False positivity of ETV6/RUNX1 detected by FISH in healthy newborns and adults

Kusk, Maria Schioldan; Lausten-Thomsen, Ulrik; Andersen, Mette Klarskov; Olsen, Marianne; Hjalgrim, Henrik; Schmiegelow, Kjeld

doi:10.1002/pbc.25050

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…Initially identified in umbilical cord blood of one healthy newborn and the peripheral blood of 13 healthy children and adults [32], ETV6-RUNX1 was shown to be present in~1% of newborns [6]. Several Danish studies later challenged these findings [36,[44][45][46][47], but newer reports confirmed the original results [7,[37][38][39].…”

Section: Etv6-runx1mentioning

confidence: 95%

Insights into the prenatal origin of childhood acute lymphoblastic leukemia

Hein

Borkhardt

Fischer

2020

Cancer Metastasis Rev

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Pediatric acute lymphoblastic leukemia (ALL) is defined by recurrent chromosomal aberrations including hyperdiploidy and chromosomal translocations. Many of these aberrations originate in utero and the cells transform in early childhood through acquired secondary mutations. In this review, we will discuss the most common prenatal lesions that can lead to childhood ALL, with a special emphasis on the most common translocation in childhood ALL, t(12;21), which results in the ETV6-RUNX1 gene fusion. The ETV6-RUNX1 fusion arises prenatally and at a 500-fold higher frequency than the corresponding ALL. Even though the findings regarding the frequency of ETV6-RUNX1 were originally challenged, newer studies have confirmed the higher frequency. The prenatal origin has also been proven for other gene fusions, including KMT2A, the translocations t(1;19) and t(9;22) leading to TCF3-PBX1 and BCR-ABL1, respectively, as well as high hyperdiploidy. For most of these aberrations, there is evidence for more frequent occurrence than the corresponding leukemia incidences. We will briefly discuss what is known about the cells of origin, the mechanisms of leukemic transformation through lack of immunosurveillance, and why only a part of the carriers develops ALL.

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Section: Etv6-runx1mentioning

confidence: 95%

Insights into the prenatal origin of childhood acute lymphoblastic leukemia

Hein

Borkhardt

Fischer

2020

Cancer Metastasis Rev

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“…However, there were possibilities that RT-PCR and FISH produce false positive results due to technical problems although it might be rare, [33][34][35][36][37][38] and that not all the best probes and primers for known fusion genes with optimal conditions were used in all 13 cases. Thus, we are still unable to definitively conclude that all thirteen reported cases were primary CCS of the bone.…”

Section: Discussionmentioning

confidence: 99%

Primary clear cell sarcoma of the femur: a unique case with RT-PCR and direct sequencing confirmation of EWSR1/ATF1 fusion gene

Kubota

Tanaka

Hisaoka

et al. 2021

BMC Musculoskelet Disord

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Background It is very rare for clear cell sarcomas (CCS) to arise in the bone. During diagnosis, it is important to distinguish primary CCS of bone from bone metastasis of melanoma because this difference fundamentally changes the therapeutic options. Recently, characteristic fusion genes of CCS have been detected using reverse transcription polymerase chain reaction (RT-PCR) or direct sequencing which allowed to distinguish CCS from melanoma. However, there was no study applying these analyses with positive results. In this case, we describe the use of fusion gene analysis to diagnose a primary CCS of the bone. Case presentation A 36-year-old male presented with a four-months history of left knee pain. Magnetic resonance imaging showed a lesion in the left femoral medial epicondyle. Histological examination of the biopsy specimen revealed proliferating oval or rounded cells. These cells had clear cytoplasm arranged in fascicles or compact nests with frequent deposits of brown pigment. Furthermore, immunohistochemistry analysis revealed that tumor cells were positive for S-100 protein, HMB-45, Melan-A, and SOX10. It stained negative for CD34 and BRAF v600e. Conclusively, detection of the EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing confirmed that the lesion was a primary CCS of the bone. Wide-margin resection and reconstruction with a tumor endoprosthesis were performed. Conclusions Herein, we diagnosed a rare case of primary CCS of the bone by detecting EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing. Since fluorescence-in situ hybridization (FISH) and RT-PCR could show false positive by mainly due to technical problems, it is better to perform direct sequencing to confidently diagnose the tumor as a primary CCS especially at very rare site such as bone.

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“…Moreover, a technical detail deserves mentioning that in recent years fusion genes are not usually studied using traditional hybridization-based techniques such as FISH (fluorescent in-situ hybridization) and southern blots but, instead, are mainly studied using RT-PCR and RNA-seq technologies that detect fusion RNAs, but not fusion genes per se . RT and PCR may create many artifacts, as to be discussed later, making it possible that some so detected are artifacts 75 , 94 , 100 , 101 . Also, in many cases it is actually unclear whether the detected chimeric RNAs, even if they are authentic, are associated with a corresponding fusion gene or are just formed at the RNA level.…”

Section: From Fusion Rna To Fusion Gene: the Cart Before The Horse Inmentioning

confidence: 99%

Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers

Peng¹,

Yuan²,

Zellmer³

et al. 2015

J. Cancer

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Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine “two neighboring genes” as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of “consecutive reverse transcriptions (RTs)”, i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR.

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False positivity of ETV6/RUNX1 detected by FISH in healthy newborns and adults

Cited by 5 publications

References 11 publications

Insights into the prenatal origin of childhood acute lymphoblastic leukemia

Insights into the prenatal origin of childhood acute lymphoblastic leukemia

Primary clear cell sarcoma of the femur: a unique case with RT-PCR and direct sequencing confirmation of EWSR1/ATF1 fusion gene

Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers

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