The effect of an abdominal binder on postoperative outcome after open incisional hernia repair in sublay technique: a multicenter, randomized pilot trial (ABIHR-II).

INTRODUCTION: Although the evidence is minimal, an abdominal binder is commonly prescribed after open incisional hernia repair (IHR) to reduce pain. This study aimed to investigate this common postoperative treatment. METHODS: The ABIHR-II trial was a national prospective, randomized, multicenter non-AMG/MPG pilot study with two groups of patients (wearing an abdominal binder (AB) for 2 weeks during daytime vs. not wearing an AB following open IHR with the sublay technique). Patient enrollment took place from July 2020 to February 2022. The primary endpoint was pain at rest on the 14th postoperative day (POD) using the visual analog scale (VAS). The use of analgesics was not systematically recorded. Mixed-effects linear regression models were used. RESULTS: A total of 51 individuals were recruited (25 women, 26 men; mean age 61.4 years; mean body mass index 30.65 kg/m2). The per-protocol analysis included 40 cases (AB group, n = 21; No-AB group, n = 19). Neither group showed a significant difference in terms of pain at rest, limited mobility, general well-being, and seroma formation and rate. Patients among the AB group had a significantly lower rate of surgical site infection (SSI) on the 14th POD (AB group 4.8% (n = 1) vs. No-AB group 27.8% (n = 5), p = 0.004). CONCLUSION: Wearing an AB did not have an impact on pain and seroma formation rate but it may reduce the rate of postoperative SSI within the first 14 days after surgery. Further trials are mandatory to confirm these findings.

Gene expression profiling of CD34+ cells identifies a molecular signature of chronic myeloid leukemia blast crisis.

Despite recent success in the treatment of early-stage disease, blastic phase (BP) of chronic myeloid leukemia (CML) that is characterized by rapid expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. The development of resistance upon continuous administration of imatinib mesylate is associated with poor prognosis pointing to the need for alternative therapeutic strategies and a better understanding of the molecular mechanisms underlying disease progression. To identify transcriptional signatures that may explain pathological characteristics and aggressive behavior of BP blasts, we performed comparative gene expression profiling on CD34+ Ph+ cells purified from patients with untreated newly diagnosed chronic phase CML (CP, n=11) and from patients in BP (n=9) using Affymetrix oligonucleotide arrays. Supervised microarray data analysis revealed 114 differentially expressed genes (P<10(-4)), 34 genes displaying more than two-fold transcriptional changes when comparing CP and BP groups. While 24 of these genes were downregulated, 10 genes, especially suppressor of cytokine signalling 2 (SOCS2), CAMPATH-1 antigen (CD52), and four human leukocyte antigen-related genes were strongly overexpressed in BP. Expression of selected genes was validated by real-time-polymerase chain reaction and flow cytometry. Our data suggest the existence of a common gene expression profile of CML-BP and provide new insight into the molecular phenotype of blasts associated with disease progression and high malignancy.

The t(8;17)(p11;q23) in the 8p11 myeloproliferative syndrome fuses MYO18A to FGFR1.

The 8p11 myeloproliferative syndrome (EMS) also known as stem cell leukemia-lymphoma syndrome (SCLL) is associated with translocations that disrupt FGFR1. The resultant fusion proteins are constitutively active tyrosine kinases, and different FGFR1 fusions are associated with subtly different disease phenotypes. We report here a patient with a t(8;17)(p11;q23) and an unusual myelodysplastic/myeloproliferative disease (MDS/MPD) characterized by thrombocytopenia due to markedly reduced size and numbers of megakaryocytes, with elevated numbers of monocytes, eosinophils and basophils. A novel mRNA fusion between exon 32 of the myosin XVIIIA gene (MYO18A) at chromosome band 17q11 and exon 9 of FGFR1 was identified. Partial characterization of the genomic breakpoints in combination of bubble-PCR with fluorescence in situ hybridization revealed that the t(8;17) arose from a three-way translocation with breaks at 8p11, 17q11 and 17q23. MYO18A-FGFR1 is structurally similar to other fusion tyrosine kinases and is likely to be the causative transforming lesion in this unusual MDS/MPD.

Pegylated liposomal doxorubicin in combination with mitomycin C, infusional 5-fluorouracil and sodium folinic acid. A phase-I-study in patients with upper gastrointestinal cancer.

Mitomycin C (MMC) in combination with infusional 5-fluorouracil (FU) plus folinic acid (FA) is an effective treatment for metastatic gastrointestinal cancer. Anthracyclines are commonly used in the treatment of upper gastrointestinal cancer. The aim of this study was to determine the maximum tolerated dose of liposomal, pegylated doxorubicin (Caelyx) in combination with infusional 5-FU/sodium FA and MMC. Escalating doses of Caelyx (15-25-30-35 mg m(-2) corresponding to dose levels I-IV) were applied on days 1 and 29, given to fixed doses of 24-h 5-FU (2000 mg m(-2)) and sodium FA (500 mg m(-2), mixed with 5-FU in one pump) weekly for 6 weeks, and MMC 7 mg m(-2) on days 8 and 36. At least three patients were treated at each dose level. A total of 25 patients are evaluable. No dose-limiting toxicity (DLT) was observed on level I (n=3). On level II, DLT occurred in three out of five patients (mucositis and leucopenia). Owing to the early DLTs at this dose, we added a 20 mg m(-2) Caelyx dose level (Ia). In total, 17 patients were treated at this dose level. Among these, only two patients experienced DLT in cycle one and 37 complete cycles have been administered in association with a low toxicity profile. The median dose intensity was 100% for each drug during the first course and no treatment delay exceeding 7 days was required. The recommended dose of 4-weekly Caelyx in combination with weekly 24-h 5-FU/sodium FA and 4-weekly MMC is 20 mg m(-2). Preliminary antitumour activity has been observed in patients with pretreated pancreatic cancer and in untreated gastric cancer.

Molecular monitoring of response to imatinib (Glivec) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission.

A significant proportion of chronic myeloid leukemia (CML) patients achieve a major cytogenetic remission (MCR) to imatinib therapy after failing interferon (IFN) alpha-based protocols. We sought to determine levels of residual disease in patients with MCR using various molecular methods and to establish a relation between residual BCR-ABL transcript levels and rate of relapse in complete cytogenetic remission (CCR). Response was measured by conventional cytogenetic analysis, hypermetaphase and interphase fluorescence in situ hybridization (HM-FISH, IP-FISH) of bone marrow (BM) cells, qualitative nested and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for BCR-ABL transcripts. We investigated 323 peripheral blood (PB) and BM samples from 48 CML patients who achieved a complete (Ph+ 0%; n=41) or partial (Ph+ 1-34%; n=7) cytogenetic remission after 3-20 months of imatinib therapy. Prior to imatinib, 35 patients were in chronic phase (CP), eight in accelerated phase (AP), four in myeloid and one in lymphoid blast crisis. HM-FISH results correlated with ratios BCR-ABL/ABL in PB and BM. In patients with CCR, residual disease was detectable by HM-FISH (31%), IP-FISH (18%), and RT-PCR (100%). During follow-up, BCR-ABL became undetectable in two patients (one CP, one AP) by both nested and quantitative RT-PCR. CCR is ongoing in 30 evaluable patients, 11 patients have relapsed. At the time of best response, median ratios BCR-ABL/ABL were 2.1% (range 0.82-7.8) in patients with subsequent relapse and 0.075% (range 0-3.9) in patients with ongoing remission (P=0.0011). All 16 CP patients, who achieved ratios BCR-ABL/ABL <0.1% as best molecular response are in continuous remission, while 6/13 patients (46%) with ratios >/=0.1% have relapsed (P=0.0036). We conclude that: (i) in patients with CCR to imatinib, HM-FISH and RT-PCR usually reveal residual BCR-ABL+ cells; (ii) RT-PCR results derived from PB and BM are comparable in CP CML; and (iii) low levels of residual disease with ratios BCR-ABL/ABL &<0.1% are associated with continuous remission.

Treatment of a patient with advanced esophageal cancer with a combination of mitomycin C and capecitabine: activation of the thymidine phosphorylase as active principle?

BACKGROUND: Both capecitabine, an oral prodrug of 5-fluorouracil (5-FU), and mitomycin C (MMC) have demonstrated activity as single agents in patients with gastrointestinal cancer. Furthermore, a combination of MMC with infusional 5-FU can induce tumor remission even in patients pretreated with 5-FU. Capecitabine and MMC act synergistically due to an upregulation of the thymidine phosphorylase activity by MMC in a human xenograft model. PATIENT: We sought to exploit these preclinically observed effects in a patient with esophageal cancer who was progressive after a first-line radiochemotherapy with 5-FU and cisplatin. He was treated with a combination of MMC and capecitabine on a compassionate use basis. A rapid remission lasting for about 6 months was observed. CONCLUSION: This is the first report on a combination therapy with capecitabine and MMC. The remission observed in our patient suggests that the preclinically observed synergy has clinical impact. This combination should be further investigated in prospective clinical trials.

Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization.

The sensitivity of assays designed to monitor minimal residual disease (MRD) by RT-PCR in leukemia depend on quality and quantity of RNA derived from peripheral blood (PB) and bone marrow (BM) leukocytes. Shipment of material may lead to RNA degradation resulting in a loss of sensitivity and, potentially, false negative results. Furthermore, degradation may lead to inaccurate estimates of MRD in positive specimens. We sought to determine feasibility and efficacy of a novel blood collection and processing system which is based on integrated RNA stabilization at the time of phlebotomy (PAXgene Blood RNA Kit) by comparison with standard methods of RNA extraction (cesium chloride gradient ultracentrifugation and RNeasy Mini Kit) using unstabilized EDTA anticoagulated PB. In 26 patients with chronic myelogenous leukemia (CML) on therapy, PB was processed after a storage time at room temperature of 2 and 72 h according to these protocols. BCR-ABL, total ABL and glucose-6-phosphate dehydrogenase (G6PD) mRNA transcripts of PB samples were quantified as a measure for response to therapy and RNA integrity. RNA yield expressed as the ratio of ABL transcripts after a storage time of 72 h/ABL transcripts after a storage time of 2 h at room temperature was significantly higher with the stabilizing method (median 0.40) compared to the RNeasy method using unstabilized PB (median 0.13, P = 0.01). Furthermore, ratios BCR-ABL/ABL after 72 vs 2 h still correlated well using the PAXgene method (r = 0.99, P < 0.0001) in contrast to the standard method which did not (r = 0.65, P = 0.03). Even investigation of complete cytogenetic responders with very low tumor burden showed a good correlation of ratios BCR-ABL/ABL compared to the reference method. Comparable results were achieved using G6PD transcripts as standard. We conclude that the new PAXgene stabilization method could improve RNA quality and the comparability of molecular monitoring within and between multicenter trials.

Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha.

The degree of tumor load reduction as measured by cytogenetic response is an important prognostic factor for chronic myelogenous leukemia (CML) patients on therapy. We sought to determine whether BCR-ABL transcript levels can predict chromosomal response. Residual disease was evaluated in 120 CML patients in chronic phase (CP) treated with the selective tyrosine kinase inhibitor imatinib after resistance or intolerance to interferon alpha (IFN). Median time of therapy was 401 days (range 111-704). BCR-ABL and total ABL transcripts were measured in 486 peripheral blood (PB) specimens with a real time RT-PCR approach using fluorescent-labeled hybridization probes (LightCycler technology) and results were expressed as the ratio BCR-ABL/ABL. Cytogenetic response was determined in 3-monthly intervals: From 101 evaluable patients, 42 achieved a complete (CR, 0% Philadelphia chromosome (Ph)- positive metaphases), 18 a partial (PR, 1-34% Ph+), 13 a minor (MR, 35-94% Ph+), and 26 no response (NR, >94% Ph+). All PB samples were RT-PCR positive. The proportion of Ph+ metaphases and simultaneous BCR-ABL/ABL ratios correlated with r = 0.74, P < 0.0001. In order to investigate whether early molecular analysis may predict cytogenetic response, quantitative RT-PCR data obtained after 1 and 2 months of therapy were compared with cytogenetic response at 6 months. BCR-ABL/ABL ratios after 1 month were not predictive, but results after 2 months correlated with the consecutive cytogenetic response (P = 0.0008). The probability for a major cytogenetic response was significantly higher in patients with a BCR-ABL/ABL ratio <20% after 2 months of imatinib therapy. We conclude that: (1) quantitative determination of residual disease with real time RT-PCR is a reliable and sensitive method to monitor CML patients on imatinib therapy; (2) BCR-ABL/ABL ratios correlate well with cytogenetic response; (3) in IFN-pretreated patients all complete responders to imatinib have evidence of residual disease with the limited follow-up available; and (4) cytogenetic response at 6 months of therapy in CP patients is predictable with real time RT-PCR at 2 months.

Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes.

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has no implication in the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. We have developed a RT-PCR-based screening method to easily identify polymorphic BCR and BCR-ABL alleles in CML patients and normal individuals in order to estimate their frequency. After amplification from cDNA, a melting curve of a specific fluorogenic probe mapping to the 3' end of BCR exon b2 and spanning the polymorphism readily discriminates between normal and polymorphic BCR and BCR-ABL alleles. This reporter probe is 3' labeled with fluorescein and placed next to 5' LC Red640-labeled anchor probes mapping to the 5' ends of BCR exon b3 or ABL exon a2 so that resonance energy transfer occurs when the probes are hybridized (LightCycler technology). T and C alleles were discriminated by a melting temperature difference of the reporter probe of 3.2 K. We have investigated cDNAs derived from leukocytes from seven cell lines and a total of 229 individuals: normal donors, n = 15; BCR-ABL negative chronic myeloproliferative disorders, n=30; BCR-ABL negative acute leukemias, n= 11; b2a2BCR-ABL positive CML, n = 93; and b3a2BCR-ABL positive CML, n= 80. The frequency of the C allele was 33.0% in BCR-ABL negative individuals, 30.6% in b2a2BCR-ABL, and 23.8% in b3a2BCR-ABL positive CML. In CML patients, 27.7% of BCR-ABL and 27.2% of BCR alleles had the C allele (NS). In total, 132 of 458 (28.8%) exons b2 of BCR or BCR-ABL alleles demonstrated this polymorphism. We conclude that a thymidine/cytosine replacement occurs frequently in BCR exon b2. Probes for real time quantitative RT-PCR should be designed not to map to the critical region in order to avoid underestimation of the number of BCR-ABL transcripts.

Detection and quantification of residual disease in chronic myelogenous leukemia.

The degree of tumor load reduction after therapy is an important prognostic factor for patients with CML. Conventional metaphase analysis has been considered to be the 'gold standard' for evaluating patient response to treatment but this technique normally requires bone marrow aspiration and is therefore invasive. The frequency of cytogenetic analyses can be considerably reduced if patients are also monitored by molecular methods, which can be performed on peripheral blood specimens. Of the various techniques available, most attention has been paid to RT-PCR for BCR-ABL mRNA since this is by far the most sensitive. Simple, non-quantitative RT-PCR analysis gives only limited information on patients after treatment. Quantitative RT-PCR assays have been developed to monitor the kinetics of residual BCR-ABL transcripts over time. Variables in the quantitative PCR assay may be controlled for by quantification of transcripts of a normal gene (eg ABL or glucose-6-phosphate dehydrogenase, G6PD) as an internal standard. After allogeneic stem cell transplantation, most patients become RT-PCR negative, often after a period of low level positivity that may persist for several months. Those patients destined to relapse are characterized by the reappearance and/or rising levels of BCR-ABL transcripts. In contrast, for patients treated with interferon-alpha (IFN) residual disease is rarely, if ever, eliminated. The actual level of minimal residual disease in complete cytogenetic responders to IFN correlates with the probability of relapse. New quantitative real time procedures promise to simplify the protocols that are currently in use, but standardization and the introduction of rigorous, internationally accepted controls are required to enable RT-PCR to become a robust and routine basis for therapeutic decisions.

Reduced (1)H-NMR visibility of creatine in isolated rat hearts.

The aim of this study was to measure the concentration of creatine in Langendorff perfused rat hearts, both by quantitative (1)H-MRS and by high-pressure liquid chromatography (HPLC). First, the relaxation times and other parameters affecting absolute quantification by MRS were determined. At 11.75 T, the relaxation times of myocardial creatine were T(1) = 1.1+/-0.29 sec (mean +/- SD, n = 5) and T(2) = 56.4+/-6.2 ms (n = 9). In phantom experiments the MRS measurements gave accurate values for the known relative concentrations of the detected substances. In glucose-perfused rat hearts, the creatine concentration measured by HPLC was 14.2+/-1.9 mmol/kg wet weight (n = 8), in good agreement with literature values. The (1)H-MRS measurements, however, resulted in creatine concentrations of only approximately 60% of this value. The application of CHESS-pulses for water suppression led to a further 30% reduction of the creatine MRS signal. These results indicate a reduced (1)H-NMR visibility of creatine in the myocardium, which suggests a compartmentation of myocardial creatine into various pools.

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR.

We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 105 cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-alpha (n = 219), allogeneic BMT (n = 17), chemotherapy (n = 11), or at diagnosis (n = 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n = 201, r = 0.90, P < 0. 0001); (2) the proportion of Philadelphia chromosome positive metaphases determined by cytogenetics (n = 81, P < 0.0001); and (3) the BCR ratio determined by Southern blot analysis (n = 122, P < 0. 0001). We conclude that real-time PCR with hybridization probes is a reliable and sensitive method to monitor CML patients after therapy. The major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete PCR analysis takes less than 60 min.

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

Persistence of cytomegalovirus in human lymphoblasts and peripheral leukocyte cultures.

The in vitro susceptibility of human peripheral lymhpocytes and lymphoblastoid (F265) cells to infection by human cytomegalovirus was examined. Infection of these cell types with cytomegalovirus resulted in a persistent type of infection rather than the typical growth curve observed with permissive fibroblastic cells. When infection of peripheral lymphocytes was associated with a blastogenic response, the virus persisted for a longer time and at a higher titer than in cells in which a blastogenic response did not occur. Autoradiographic studies and infectious-center assays indicated that only a small number of cells, resembling lymphocytes, were involved in virus persistence. Whether or not the persistence of the virus indicates release of input virus or synthesis or new virus was not determined.