Gene conversion is a likely cause of mutation in PKD1

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.      

Phase I/II trial of capecitabine, oxaliplatin, and radiation for rectal cancer.

PURPOSE: The purpose of this study was to establish the feasibility and efficacy of preoperative radiotherapy (RT) with concurrent capecitabine and oxaliplatin (XELOX-RT) in patients with rectal cancer. PATIENTS AND METHODS: Thirty-two patients with locally advanced (T3/T4) or low-lying rectal cancer received preoperative RT (total dose, 50.4 Gy). Capecitabine was administered concurrently at 825 mg/m2 bid on days 1 to 14 and 22 to 35, with oxaliplatin starting at 50 mg/m2 on days 1, 8, 22, and 29 with planned escalation steps of 10 mg/m2. End points of the phase II study included downstaging, histopathologic tumor regression, resectability of T4 disease, and sphincter preservation in patients with low-lying tumors. RESULTS: Dose-limiting grade 3 gastrointestinal toxicity was observed in two of six patients treated with 60 mg/m2 of oxaliplatin. Thus, 50 mg/m2 was the recommended dose for the phase II study. Toxicities observed at this dose level were generally mild, with only two cases of short-lived grade 3 diarrhea. Myelosuppression, mainly leukopenia, was restricted to grade 2 in 19% of patients. T-category downstaging was achieved in 17 (55%) of 31 operated patients, and 68% of patients had negative lymph nodes. Pathologic complete response was found in 19% of the resected specimens. Radical surgery with free margins could be performed in 79% of patients with T4 disease, and 36% of patients with tumors < or = 2 cm from the dentate line had sphincter-saving surgery. CONCLUSION: Preoperative XELOX-RT is a feasible and well tolerated treatment. This regimen is proposed for phase III evaluation comparing standard fluorouracil-based therapy with XELOX chemoradiotherapy.     

Shwachman syndrome: Exocrine pancreatic dysfunction and variable phenotypic expression

BACKGROUND & AIMS: Shwachman syndrome is an inherited condition with multisystemic abnormalities, including exocrine pancreatic dysfunction. The aim of this study was to evaluate the occurrence and progression of features in a large cohort of patients. METHODS: Clinical records of 25 patients with Shwachman syndrome were reviewed. RESULTS: Mean birth weight (2.92 +/- 0.51 kg) was at the 25th percentile. However, by 6 months of age, mean heights and weights were less than the 5th percentile. After 6 months of age, growth velocity was normal. Severe fat maldigestion due to pancreatic insufficiency was present in early life (fecal fat, 26% +/- 17% of fat intake; age, < 2 years). Serial assessment of exocrine pancreatic function showed persistent deficits of enzyme secretion, but 45% of patients showed moderate age-related improvements leading to pancreatic sufficiency. Neutropenia was the most common hematologic abnormality (88%), but leukopenia, thrombocytopenia, and anemia were also frequently encountered. Patients with hypoplasia of all three bone marrow cellular lines (n = 11) had the worst prognosis; 5 patients died, 2 of sepsis and 3 of acute myelogenous leukemia. Other findings included hepatomegaly and/or abnormal liver function test results and skeletal abnormalities. CONCLUSIONS: A wide and varied spectrum of phenotypic abnormalities among patients with Shwachman syndrome is described. Pancreatic acinar dysfunction is an invariable abnormality. Patients with severe bone marrow involvement may have a guarded prognosis. (Gastroenterology 1996 Dec;111(6):1593-602)     

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.     

Interleukin-6 enhances growth factor-dependent proliferation of the blast cells of acute myeloblastic leukemia

The effects of recombinant interleukin-6 (IL-6) on the proliferation of blast precursors present in the peripheral blood of patients with acute myeloblastic leukemia (AML) was investigated. IL-6 had little effect by itself; however, it synergized with granulocyte macrophage colony- stimulating factor (GM-CSF) and interleukin-3 (IL-3) in the stimulation of AML blast colony formation. Responsiveness of blast progenitors to IL-6 was heterogeneous. On normal bone marrow cells the same synergy was observed on granulocyte and monocyte precursors (GM-CFC), while there was no significant effect on erythroid and multipotential precursors.     

Radiochemotherapie des Analkarzinoms

The implementation of intensity modulated radiation therapy (IMRT) in the total concept of radiochemotherapy for anal cancer means that tolerance of the treatment can be decisively improved. However, modern techniques such as IMRT require an exact imaging procedure preferentially using fluorodeoxyglucose positron emission tomography computed tomography (FDG-PET-CT) and a standardized, quality assurance defined target volume. The following elective target areas should also principally be included in addition to the primary tumor together with affected lymph node locations: mesorectum, presacral space, internal iliac lymph nodes, ischiorectal fossa, obturator crest lymph nodes, external iliac lymph nodes and inguinal lymph nodes. The total dose of IMRT should be 45?Gy for elective lymph nodes, 50.4-54?Gy for positive lymph nodes and 50.4-54?Gy for T1-2 and 54–59.4?Gy for primary tumors larger than T2.     

Organ preservation in patients with invasive bladder cancer: initial results of an intensified protocol of transurethral surgery and radiation therapy plus concurrent cisplatin and 5-fluorouracil

To assess safety, tolerance, and disease control of transurethral resection of the bladder tumor (TURB) plus concurrent cisplatin, 5-fluorouracil (5-FU), and radiation therapy (RT) with selective organ preservation in patients with bladder cancer.

Forty-five patients with muscle-invading or high-risk T1 (G3, associated carcinoma in situ, multifocality, >5 cm) bladder cancer were entered into a protocol of TURB followed by concurrent cisplatin (20 mg/m²/day, 20-min infusion) and 5-FU (600 mg/m²/day, 120-hour continuous infusion), administered on Day 1–5 and 29–33 of RT (single dose 1.8 Gy, total dose to the bladder 54–59.4 Gy). Response was evaluated by restaging TURB 6 weeks later. In case of invasive residual or recurrent tumor, salvage cystectomy was recommended. Median follow-up was 35 months (range: 8–80 months).

Thirty-nine patients (87%) had no detectable tumor at restaging TURB; 29 patients (64%) have been continuously free of tumor in their bladders. A superficial relapse occurred in 4 patients, a muscle-invasive relapse in 6 patients. Overall survival and survival with preserved bladder was 67% and 54%, respectively, at 5 years. Hematologic Grade 3/4 toxicity occurred in 10%/4%; Grade 3 diarrhea occurred in 9%. Thirty-four patients (76%) completed the protocol as scheduled or with only minor deviations. One patient required salvage cystectomy because of a shrinking bladder.

Conclusion: This protocol of concurrent cisplatin/5-FU and RT has been associated with acceptable toxicity. The complete response rate of 87% and the 5-year survival with intact bladder of 54% are encouraging and compare favorably with our historical control series using RT with carboplatin and cisplatin alone.     

Is 24-Color FISH Detection of In-Vitro Radiation-Induced Chromosomal Aberrations Suited to Determine Individual Intrinsic Radiosensitivity?

Background: Reliable determination of intrinsic radiosensitivity in individual patients is a serious need in radiation oncology. Chromosomal aberrations are sensitive indicators of a previous exposure to ionizing irradiation. Former molecular cytogenetic studies showed that such aberrations as an equivalent of intrinsic radiosensitivity can be detected by fluoroscence in-situ hybridization (FISH) techniques using whole chromosome painting (wcp) probes. However, only one up to three randomly chosen wcp probes have been applied for such approaches until now. As a random distribution of chromosomal rearrangements along the chromosomes is up to now still controversial, the power of the 24-color FISH approach should be elucidated in the present study. Methods and Material: Lymphocytes derived from lymphoblastoid cell lines of one patient with Nijmegen breakage syndrome (NBS homozygote) and of two NBS heterozygotes and peripheral blood lymphocytes of two controls were analyzed. Samples of each patient/control were irradiated in vitro with 0.0 Gy, 0.7 Gy or 2.0 Gy prior to cultivation. Chromosomal aberrations were analyzed in detail and quantified by means of 24-color FISH as an expression of the individual intrinsic radiosensitivity. Results: 24-color FISH analyses were done in a total of 1,674 metaphases. After in-vitro irradiation, 21% (0.7 Gy) or 57% (2.0 Gy) of the controls' cells, 15% (0.7 Gy) or 53% (2.0 Gy) of the heterozygotes' cells and 54% (0.7 Gy) or 79% (2.0 Gy) of the homozygotes' cells contained aberrations. The highest average rates of breaks per mitosis [B/M] (0.7 Gy: 1.80 B/M, 2.0 Gy: 4,03 B/M) and complex chromosomal rearrangements [CCR] (0.7 Gy: 0.20 CCR/M, 2.0 Gy: 0.47 CCR/M) were observed in the NBS patient. Moreover, the proportion of different aberration types after irradiation showed a distinct increase in the rate of CCR combined with a decrease in dicentrics in the NBS homozygote. Conclusion: To come to a more complete picture of radiation-induced aberrations and to detect and quantify genetically determined intrinsic radiosensitivity, a 24-color FISH approach using all human chromosome painting probes has been successfully applied on cytogenic preparation lymphocytes. The controls and NBS heterozygotes were clearly distinguished from the NBS homozygote subject. Hintergrund: Die Verfügbarkeit eines verlässlichen und schnellen prädiktiven Testsystems zur Bestimmung individueller Radiosensitivität von Tumorpatienten ist in der onkologischen Strahlentherapie von enormer Bedeutung. Molekularzytogenetische Studien haben gezeigt, dass intrinsische Radiosensitivität in Form von chromosomalen Aberrationen mittels Fluoreszenz-in-situ-Hybridisierungs-(FISH-)Techniken unter Verwendung von sog. "whole chromosome painting"-(wcp-)Sonden nachgewiesen werden kann. Bisher wurden für solche Ansätze allerdings lediglich maximal drei zufällig ausgewählte wcp-Sonden gleichzeitig eingesetzt. Da eine zufällige Verteilung der induzierten Schäden über das Genom bisher immer noch ungeklärt ist, sollten die Grenzen und Möglichkeiten der 24-Farben-FISH-Methode für die Bestimmung individueller Strahlenempfindlichkeit mit der vorliegenden Studie ermittelt werden. Methoden und Material: Lymphozyten aus lymphoblastoiden Zelllinien von einem Patienten mit Nijmegen-Breakage-Syndrom (NBS-homozygoter Genträger) und von zwei NBS-heterozygoten Genträgern sowie Lymphozyten aus peripheren Blut von zwei Kontrollen wurden untersucht. Die Proben wurden in vitro mit 0,0 Gy, 0,7 Gy oder 2,0 Gy bestrahlt und anschließend kultiviert. Die chromosomalen Aberrationen wurden mittels 24-Farben-FISH analysiert und können als Maß für die individuelle intrinsische Strahlenempfindlichkeit interpretiert werden. Ergebnisse: Mittels der 24-Farben-FISH Methode wurden insgesamt 1674 Metaphaseplatten analysiert. Nach In-vitro-Bestrahlung mit 0,7 bzw. 2,0 Gy zeigten 21% bzw. 57% der Zellen der beiden Kontrollen, 15% bzw. 53% der Zellen der beiden NBS-Heterozygoten und 54% bzw. 79% der Zellen des NBS-Homozygoten Aberrationen. Die höchsten durchschnittlichen Bruchraten (Brüche pro Mitose = B/M) (0,7 Gy: 1,80B/M, 2,0 Gy: 4,03 B/M) und Raten an komplexen chromosomalen Rearrangements (= CCR) (0,7 Gy: 0,20 CCR/M, 2,0 Gy: 0,47 CCR/M) wurden in den Zellen des NBS-homozygoten Patienten nachgewiesen. Nach Bestrahlung zeigte sich ein deutlicher Anstieg bezüglich der Anzahl an CCR bei einem gleichzeitigen Abfall der Anzal an dizentrischen Chromosomen beim NBS-Homozygoten. Schlussfolgerung: Die Methoden der 24-Farben-FIS unter Verwendung aller menschlischen wcp-Sonden wurde erfolgreich zu Nachweis, Charakterisierung und Quantifizierung strahleninduzierter chromosomaler Aberrationen in Lymphozyten eingesetzt. Darüber hinaus ist diese Methode geeignet, um ein vollständigeres Bild der entstandenen chromosomalen Rearrangements zu erhalten. Kontrollen und NBS-Heterozygote konnten klar von einem NBS-homozytogen Genträger unterschieden werden.