In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point ...mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.
Next‐generation sequencing is increasingly being chosen as a diagnostic tool for cases of expected genetic, but unresolved origin. The consequential increased need for decisions on disclosure of ...unsolicited findings poses a challenge for the informed consent procedure. This study explored the first experiences with, and needs for, the informed consent procedure in diagnostic exome sequencing, with the stakeholders involved. Semi‐structured interviews were conducted with 11 professional experts and one professional gave a written response. Furthermore, the counseling process was observed in three cases where exome sequencing was offered, followed by interviews with the patient (representative) and the genetic counselor. The respondents not only preferred an opt‐out for unsolicited findings but also identified many challenges and therefore more experiences with exome sequencing was considered needed. Context‐dependent decision‐making was observed and an Advisory Board for unsolicited findings was considered helpful while doubts were raised about the feasibility and the possibility of undermining patients' autonomy. Finally, respondents brought up the complexity of information provision, and division of responsibilities between clinicians and the lab. These challenges and needs, raised by stakeholders involved, provide more insight in the next steps needed for an optimal informed consent procedure for exome sequencing in diagnostics.
Section Editor:
Aad Tibben, email: a.tibben@lumc.nl
PALB2 analysis in BRCA2-like families Adank, M. A.; van Mil, S. E.; Gille, J. J. P. ...
Breast cancer research and treatment,
06/2011, Letnik:
127, Številka:
2
Journal Article
Recenzirano
Odprti dostop
BRCA2 and PALB2 function together in the Fanconi anemia (FA)–Breast Cancer (BRCA) pathway. Mono-allelic and bi-allelic
BRCA2
and
PALB2
mutation carriers share many clinical characteristics. ...Mono-allelic germline mutations of
BRCA2
and
PALB2
are risk alleles of female breast cancer and have also been reported in familial pancreatic cancer, and bi-allelic mutations cause a severe form of Fanconi anemia. In view of these similarities, we investigated whether the prevalence of
PALB2
mutations was increased in breast cancer families with the occurrence of
BRCA2
associated tumours other than female breast cancer.
PALB2
mutation analysis was performed in 110 non-
BRCA1/2
cancer patients: (a) 53 ovarian cancer patients from female breast-and/or ovarian cancer families; (b) 45 breast cancer patients with a first or second degree relative with pancreatic cancer; and (c) 12 male breast cancer patients from female breast cancer families. One truncating
PALB2
mutation
,
c.509_510delGA, resulting in p.Arg170X, was found in a male breast cancer patient. We conclude that germline mutations of
PALB2
do not significantly contribute to cancer risk in non-
BRCA1/2
cancer families with at least one patient with ovarian cancer, male breast cancer, and/or pancreatic cancer.
Objectives Inherited leukodystrophies represent a diagnostic challenge, as many patients remain without definitive diagnosis. In this study we investigated the genetic etiology of the recently ...described X-linked childhood disorder “Hypomyelination of Early Myelinating Structures” (HEMS). In HEMS, brain structures normally myelinating early, are hypomyelinated, which is in contrast to known hypomyelination disorders, like Pelizaeus-Merzbacher disease (PMD). Methods We included patients diagnosed with HEMS by brain MRI criteria and affected siblings. Exome sequencing was used to search for causal mutations in 16 patients of 10 families. In silico analysis of effects of the mutations on splicing and secondary RNA folding was performed. Also, gene splicing was examined in RNA prepared from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations in a specific region of the PLP1 gene. These mutations were located in exon 3B (2 silent, 1 missense and 1 deletion), which is splice out in isoform DM20 or in intron 3 outside the splice donor sequence (5 mutations). Four mutations are located deep in intron 3; a region that is not sequenced with Sanger sequencing. These mutations effect the secondary PLP1 RNA structure and decreased the PLP1/DM20 ratio. The other mutations, except for the deletion that truncated PLP1 but not DM20, were also predicted to alter PLP1/DM20 alternative splicing. Splicing studies in fibroblasts' and transfected cells confirmed a decreased PLP1/DM20 ratio. Conclusions We show that specific mutations in the PLP1 gene cause a hypomyelination pattern which contrasts the pattern seen in PMD, also caused by PLP1 mutations. This indicates that the PLP1/DM20 ratio influences early myelination. The mutations present in HEMS patients provide a challenge for PLP1 diagnostic sequencing and support the need to include intron 3.
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.
The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we ...identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.
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.
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.
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.
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.
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