Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.     

KTP Laser and Neutral Red Phototherapy of Human Squamous Cell Carcinoma

Neutral red (NR) is a cationic, nontoxic vital dye employed as a histologic stain for proliferating cells; it has been used clinically for photodynamic treatment of herpes simplex virus lesions. NR is selectively taken up and concentrated by mitotic cells, an important characteristic for more effective antineoplastic agents. In the present study, UCLA-SO-P3 human squamous carcinoma cells displayed minimal toxicity when incubated with up to 50 μg/ml NR in the absence of light. However, cells incubated with greater than 0.5μg/ml NR followed by exposure to KTP laser light at 532 nm exhibited nearly 100% tumor cell death. The degree of cell toxicity was proportional to NR dose and laser light fluence. This study demonstrates that NR is an excellent cancer cell photosensitizer in vitro, and, after adding additional in vivo preclinical testing, may prove to be a useful agent in photodynamic destruction of head and neck tumors.     

High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.

PURPOSE: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34(+)CD38(-) stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. EXPERIMENTAL DESIGN: First, the leukemogenic potential of unpurified CD34(+)CD38(-) cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34(+)CD38(-) compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. RESULTS: In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34(+)CD38(-) stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34(+) percentage showed no such correlations. CONCLUSIONS: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34(+)CD38(-) population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.     

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.     

Measuring growth of residual cholesteatoma in subtotal petrosectomy

Background: Little is known about the growth rate of cholesteatoma in patients.

Objective: Investigate the growth of residual cholesteatoma in subtotal petrosectomy based on volume measured in MRI scans.

Materials and methods: Retrospective case series in a Tertiary Medical Centre. Thirteen residual cholesteatomas were identified in 10 patients after subtotal petrosectomy for which a wait-and-scan policy was adopted. Volume of the residual cholesteatoma was calculated by manual segmentation as well as the ‘box method’.

Results: Mean growth rate was 27.9 mm3/month (SD 22.8), with a large individual variation ranging from 2.2 to 69.8 mm3/month. No complications were reported in 10 patients with a wait-and-scan policy for residual cholesteatoma in subtotal petrosectomy. The box method overestimates growth rate compared to the reference method manual segmentation and a linear increase of this systematic error was seen with increasing size of the cholesteatoma.

Conclusions: Residual cholesteatoma growth rate shows a large individual variation. A wait-and-scan policy could be considered in case of a (small) residual in subtotal petrosectomy with ample room to grow before destroying any remaining structures. Furthermore, the clinically more applicable and less time-consuming box method can be used to accurately measure volumes of small cholesteatomasup to a volume of 500?mm³.