Guido Lammering

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease.

BackgroundTumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.Materials and methodsWe prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.ResultsWe found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).ConclusionThe detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Curcumin inhibits ultraviolet light induced human immunodeficiency virus gene expression.

Recently, we reported that the herbal drug St. Johns Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat/HeLa cells [35]. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-κB are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism by which curcumin inhibits UV-activated HIV-LTR gene expression. We found that treatment of HIVcat/HeLa cells with micromolar concentrations of curcumin completely abolished UV activation of HIV gene expression. Curcumin treatment at similar doses as those used to inhibit HIV gene expression also effectively blocked UV activation of NF-κB, as demonstrated by electrophoretic mobility shift assay. In contrast, curcumin did not inhibit UV-induced phosphorylation of p38 MAP kinase. This observation was also supported by findings that curcumin did not inhibit UV-induced phosphorylation of CREB/ATF-1 and ATF-2. Although curcumin was ineffective in preventing UV-induced p44/42 MAP kinase phosphorylation, the JNK (1 and 2) and AP-1 activation were efficiently blocked by curcumin in HeLa cells. We conclude that the mechanism by which curcumin modulates UV activation of HIV-LTR gene expression mainly involves the inhibition of NF-κB activation.

Mood‐Enhancing Antidepressant St. John's Wort Inhibits the Activation of Human Immunodeficiency Virus Gene Expression by Ultraviolet Light

Ultraviolet (UV) radiation is a potent activator of the human immunodeficiency virus (HIV) gene expression in a HeLa cell clone with stably integrated copies of the HIVcat reporter construct. Recently, we have shown that activation of p38 MAP kinase and NF‐ κB is necessary but not sufficient for triggering efficient HIV gene expression in response to UV. Here we demonstrate that St. Johns wort is a potent inhibitor of the UV‐induced activation of HIV gene expression in HeLa cells. Stably transfected HIVcat/HeLa cells were preincubated with different amounts (25‐100 μl) of St. Johns wort or gingko biloba extracts for 30 min, then irradiated with UV (30 J/m 2 ). In contrast to ginkgo biloba, St. Johns wort inhibited the UV‐induced HIV gene expression in a dose‐dependent manner. Furthermore, preincubation with St. Johns wort (10, 20, and 30 μl) for 30 min before UV (30 J/m 2 ) irradiation, PMA‐ and UV‐induced NF‐ κB activation was completely blocked, whereas ginkgo biloba did not affect the PMA‐ and UV‐induced NF‐ κB activation in HeLa cells. UV activation of p38 MAP kinase was not inhibited by St. Johns wort or by ginkgo biloba. However, we found that p38 MAP kinase and JNK1 and ‐2 were activated by St. Johns wort, but p44/42 MAP kinase was not activated by St. Johns wort in HeLa cells. Hypericin an active ingredient in St. Johns wort also inhibited the UV activation of HIV gene expression in HeLa cells. These results firmly confirm that St. Johns wort is a potent inhibitor of the UV‐induced activation of HIV gene expression in HeLa cells.

Cystatin C - a fast and reliable biomarker for glomerular filtration rate in head and neck cancer patients.

Purpose:Determination of renal function is a prerequisite for planning therapy in cancer patients. Limitations of creatinine as marker for the glomerular filtration rate (GFR) led to the proposal of cystatin C as a more accurate biomarker especially in mild renal insufficiency or in patients with low muscle mass. We compared the accuracy of cystatin C- and creatinine-based equations to estimate GFR in head and neck cancer (HNC) patients receiving platinum-based radiochemotherapy.Patients and Methods:The study population consisted of 52 HNC patients (GFR range, 37–105 mL/min/1.73 m2 complemented by 17 patients with known renal insufficiency (GFR range, 10–60 mL/min/1.73 m2). Intraclass correlation coefficients were calculated between the reference method (51)Cr-EDTA clearance and estimated GFR by creatinine clearance and equations based on creatinine (Cockroft-Gault, modification of diet in renal disease (MDRD), Wright) or cystatin C (Larsson, Dade-Behring, Hoek). In addition, sensitivity and specificity to discriminate GFR > 60 mL/min/1.73 m2 were evaluated by receiver operating characteristic curve (ROC).Results:The highest correlation coefficients were found for the cystatin C-based estimates in comparison with creatinine-based estimates or creatinine clearance, even though Bland-Altman plots revealed GFR overestimation for all equations tested. The cystatin C-based Hoek formula exhibited the highest overall precision and accuracy. GFR of < 60 mL/min/1.73 m2 was assumed as a cut-off for chemotherapy. ROC analyses revealed the highest AUC to predict a GFR > 60 mL/min/1.73 m2 for the creatinine-based Wright formula, closely followed by the MDRD formula and cystatin C-based equations of Larsson, Dade-Behring, and Hoek.Conclusion:Cystatin C-based GFR estimates showed the overall strongest correlation to the reference method. Thus, we recommend cystatin C for GFR estimation in HNC patients as an alternative method to the estimated creatinine clearance in clinical practice.ZusammenfassungZielsetzung:Bei Tumorpatienten ist die exakte Bestimmung der Nierenfunktion eine wichtige Voraussetzung für die individuelle Therapieplanung. Die Aussagefähigkeit des Serumkreatinins als Marker der glomerulären Filtrationsrate (GFR) ist jedoch limitiert aufgrund seiner Abhängigkeit von der Muskelmasse sowie fehlendem Anstieg der Serumkonzentration bei einer GFR > 60 ml/ min/1,73 m2. Cystatin C wird als sensitiverer Parameter für das Vorliegen einer Niereninsuffizienz in den Frühstadien sowie bei Muskelschwachen Patienten diskutiert. In der vorliegenden Studie wurde die Präzision von Cystatin-C-basierten sowie Kreatinin-basierten Formeln zur Berechung der GFR in Kopf-Hals-Tumorpatienten verglichen.Patienten und Methodik:Die Studienkohorte bestand aus 52 Kopf-Hals-Tumorpatienten (GFR 37–105 mL/min/1,73 m2) sowie 17 Patienten mit bekannter Niereninsuffizienz Stadium 3–5 (GFR 10–60 mL/min/1,73 m2). Es wurde der Intra-Class-Correlation factor berechnet zwischen der Referenzmethode (51)Cr-EDTA-Clearance und der Kreatinin-Clearance, den Kreatinin-basierten Formeln (Cockroft-Gault, Modified Diet in Renal Disease, Wright) und den Cystatin-C-basierten Formeln (Larsson, Dade-Behring, Hoek) zur GFR-Berechnung. Zusätzlich ermittelten wir Sensitivität und Spezifität der verschiedenen Clearance-Bestimmungen zur Erkennung einer GFR > 60 ml/min/1,73 m2 mittels Receiver Operating Characteristic Curve (ROC).Ergebnisse:Die beste Korrelation zur Referenzmethode wurde für die Cystatin-C-basierten Formeln zur GFR-Bestimmung im Vergleich zu Kreatinin-basierten Formeln oder der Kreatinin-Clearance ermittelt. Jedoch zeigte der Bland-Altman-Plot, dass im Vergleich zur Referenzmethode alle Formeln – sowohl Cystatin-C- als auch Kreatinin-basiert – die Nierenfunktion überschätzen. Die höchste Genauigkeit und Präzision wurde bei der Hoek-Formel beobachtet. Da eine GFR < 60 ml/min/1,73 m2 häufig als Grenze angesehen wird zur Durchführung einer Chemotherapie, ermittelten wir die Präzision, mit der die verschiedenen GFR-Bestimmungen dies für den Einzelnen voraussagen konnten. Bei der ROC-Analyse zeigte die Kreatinin-basierte Formel nach Wright die höchste Area Under the Curve, dicht gefolgt von der Modified-Diet-in-Renal-Disease-Formel und den Cystatin-C-basierten Formeln nach Larsson, Dade-Behring und Hoek.Schlussfolgerung:Die Cystatin-C-basierten Formeln zur GFR-Berechnung zeigten insgesamt die beste Präzision und Korrelation zur Referenzmethode in Kopf-Hals-Tumorpatienten. Daher empfehlen wir Cystatin-C-basierte Formeln zur GFR-Berechnung im klinischen Alltag bei diesen Patienten.

Kleinvolumige hypofraktionierte Radiotherapie vor totaler mesorektaler Exzision

Fragestellung: Ist die präoperative hypofraktionierte Strahlentherapie des Rektumkarzinoms mit 5-mal 5 Gy eine gut verträgliche neoadjuvante Behandlungsmodalität? Methodik: Von Dezember 1996 bis März 1999 wurden 34 Patienten mit lokal fortgeschrittenem Rektumkarzinom einer präoperativen Strahlentherapie mit 5-mal 5 Gy zugeführt. Die kleinvolumige Bestrahlung erfolgte in CT-geplanter Drei- oder Vier-Felder-Technik mit ventrodorsalen Gegenfeldern bzw. dorsalen Feldern der Größe 9 ± 2 cm × 11,5 ± 2,4 cm und bilateralen opponierendenFeldern der Größe 9 ± 1,5 cm × 11,5 ± 2 cm mit 6- bis 25-MV-Photonen. Die Operation erfolgte nach 14 ± 6 Tagen als anteriore Resektion mit totaler mesorektaler Exzision (82%) oder als abdomino-perineale Amputation (18%) bei 33/34 Patienten. Bei Lymphknotenbefall und/oder pT3/4-Läsionen wurde eine adjuvante Chemotherapie mit 5-Fluoruracil (5-FU) durchgeführt. Die mediane Nachbeobachtungszeit beträgt 189 Tage (Streuung: 15 bis 548 Tage). Ergebnisse: Akute Nebenwirkungen waren: erhöhte Stuhlfrequenz (4/34), Abgespanntheit (2/34), Schmerzen in den Leisten (1/34), Übelkeit und perianales Brennen (1/34), Schwindel (1/34), leichter Harnverhalt (1/34). Ein Patient verstarb bei bekanter Herzinsuffizienz NYHA-Grad III nach 21 Tagen an einem Myokardinfarkt. Die klinischen T- und N-Kategorien ergaben 3, 29 und 2 für T4, T3 und unbekannt und 20, 11 und 3 für N+, N− und unbekannt. Die pathologischen T- und N-Kategorien ergaben 3, 19 und 11 für T4, T3 und T2 und 19 und 14 für N+ und N−. Bei 32/33 operierten Patienten erfolgte eine R0-Resektion, ein weiterer Patient wurde bei Peritonealbefall R1-reseziert. Das intraoperative Staging zeigte bei drei Patienten eine viszerale Metastasierung. Postoperativ kam es bei jeweils zwei Patienten zu Analstomoseninsuffizienzen und Magen- bzw. Darmatonie. In jeweils einem Fall traten eine Dünndarmhernierung, Wundheilungssörung, Wunddehiszenz und passagere Niereninsuffizienz auf. Schlussfolgerung: Die präoperative kleinvolumige hypofraktionierte Strahlentherapie lokal fortgeschrittener Rektumtumoren ist bei moderater Toxizität gut durchführbar. In Einzelfällen wurde trotz des kurzen Zeitintervalls bis zur Operation ein Downstaging erreicht.Purpose: Is preoperative short-term radiotherapy of operable rectal carcinoma feasible with regard to early side effects and perioperative complications? Patients and Methods: As of December 1996 to March 1999, 34 patients with locally advanced rectal cancer have been irradiated preoperatively with 5 times 5 Gy. After CT-planning, radiotherapy was administered using a 3-field or 4-field box technique with 2 anterior-posterior fields or a posterior field of 9 ± 2 cm × 11.5 ± 2.4 cm and 2 opposed bilateral fields of 9 ± 1.5 cm × 11.5 ± 2 cm with 6- to 25-MV photons. Surgery was performed 14 ± 6 days after irradiation in 33/34 patients (82% anterior resection with total mesorectal excision, 18% abdomino-perineal resection). Patients with a positive lymph node status or pT3/4 lesions underwent adjuvant chemotherapy with 5-Fluorouracil (5-FU). The median follow-up period is 189 days (range: 15 to 548 days). Results: The following early side reactions were registered: increased bowel movements (4/34), fatigue (2/34), pain in the groins (1/34), nausea and perianal smart (1/34), vertigo (1/34), temporary urinary obstruction (1/34). One patient with heart failure NYHA Grade III died of a heart attack after 21 days. Preoperative T and N categories showed a distribution of 3, 29 and 2 for T4, T3 and unknown and 20, 11 and 3 for N+, N− and unknown; postoperative T and N categories showed a distribution of 3, 19 and 11 for T4, T3 and T2 and 19 and 14 for N+ and N−. In 32 of 33 patients tumorfree margins were achieved. One patient with peritoneal metastases had a R1 resection. In 3 patients metastases were detected intraoperatively. Perioperative complications were: 2 cases of leaking anastomosis and postoperative bowel atonia, 1 case with bowel obstruction, delayed wound healing, wound dehiscence and temporary renal dysfunction. Conclusion: Preoperative radiotherapy is feasible with moderate toxicity and is able to induce down staging despite the short time interval between radiotherapy and surgery.