Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Standardized MRD flow and ASO IGH RQ-PCR for MRD quantification in CLL patients after rituximab-containing immunochemotherapy: a comparative analysis.

Rituximab-containing regimens are becoming a therapeutic standard in chronic lymphocytic leukemia (CLL), so that a validation of flow cytometric minimal residual disease (MRD) quantification (MRD flow) in the presence of this antibody is necessary. We therefore compared results obtained by real-time quantitative (RQ)-PCR to MRD flow in 530 samples from 69 patients randomized to receive chemotherapy or chemotherapy plus rituximab. Quantitative MRD levels assessed by both techniques were closely correlated irrespective of therapy (r=0.95). The sensitivity and specificity of MRD flow was not influenced by the presence of rituximab. With 58.9% positive and 26.4% negative samples by both techniques, 85.3% of assessments (452/530) were qualitatively concordant between MRD flow and RQ-PCR. Discordant samples were typically negative by MRD flow and simultaneously positive close to the detection limit of the PCR assays, indicating a higher sensitivity of PCR for very low MRD levels. However, 93.8% of all samples were concordantly classified by both methods using a threshold of 10(-4) to determine MRD positivity. MRD flow and PCR are equally effective for MRD quantification in rituximab-treated CLL patients within a sensitivity range of up to 10(-4), whereas PCR is more sensitive for detecting MRD below that level.

[Secondary thumb reconstruction after avulsion or amputation injury with free wrap-around toe flap]. / Die sekundäre rekonstruktion des daumens nach avulsions- oder amputationsverletzung mit dem wrap-around-toe-flap.

The free wrap-around toe flap, first described by Morrison in 1980, represents an option for a tailor-made reconstruction of the thumb. This technique ensures flexibility, length, sensibility and an excellent aesthetic outcome. The morbidity rate of the donor site is negligibly small. Unfavourable and therefore limiting to the indication is the absence of a possibility for a joint transfer, the lack of growth potential and the risk of bone absorption when using an iliac bone graft. Our small series includes 5 patients with an average follow-up time of 11.4 months (range: 6 - 22 months) treated from 2003 - 2006. The static two-point discrimination on average was 19 mm (range: 4 - 39 mm). All patients describe coldness intolerance. As there is no deficit in performing opposition and pinch power, there is an acceptable motion of all thumb joints. All patients report a good subjective outcome and would undergo the operation once again. In spite of the short follow-up period with moderate to good results, the wrap-around toe flap can be recommended as an appropriate and safe procedure for thumb reconstruction after an avulsion and traumatic amputation.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data.

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

TCRB gene rearrangements in childhood and adult precursor-B-ALL: frequency, applicability as MRD-PCR target, and stability between diagnosis and relapse.

Using the multiplex PCR tubes of the BIOMED-2 Concerted Action, TCRB gene rearrangements were detected in 35% of childhood (n=161) and adult (n=172) precursor-B-ALL patients (Vbeta-(Dbeta)-Jbeta in 25%; Dbeta-Jbeta in 15%). The presence of TCRB rearrangements showed a significant relation with age (highest frequency of 46% between 5 and 10 years of age) and the presence of TEL-AML1 transcripts, and was associated with relatively high frequencies of IGK-Kde, TCRG, and Vdelta2-Jalpha rearrangements. In 62 out of 65 patients with Southern blot-detected Vbeta-(Dbeta)-Jbeta and/or Dbeta-Jbeta rearrangements, at least one TCRB gene rearrangement was detected by PCR. Based on combined Southern blot and PCR analysis, oligoclonal TCRB gene rearrangements were observed in only 12% of patients. Analysis of paired diagnosis and relapse samples (n=26) showed that 20 out of 24 (83%) Vbeta-(Dbeta)-Jbeta rearrangements and eight out of 14 (57%) Dbeta-Jbeta rearrangements remained stable. Using real-time quantitative PCR, a quantitative range < or =10(-4) was obtained in 64% of TCRB gene rearrangements and in 86% of cases a sensitivity < or =10(-4) was obtained. In conclusion, TCRB gene rearrangements occur in 35% of precursor-B-ALL patients and are relatively stable and sensitive PCR targets for detection of minimal residual disease, particularly if this concerns complete Vbeta-(Dbeta)-Jbeta rearrangements.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Rearranged T-cell receptor beta genes represent powerful targets for quantification of minimal residual disease in childhood and adult T-cell acute lymphoblastic leukemia.

Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 80%, Dbeta-Jbeta rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jbeta2.7 in childhood T-ALL (27%), whereas Jbeta2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vbeta segments (19-1/20-1/21-1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jbeta primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 10(4) polyclonal cells in 93% of cases and will be of high value for future MRD studies.

Comparative analysis of T-cell receptor gene rearrangements at diagnosis and relapse of T-cell acute lymphoblastic leukemia (T-ALL) shows high stability of clonal markers for monitoring of minimal residual disease and reveals the occurrence of second T-ALL.

A total of 28 children and nine adults with relapsed T-ALL were analyzed for the configuration of their T-cell receptor (TCR) and TAL1 genes at diagnosis and relapse to evaluate their stability throughout the disease course. A total of 150 clonal TCR and TAL1 gene rearrangements were identified in the 37 patients at diagnosis. In 65% of cases all rearrangements and in 27% of cases most rearrangements found at diagnosis were preserved at relapse. Two children with unusually late T-ALL recurrences displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. This indicates that a proportion of very late T-ALL recurrences might represent second T-ALL. Specifically, 88% of clonal rearrangements identified at diagnosis in truly relapsed T-ALL were preserved at relapse. This is significantly higher as compared to previously studied precursor-B-ALL ( approximately 70%). Thus, from biological point of view, immunogenotype of T-ALL is more stable as compared with precursor-B-ALL. The overall stability of TCR gene rearrangements was higher in adult T-ALL (97%) than in childhood T-ALL (86%). Based on the stability of TCR gene rearrangements, we propose a strategy for PCR target selection (TCRD+TAL1 --> TCRB --> TCRG), which probably allows reliable minimal residual disease detection in all T-ALL patients.

[Drug-induced toxic epidermal necrolysis with involvement of the intestinal and respiratory tract. A case report]. / Toxisch epidermale Nekrolyse Arzneimittelinduzierte toxisch epidermale Nekrolyse mit Beteiligung des Intestinal- und Respirationstraktes.

Toxic epidermal necrolysis (TEN) is a disease occurring with low-incidence but has a relatively high mortality rate. Sepsis is the predominant cause for life-threatening complications in TEN but severe mucosal damage represents a further complication which may delay convalescence. We report a case of TEN in a 51-year-old man which eventually spread to include the whole skin surface. The long-term and comprehensive treatment focused on support of the organ failure as well as wound treatment. The extent of involvement of the intestinal tract, the sustained laryngeal stenosis and the pronounced saddle-nose were unusual. It appears necessary to treat TEN in facilities which offer intensive care and are able to manage extensive skin damage. Burns units offer the best conditions for its management.

Clinical practice of glycerol preserved allograft skin coverage.

This retrospective study examines the use and advantages/disadvantages of glycerol preserved human allograft skin in our burn care facility between February 1997 and December 1999. Three hundred and twenty patients were included into the study, 85 of whom were treated with human cadaver skin. The usage of allograft slightly increased the number of operative procedures per percent of the total body surface area burn. There were no adverse effects noted from the use of allograft. The group of patients with allograft use had a significantly larger burn size, ABSI score and length of ICU stay. Demographically the groups were comparable. The considerably easier handling and storage of glycerol preserved allograft skin make it preferable to cryopreserved allograft skin in all indications where it is used as a temporary wound closure. We recommend the usage of cryopreserved skin in cases where the integration of a dermal component as a permanent part of wound closure is desired.

[Effect of recombinant growth hormone on wound healing in severely burned adults. A placebo controlled, randomized double-blind phase II study]. / Der Effekt rekombinanten Wachstumshormons auf die Wundheilung bei schwerverbrannten Erwachsenen. Eine placebo-kontrollierte, randomisierte doppelblinde Phase II-Studie.

INTRODUCTION: Recombinant growth hormone (rGH) has been used successfully in burned children with a shortened donor-site healing time and length of hospital stay as well as a protein-sparing effect. In adult burn patients, no comparable study exists to date. MATERIAL AND METHODS: The study was performed on 49 adults, aged 18-60, with an Abbreviated Burn Severity Index (ABSI) score of 7-I1 as a randomized, placebo-controlled, double-blind study. The treatment period was 28 days and follow-up period 1 year. rGH was administered subcutaneously at a dose of 0.5 lU/kg per day in 26 patients, 23 patients were in the placebo group. Wound-closure assessment was performed on the day of admission and on each day of dressing change. A wound-closure index (WCI) was calculated. RESULTS: Thirty-seven patients, 19 in the rGH group and 18 in the placebo group, survived and were available for primary efficacy analysis. The mean total body surface area (TBSA) burned was 41.5% (rGH) versus 36.7% (placebo); the average ABSI score was 8.27 (rGH) versus 7.9 (placebo). The wound-closure index was not significantly different in patients treated with rGH (1.92) compared with patients treated with placebo (1.72). WCI for partial thickness-loss burn wounds did not significantly differ from rGH (0.9) to placebo (0.69). The donor site healing time in rGH-treated patients (12 days) was not significantly different compared to placebo patients (10.4 days). CONCLUSION: In severely burned adult patients rGH has no positive effect on burn wound or donor-site healing.

The radial forearm fascial flap as a salvage procedure for chronic tuberculosis of the hand and wrist.

A patient with tuberculous infection of the hand and wrist developed a recurrent draining wound of the right forearm. After recurrent failure of surgical debridement and wound closure under antituberculous therapy, wound closure was established by means of a radial forearm fascial flap with an excellent functional and cosmetic result. Extra-pulmonary tuberculosis must be kept in mind in the diagnosis of slowly growing tumours and chronic wounds in the upper extremity.

Design and testing of beta-actin primers for RT-PCR that do not co-amplify processed pseudogenes.

Quantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene expression. To quantify different samples, measurements are often normalized using the expression of so-called "housekeeping" genes, such as cytoplasmic beta-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach can produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcripts of housekeeping genes, leads to co-amplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expression is prone to error. In this paper, we report the results of using three sets of beta-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new pairs of oligonucleotide primers that specifically amplify the human and rat beta-actin reverse-transcribed mRNA but not pseudogene sequences. These primers are especially suitable for quantitation of mRNA in small tissue samples (e.g., biopsies), where DNase digestion is not feasible, and therefore DNA contamination cannot be avoided.

Modulation of interleukin-6 expression in Hodgkin and Reed-Sternberg cells by Epstein-Barr virus.

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein-Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed-Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce interleukin (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (31 of 37, 84 per cent) as compared with LMP1-negative HD cases (35 of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD.

The value of early enteral nutrition in the prophylaxis of stress ulceration in the severely burned patient.

A retrospective analysis of prospectively collected data was performed to compare the frequency of upper gastrointestinal bleeding (GIB) in seriously burned patients treated with either cimetidine and antacids or enteral nutrition for ulcer prophylaxis. Five hundred and twenty-six seriously burned patients admitted to the burn intensive care unit of the BG Trauma Centre Ludwigshafen during a 4-year period were included into the study. All patients admitted to the burn unit from 1989 to 1991 received i.v. cimetidine (400 mg q4) for ulcer prophylaxis. If the intragastric pH dropped below 3.5, gastric pH was titrated with antacids up to > or = 4 via nasogastric tube. During the second 2-year period (1992-1993) early enteral nutrition alone was regarded to be ulcer protective and no further interventions for ulcer prophylaxis were routinely performed. Signs of overt upper GIB were monitored and documented through the entire study period. The overall occurrence rate of upper GIB in the cimetidine/antacids (C/A) group (n = 253) was 8.3 per cent with six cases of serious bleeding in five patients (1.98 per cent). In the enteral nutrition (EN) group (n = 273) the overall incidence of GIB was 3.3 per cent with two cases of serious bleeding (0.73 per cent). There were no deaths directly related to ulcer haemorrhage. The difference in the overall frequency of overt GIB between the groups studied was statistically significant (< 0.05). In our experience, early enteral nutrition is effective in the prevention of stress haemorrhage in the upper gastrointestinal tract. Additional medicinal prophylaxis is not required in burn patients.

The impact of risk factors and pre-existing conditions on the mortality of burn patients and the precision of predictive admission-scoring systems.

Clinical experience has shown that concomitant diseases and risk factors have a significant influence on the patient's outcome. Since none of the currently available score systems consider the impact of concomitant diseases or risk factors on burn trauma mortality, the present study was planned to further evaluate the role of these factors. Four hundred and ninety-eight patients could be included in this retrospective analysis of prospectively collected data. Parameters documented were: sex, age, weight, height, laboratory data, TBSA, inhalation trauma (IHT), full thickness (3 degrees) burn and pre-existing conditions. Single-variable analysis (SVA), logistic regression and CART analysis were performed. The data confirm the role of age and TBSA as the strongest prognostic variables. Chronic alcohol abuse and smoking, IHT and pre-existing cardiac and neurologic conditions were also found to be significant. Borderline groups could be identified in the ABSI score (7-10), where the risk factors cause 'mortality-shifting'. It can be concluded that risk factors and pre-existing conditions have a significant impact on the prognosis of burn mortality and should be incorporated into further refinements of burn admission scores.

Salvage of an avascular thumb by arteriovenous flow reversal and a microvascular "kite" flap: case report.

This case report describes the salvage of an avascular thumb by retrograde venous arterialization. The thumb survived with a 30 percent tissue loss at the radial aspect. Soft-tissue reconstruction and contour correction were achieved with a new microvascular free flap--the free "kite" flap--from the contralateral index finger. Aesthetic and functional results were excellent, and the patient returned to his original occupation. It can be concluded that retrograde arterialization can provide successful salvage in replantation and revascularization under favorable circumstances.

The reverse pedicle heterodigital cross-finger island flap.

The use of the reverse pedicle island flap as a heterodigital cross-finger flap is reported in five patients in whom conventional cross-finger flaps or homodigital flaps could not be used. All flaps survived and patient satisfaction was high. However, the indication for the flap must be considered carefully as the dissection is technically demanding and there is some donor site morbidity.

Early intragastric feeding of seriously burned and long-term ventilated patients: a review of 55 patients.

Aims of this retrospective study were: (1) to evaluate the feasibility of a protocol for early intragastric feeding: (2) to examine whether the caloric needs can be provided via the intragastric route within 72 h postburn; (3) to determine the influence of a delayed onset of feeding on the success of the feeding protocol; (4) to evaluate whether delayed feeding has any impact on morbidity and mortality. Fifty-five long-term ventilated patients were enrolled in the study. Their mean age was 37.6 years and mean total body surface area burned 44.2 per cent. The mean duration of ventilation amounted to 24.8 days. Intragastric tube feeding was initiated as early as possible after admission. Forty-five patients (81.8 per cent) could be fed successfully and their caloric needs were met within 72 h. In these patients the mean interval between burn injury and the initiation of tube feeding was 11.5 h. The mean interval was 32.4 h in the 10 patients, demonstrating failure of early intragastric feeding. Only four of the 48 patients who had enteral feeding within 18 h postburn failed to meet the caloric needs. The patients fed successfully showed a significantly decreased mortality. Early intragastric feeding after serious burns can be initiated successfully. A time interval of more than 18 h is unfavourable and significantly decreases the success rate.