD8+ T‐lymphocytes which express low levels of P‐glycoprotein. Inhibition of Rhl23 efflux by R‐verapamil, dexnigludipine‐HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum‐free medium and in serum at concentrations from 0.1 to 50μmol/l. With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P‐glycoprotein under serum conditions at concentrations achievable in vivo. The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5μxmol/1 a sufficient inhibitory effect was observed. Subsequently this approach was applied to patients suffering from B‐cell chronic lymphocytic leukaemia (B‐CLL; n= 3) and acute myeloid leukaemia (AML; n= 2) which were positive in the Rh l23 efflux assay. As for normal CD8+ T‐lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rhl23 efflux from the leukaemic cells. Thus the interpretation of results of clinical‘modulator’trials should consider the decreased bioavailability of MDR‐reversing agents.

Preoperative Oxaliplatin, Capecitabine, and External Beam Radiotherapy in Patients with Newly Diagnosed, Primary Operable, cT3NxM0, Low Rectal Cancer

AbstractPurpose:In patients with locally advanced rectal cancer (LARC), preoperative chemoradiation is known to improve local control, and down-staging of the tumor serves as a surrogate for survival. Intensification of the systemic therapy may lead to higher downstaging rates and, thus, enhance survival. This phase II study investigated the efficacy and safety of preoperative capecitabine and oxaliplatin in combination with radiotherapy.Patients and Methods:Patients with LARC of the mid and lower rectum, T3NxM0 staged by MRI received radiotherapy (total dose 45 Gy) in combination with oral capecitabine (825 mg/m2 twice a day on radiotherapy days; weeks 1–4) and oxaliplatin 50 mg/m2 intravenously (days 1, 8, 15, and 22). Efficacy was evaluated as rate of tumor down-categorization at the T level.Results:A total of 59 patients were enrolled (19 women, 40 men; median age of 61 years) and all were evaluable for efficacy and toxicity. Down-categorization at the T level was observed in 53% with pathological complete response in 6 patients (10%). Actual total radiotherapy, oxaliplatin and capecitabine doses received were 97%, 90%, and 93% of the protocol-specified preplanned doses, respectively. Grade 3/4 toxicity was observed in 15 patients (25%). The most frequent was diarrhea (12%).Conclusions:Preoperative chemoradiation with capecitabine and oxaliplatin is feasible in patients with MRI-proven cT3 LARC. The only clinically relevant toxicity was diarrhea. Overall, efficacy of the multimodality treatment was good, but not markedly exceeding that of 5-FU- or capecitabine-based chemoradiation approaches.ZusammenfassungZiel:Eine präoperative Radiochemotherapie verbessert bei Patienten mit einem tief sitzenden Rektumkarzinom (LARC) die lokale Tumorkontrolle und ein so genanntes „down-staging“ dient als Überlebenssurrogatparameter. Von einer Dosisintensivierung der systemischen Therapie kann man sich höhere Down-Staging-Raten erwarten und damit das Überleben verbessern. Diese multizentrische Phase-II-Studie soll die Wirksamkeit und Toxizität einer neoadjuvanten durch Capecitabin und Oxaliplatin intensivierten Radiochemotherapie prüfen.Patienten und Methodik:Patienten mit einem LARC, das mittels MRI als cT3NxM0 klassifizierten wurde, erhielten eine Radiotherapie (45 Gy in konventioneller Fraktionierung) mit konkomitanter Gabe von Capecitabin (oral 2 x täglich 825 mg an den Bestrahlungstagen, Woche 1–4) und Oxaliplatin intravenös 50mg/m2 (an den Tagen 1, 8, 15 und 22). Die Rate an Tumor-Down- Categorization dient als Parameter der Wirksamkeit.Ergebnisse:59 Patienten (davon 68% männlich, mittleres Alter 61 Jahre) wurden in die Studie eingeschlossen. Eine Down- Categorization in der T-Kategorie wurde in 53% der Patienten beobachtet, wobei 6 Patienten (10%) eine komplette pathologische Remission zeigten. Die tatsächlich verabreichte Strahlendosis betrug 97%, die Capecitabindosis 93% und die Oxaliplatindosis 90% der im Protokoll festgelegten Gesamtdosis. Akute Nebenwirkungen CTC-Grad ≥3 (Common Toxicity Criteria) wurden in 15 Patienten (25%) registriert, wobei mit 12% eine Diarrhoe am häufigsten vorkam.Schlussfolgerung:Eine präoperative Radiochemotherapie mit Capecitabin und Oxaliplatin ist bei Patienten mit mittels MRI diagnostiziertem cT3 LARC gut durchführbar. Die einzige klinisch relevante Nebenwirkung war eine Diarrhoe. Allerdings übertrifft die Wirksamkeit nicht wesentlich die bisherigen Erkenntnisse von Studien mit kontinuierlicher 5-Fluorouracil- oder alleiniger Capecitabingabe.

A randomized study comparing interferon (IFNα) plus low-dose cytarabine and interferon plus hydroxyurea (HU) in early chronic-phase chronic myeloid leukemia (CML)

This multicenter randomized phase III study was designed to compare the efficacy and toxicity of IFN alpha-2c (3.5 MU/d) in combination with either araC (10 mg/m(2) d1-10) or hydroxyurea (HU: 25 mg/kg per day) in newly diagnosed CML patients. A total of 114 patients were randomized. Following a median observation period of 36 (range 1-73) months the major cytogenetic response rates were 25 and 27% and the 4-year survival probabilities 62.5 and 63% for the araC and HU group, respectively. While the overall toxicity profile was comparable between both groups, patients in the HU arm exhibited a slightly higher degree of WHO grades 3 and 4 non-hematological toxicities.

Postoperative Morbidity Following Chemoradiation for Locally Advanced Low Rectal Cancer

BackgroundPostoperative morbidity remains a significant clinical problem and may alter long-term outcome particularly after neoadjuvant chemoradiation in patients with locally advanced low rectal cancer. The aim of the present study was to identify a potential long-term effect of postoperative morbidity.MethodsAnalysis of prospectively collected data of 90 consecutive patients who underwent neoadjuvant chemoradiation and curative mesorectal excision for locally advanced (cT3/4, Nx, M0/1) adenocarcinoma of the mid and lower third of the rectum during a 7-year period (1996–2002).ResultsMajor postoperative complications occurred in 17.8% and minor complications in 26.6% of patients. Hospital mortality and 30-day mortality was 0%. Infectious complications were seen in 34.5%. The leading causes of infectious complications were anastomotic leakage and perineal wound infection. Postoperative morbidity was statistically significantly associated with gender (P < 0.05), pre-therapeutic haemoglobin level (P < 0.05), ASA score (P < 0.05), hospitalisation (P < 0.001) and clinical long-time course (P < 0.01). Moreover, early postoperative morbidity was proven as an independent prognostic factor concerning disease-free (P < 0.05) and overall survival (P < 0.05).ConclusionEarly postoperative morbidity in patients with preoperative chemoradiation due to locally advanced low rectal cancer is demonstrated as an independent prognosticator. Gender, pre-therapeutic haemoglobin level and ASA score indicate patients at risk for early postoperative complications and may therefore serve as predictive features.

Interferon-α for the treatment of elderly patients with chronic myeloid leukaemia

The present retrospective analysis is based on data of 213 patients with chronic myeloid leukaemia (CML). They were treated with interferon (IFN)a-2C (Berofor ® ) at daily doses of 3.5 MU subcutaneously (s.c.), alone or in combination with low-dose ara-C or hydroxyurea, according to four consecutive studies of the Austrian CML Study Group. Comparisons were made between 41 patients aged ]60 years and 172 younger patients. The elderly patients (median: 64 years; range: 60‐73) showed similar pretreatment characteristics compared with the younger group, but included a higher percentage of Sokal Stage three (51 vs 20%). Median observation periods were similar (38 vs 39 months), whereas the duration of IFNa treatment was shorter in the elderly group (median 57 vs 42 weeks). The rate of overall haematological responses (73 vs 78%) and complete haematological response (44 vs 54%), was similar in both cohorts. Differences seen in partial (5 vs 12%) and complete cytogenetic response (10 vs 13%), were not statistically significant, but a tendency in favour of the younger cohort had to be noted. Summing up, in elderly patients acceptable rates of haematological and cytogentic response can be expected after treatment with IFNa alone or in combination with LD ara-C or HU.

CYTOKINE PRIMING OF THE GRANULOCYTE RESPIRATORY BURST IN MYELODYSPLASTIC SYNDROMES

Increased susceptibility to infections in patients with myelodysplastic syndromes (MDS) is thought to be due to neutropenia as well as functional abnormalities of neutrophils. In the present study we examined the effect of two different stimulants (fMLP, PMA) and three cytokines (alphaTNF, G-CSF and GM-CSF), both singly and in combination on granulocyte (RB) in 25 MDS patients compared to seven healthy controls. Single fMLP and PMA-stimulation showed similar results for both groups. Preincubation with cytokines enhanced fMLP-stimulated RB in most MDS patients and controls, but in patients to a significantly lesser extent when compared to the control group (p < or = 0,05). Combinations of alphaTNF + GM-CSF and alphaTNF + G-CSF were highly synergistic in priming fMLP-stimulated burst in both groups. But again, as with the single cytokine priming this effect was markedly reduced in MDS patients compared to controls (p < or = 0,05). A specific priming defect for one of the cytokines or a cytokine combination could not be demonstrated. Serum alphaTNF levels were measured in 18 and neutrophil alkaline phosphatase (NAP) index in 23 patients. Results did not correlate with variations of the RB in MDS patients. We conclude that reduced alphaTNF, GM-CSF and G-CSF priming of granulocyte RB is a frequent finding in MDS and may contribute to the enhanced susceptibility to bacterial infections.

Treatment of patients with advanced chronic myelogenous leukemia with interferon-alpha-2b and continuous oral cytarabine ocfosfate (YNK01): a pilot study

The efficacy of continuous oral cytarabine ocfosfate (YNK01) (300 mg/day) in combination with interferon alpha (IFNalpha, 5x10(6) IU/day) was evaluated in patients with advanced chronic myelogenous leukemia, who previously failed to respond to IFNalpha-based therapies. Dose escalations up to 900 mg YNK01 were allowed in patients who failed to respond. In view of our results, four patients developed a complete hematological response after YNK01 was started. Among those who initially responded to YNK01, one complete cytogenetic response was achieved 18 months later. Although the data are preliminary, this is the first study showing that continuous administration of YNK01 along with IFNalpha is effective in patients with advanced chronic myelogenous leukemia.