Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

INTRODUCTION: Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time-consuming and require experienced laboratory staff. METHODS: To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. RESULTS: Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n = 20) or after fludarabine-based therapy (n = 22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r = 0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. CONCLUSION: Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.

Increasing hematopoietic microchimerism is a reliable indicator of incipient AML relapse.

INTRODUCTION: The reoccurrence or increase in autologous hematopoiesis after allogeneic transplantation has been linked to incipient leukemia relapse. However, the importance of such an emergency regarding microchimerism (i.e. mixed chimerism below 1% of autologous cells) still remains controversial, as fluctuating microchimerism can be observed for a very long time after transplantation. METHODS: Using real-time PCR (RQ-PCR), we compare peripheral blood samples obtained from patients with acute myeloid leukemia (AML) before hematological relapse and those taken during complete remission (i.e. either complete cytogenetic remission or complete molecular remission where applicable). By comparison of these two groups, we describe microchimerism dynamics clearly connected with imminent AML relapse. Additionally, we compare applicability of RQ-PCR and conventional PCR with fragment analysis. RESULTS: Mere reappearance of autologous hematopoiesis within patients with complete donor chimerism is alarming, and another sample with further increase confirms ongoing relapse. In case of patients with continuous microchimerism, another two consecutive samples with increasing trend are required. RQ-PCR predicted a significantly higher number of hematological relapses (87%vs. 39%) with a median anticipation period of 33 days, 26 days earlier than conventional PCR (P= 0.0002). Moreover, the outcome of microchimerism dynamics was in complete agreement with monitoring of minimal residual disease when analyzed from the same cell compartment. CONCLUSION: Within this paper, we emphasize the importance of microchimerism monitoring as a reliable indicator of incipient AML relapse, especially in patients where no other specific molecular marker is available.

Telomerase as a diagnostic and predictive marker in colorectal carcinoma.

In a search for molecular markers providing both informative diagnostics of malignant disease, and rational stratification of a therapeutic strategy to achieve optimal response in a given patient, we examined the possibility of using telomerase for this purpose in colorectal cancer. Telomerase, a ribonucleoprotein enzyme complex catalysing synthesis of chromosome ends (telomeres), has been known as an almost universal tumor marker but its predictive value has been found in only a limited number of malignant tumor types. Telomerase activity and expression of its catalytic subunit hTERT was determined in 82 surgical specimens from 41 patients (a sample of tumor tissue and of adjacent morphologically normal tissue was obtained from each patient). Telomerase activity was present in tumor samples from 34 (83%) patients, reaching an average value of 47.6 telomerase units (T.U.), while adjacent tissue specimens were either negative (in 25 (61%) patients), or slightly positive (in 16 (39%) patients) showing 1.5 T.U. on average. In tumor samples from patients without lymphatic node metastases (pN0), an average of 37.1 T.U was found. In contrast, in tumor samples from patients with lymphatic node involvement (pN1 or pN2) the average activity was significantly higher (60.2 T.U., p<0.05). In patients with distant metastases a tendency towards higher telomerase activity, although lacking statistical significance, could be observed. Among patients that obtained chemotherapy with 5-fluoruracil, those with low telomerase activity showed a tendency to chemosensitivity. Expression of hTERT was detected not only in samples showing telomerase activity, but also in a considerable portion of telomerase-negative samples either from the tumor or the adjacent normal tissue. We demonstrate that some of these apparent discrepancies may be attributed to differential splicing of hTERT mRNA. We conclude that TRAP assay for telomerase activity is more informative than the common testing for hTERT expression. Telomerase activity is useful both as a diagnostic as well as a predictive factor in colorectal cancer.

[Direct DNA diagnosis of Friedreich's ataxia]. / Prímá DNA diagnostika Friedreichovy ataxie.

BACKGROUND: Friedreich's ataxia is an autosomal recessive, neurodegenerative disease with a prevalence of 1-2: 100,000. Ninety five % of cases are caused by Friedreich's ataxia expansion of GAA triplet repeat in the first intron of the X25 gene. The gene is mapped on chromosome 9q. The objective of the investigation was to introduce simple and reliable DNA diagnosis helping to specify of spinocerebellare ataxias. METHODS AND RESULTS: Our diagnosis is based on the differentiation of normal and mutant alleles of gene X25 with PCR and electrophoresis on agarose gel. Size of PCR product of normal allele is in our case 521-614 bp. It is responding to 7-38 GAA triplets. Size of mutant alleles with 200-1200 GAA triplets is as 4100 bp. After the method was introduced, we analysed 12 probands. Four of them suffered from Friedreich's ataxia. CONCLUSIONS: We introduced a fast, non-radioactive, reliable DNA diagnostic method. The contribution of this method is defection of carriers and we can screen of families with the risk of Friedreich's ataxia.

[Does insurance status have an effect on frequency of appendectomies?]. / Hat der Versicherungsstatus einen Einfluss auf die Appendektomiehäufigkeit?

We investigated the influence of insurance status on the frequency of negative appendectomy. Over 5 years 820 patients underwent surgery with the diagnosis of acute appendicitis. Private and semi-private rated patients had statistically significantly more appendectomies without inflammation in the pathological specimen than ward patients (p < 0.04). This difference arose only from a statistically highly significant difference in patients aged over 40 years (p < 0.001); there was no difference between patients aged under 40 years. Possible reasons for this phenomenon are discussed. The most important finding from our data seems that financial reasons are not at all a major factor influencing the surgeon's decision for operation.

[Long-term results of replacement of the head of the radius with a silastic prosthesis]. / Langzeitresultate nach Ersatz des Radiusköpfchens mittels Silasticprothese.

22 patients after head fracture were treated with replacement of the radial head by a silastic prosthesis. 11 patients were followed up after 91 +/- 48.8 months. The results are good to excellent. Twice a fracture of the prosthesis was detected at control. No prosthetic synovitis occurred in our patients.

Arotinoid acid (Ro 13-7410): a pilot study in dermatology.

Ro 13-7410 was given to 29 patients (a total of 38 treatment courses) for 7.5 weeks (range 4-23). This compound is one of the most potent retinoids ever synthetized and has the highest affinity to human skin cellular retinoic-acid-binding protein. At therapeutically active doses, it did not induce the commonly seen mucocutaneous signs of retinoid toxicity such as scaling and cheilitis; over 0.5 microgram/kg body weight/day, it very frequently induced an eczematous retinoid dermatitis. This pilot study provides some indications on what appears to be in several aspects a drug quite distinct from retinoids previously used in humans.