Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern ...in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5-11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions.
We assessed the prognostic impact of
TET2
mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and ...Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell’ Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of
TET2
mutations were investigated.
TET2
mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (
p
= 0.024).
TET2
and
IDH1
/
2
mutations strongly associated with aberrations in the DNA methyltransferase
DNMT3A
. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant
TET2
forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function
TET2
mutations predicted poor outcome, we questioned whether low
TET2
mRNA expression in cases of AML without
TET2
mutations would affect overall survival. Notably, also AML patients with low
TET2
mRNA expression levels showed inferior overall survival.
In normal bone marrow, WT1 expression is restricted to CD34+ cells. We assessed WT1 mRNA expression levels with quantitative, real‐time reverse transcription polymerase chain reaction in normal, ...myelodysplastic (MDS) and secondary acute myeloid leukaemia (sAML) bone marrow subfractions, based on differentiation status. The highest WT1 expression was observed in the primitive CD34+ rhodamine‐123 (rho) dull cells, both in healthy donors and MDS or sAML patients. In contrast to normal CD34‐negative bone marrow cells, WT1 was present in CD34‐negative bone marrow cells in 12 out of 13 MDS patients and two sAML samples. Further analysis of this aberrant WT1 expression was performed in the CD34‐negative subfractions of three MDS patients. In one of these, WT1 expression was found exclusively in the erythroid cells. This patient was completely transfusion dependent and showed morphological dyserythropoiesis. In another MDS patient, WT1 expression was found in a non‐erythroid compartment. We conclude that abnormal WT1 expression may contribute to the disturbed differentiation of haematopoietic cells in MDS patients.
The specificity of antisense oligonucleotides targeted to the mRNA breakpoint region of the Bcr‐Abl oncogene, found in leukaemic cells from patients with chronic myeloid leukaemia, remains ...controversial due to non‐specific effects. To prevent protein binding of oligonucleotides we designed and tested a methylphosphonate oligonucleotide with an attached 3′ soluble phosphodiester tail. Growth of chronic myeloid leukaemia (CML) cell lines BV173, KCL‐22 and cells of CML patients tested was inhibited by the b2a2 type antisense Bcr‐Abl oligonucleotide and not with controls. Also the growth of control CD34+ cells of two healthy donors, control cell lines and cells from AML patients was only moderately affected or not affected. Bcr‐Abl protein studies in combination with growth‐determination experiments indicated that the antisense methylphosphonate Bcr‐Abl oligonucleotide tested is a potent inhibitor of the growth of CML cells but works in a non‐antisense manner.
summary
Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C.The mutation is associated with an increased thrombotic ...risk and thus- far the most common genetic cause of thrombophilia.Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing.The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal,heterozygous and homozygous mutant individuals.Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring.
The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient,fast,automated and highly reproducible method that can be used in a routine laboratory setting.
The aims of Molecular Diagnosis of Cancer are to introduce scientists and physicians working in the field of diagnostics to the area of cancer molecular pathology and to highlight the possibilities ...of its application to the cancer physician in the clinic. The degree of molecular biological expertise required should be minimal, although those with more experience may also be able to benefit from the book. All of the authors have considerable practical experience in the method they describe and are working predominantly in the setting of the cancer clinic. As such, the book pulls together a number of te- niques that are already being applied to a wide range of malignancies. Mo- over, this field will continue to expand exponentially as further research leads to a greater understanding of the molecular basis of cancer. Detection of the changes to the DNA or RNA code within a diseased cell often provides pathological information for the diagnosis, prognosis, and m- agement of the disease. Such DNA-related analysis is primarily the role of molecular pathology. Methods to detect these alterations are being refined and are evolving from the research to the diagnostic laboratory. One of the single most powerful techniques in this new branch of pathology has been the polymerase chain reaction (PCR), to which much of this book is devoted.
Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to ...quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the β-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the ΔCt
2value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The β-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.
The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered
recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary ...tumor, and peripheral blood. Ten previously untreated
patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two
dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients.
Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-γ and IL-10
increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections.
A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed,
due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage
of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern
was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear
cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells
in the lymph nodes showed a 128-fold (mean) increase of IFN-γ mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes
compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and
neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose
level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.
PDF
