Accepted Manuscript Identifying molecular markers for the sensitive detection of residual AT/RT cells Tu-Lan Vu-Han , Michael C. Frühwald , Martin Hasselblatt , Kornelius Kerl , Inga Nagel , Tobias Obser , Florian Oyen , Reiner Siebert , Reinhard Schneppenheim PII:
S2210-7762(14)00094-5
DOI:
10.1016/j.cancergen.2014.05.008
Reference:
CGEN 291
To appear in:
Cancer Genetics
Received Date: 1 March 2014 Revised Date:
12 May 2014
Accepted Date: 14 May 2014
Please cite this article as: Vu-Han T-L, Frühwald MC, Hasselblatt M, Kerl K, Nagel I, Obser T, Oyen F, Siebert R, Schneppenheim R, Identifying molecular markers for the sensitive detection of residual AT/RT cells, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MRD in AT/RT
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ACCEPTED MANUSCRIPT Identifying molecular markers for the sensitive detection of residual AT/RT cells
Tu-Lan Vu-Hana, Michael C. Frühwaldb, Martin Hasselblattc, KorneliusKerld, Inga Nagele, Tobias Obsera, Florian Oyena, Reiner Sieberte, Reinhard
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Schneppenheima*
a) University Medical Center Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology, Hamburg, Germany
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b) Children’s Hospital Augsburg, Swabian Children’s Cancer Center Augsburg, Germany
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c) Institute of Neuropathology, University Hospital Münster Muenster, Germany
d) University Children's Hospital Münster, Department of Pediatric Hematology and Oncology, Münster, Germany
e) Christian-Albrechts-University Kiel, Institute for Human Genetics, Kiel,
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Germany
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Corresponding author Reinhard Schneppenheim, MD, PhD University Medical Center Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology Martinistr. 52 20246 Hamburg, Germany phone: +49 40 7410 54270 fax: +49 40 7410 54601 email: [email protected]
running title: MRD in AT/RT Key words: AT/RT, Rhabdoid tumor, SMARCB1 Words 3585 Characters 23807
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Abstract Introduction Atypical teratoid rhabdoid tumors (AT/RT), a rare and highly malignant tumor entity of the central nervous system in early childhood, have a poor prognosis.
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They are characterized by biallelic inactivating mutations of the gene SMARCB1 in 98% of the patients which may serve as molecular markers for residual tumor cell detection in liquid biopsies. We developed a markerspecific method to detect residual AT/RT cells.
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Methods
Seven of 150 patient samples were selected, each with a histological and genetically ascertained diagnosis of AT/RT. Tumor tissue was either formalin
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fixed or fresh frozen. DNA was extracted from the patients’ peripheral blood leukocytes (PBL) and cerebro-spinal fluid (CSF). MLPA, DNA sequencing and FISH were used to characterize the tumors’ mutations. Residual tumor cell detection used mutation specific primers and real-time PCR. Results
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The detection limit for residual tumor cell search ranged from 1-18 % depending on the quality of the template provided. Residual tumor cell search in PBL and CSF was negative for all seven patients. Conclusion
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The SMARCB1 region of chromosome 22 is liable to DNA double strand breaks. The individual breakpoints and breakpoint specific PCR, offer the
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option to detect minimal residual tumor cells in CSF or blood. Even if we did not detect minimal residual tumor cells in the investigated material, proof of principle for this method was confirmed.
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Introduction Atypical teratoid rhabdoid tumor (AT/RT) is a rare and highly malignant tumor of the central nervous system, which was first described as a distinct entity in 1996 [1, 2]. Currently, AT/RT are WHO-classified under embryonal tumors
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9508/3 and received a WHO-Grade IV [3]. AT/RT account for approximately 1-2% of all tumors of the central nervous system during childhood. However, data from institutional reviews and institutional cancer registries encourage
the supposition that AT/RT constitute about 50% of all malignant brain tumors
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in children below the age of one [4]. AT/RT have an unfavorable prognosis
and the actual number of cases has likely been underestimated in the past [5], due to its close resemblance to the more prevalent childhood tumor
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medulloblastoma [6]. Rhabdoid tumors may also occur at other sites of the body like the kidney, the liver and in soft tissues [7]. One unique feature that the majority of AT/RT and other Rhabdoid tumors have in common is a biallelic alteration of the gene SMARCB1, a tumor suppressor gene located on chromosome 22q11.2 corresponding with Knudson’s two-hit recessive model
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of oncogenesis [8-10]. About 20% of the patients carry heterozygous germline mutations of SMARCB1 which defines the Rhabdoid Tumor Predisposition Syndrome type 1 (RTPS1, OMIM #609322) characterized by very early onset of tumor manifestation.
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SMARCB1/INI1 is a subunit of the SWI/SNF chromatin remodeling complex, an evolutionarily conserved multi-subunit chromatin remodeling complex,
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which uses the energy of ATP hydrolysis to mobilize nucleosomes and remodel chromatin and thereby regulate transcription of target genes [11]. Recently, loss of function mutations of the ATPase subunit BRG1/SMARCA4 of the SWI/SNF chromatin remodeling complex have also been implicated in the pathogenesis of AT/RT and other Rhabdoid Tumors [12, 13]. Constitutional inactivating mutations in SMARCA4 define Rhabdoid Tumor Predisposition Syndrome type 2 (RTPS2, OMIM# 613325) [12], further underlining the importance of this complex to suppress the generation of tumors. An inactivation or loss of the SWI/SNF complex core proteins leads to increased transcription of many genes, promoting tumor genesis [8]. These
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ACCEPTED MANUSCRIPT findings indeed provided the first link between chromatin remodeling complexes and tumor suppression [14]. AT/RT have since obtained a special position among the different tumor entities because of its distinct pathogenesis, its association with SMARCB1 and its seemingly independence from further accumulation of genetic alterations, as Hasselblatt et al. [15] and
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Kieran et al. [16] have screened AT/RT for genetic alterations other than SMARCB1 and detected none. The thus suggested genetic stability [17],
makes the gene SMARCB1 an ideal molecular marker because its clones
rarely escape detection or targeted treatment by change of genetic make-up
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[17]. The most common germline mutations have been found to be nonsense and frameshift mutations that lead to a premature truncation of the protein most preferentially in the region of exons 4 and 5 [18]. Intragenic deletions are
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likewise distributed among both somatic as well as germline abnormalities. They may involve single exons or the complete SMARCB1gene and even other flanking genes. The deletions and their corresponding breakpoints, however, have not yet been examined extensively. Thus, one research objective was to identify the breakpoints of SMARCB1 deletions as the region
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is apparently liable to DNA double strand breaks. The final objective was a proof-of-principle study whether SMARCB1-mutations may serve as molecular markers for the sensitive detection of residual tumor cells by means of real-time PCR of DNA from liquid biopsies, such as peripheral blood or
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CSF. Since the risk for an early relapse of these tumors is very high, a specific and sensitive method for early detection of tumor recurrence would be
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highly desirable.
Materials and Methods Patient-Sample Selection Seven individuals out of a cohort of 150 consecutive patients were studied. The diagnosis of AT/RT was based on the histopathology and SMARCB1/INI1 staining, which was negative in all cases. The diagnosis was also confirmed by the molecular identification of biallelic SMARCB1 alterations. Patients with germline mutations were excluded since their diagnostic targets were not specific for tumor cells. Tumor material was provided by the EU-RHAB
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ACCEPTED MANUSCRIPT neuropathology reference center (Institute of Neuropathology, University Hospital Münster), or by the Department of Pediatric Hematology and Oncology at the University Medical Center Hamburg-Eppendorf. This scientific study was covered by approval of the ethics committee and informed consent by the patients’ parents.
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DNA Extraction
Genomic DNA from either formalin-fixed paraffin-embedded (FFPE) tumor or from fresh frozen (ff) tumor tissue, as well as peripheral blood leukocytes
(PBL) and cerebrospinal fluid (CSF) from the patients was extracted by the
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QIAamp DNA Mini Kit for Tissue and Blood, according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Negative wild type control DNA was
Breakpoint Localization MLPA
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extracted from peripheral blood leukocytes of tumor-negative humans.
The breakpoints of the SMARCB1 deletions were roughly localized using multiplex-ligand-dependent probe amplification (MLPA) by the SALSA MLPA
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kit P250 DiGeorge and SALSA MLPA kit P258 SMARCB1 (both by MRCHolland, Amsterdam, Netherlands) and further narrowed down using primer walking PCR. All primers were designed using the NCBI reference Sequence NT_011520.12 of chromosome 22. Primers were designed with melting
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temperatures Tm at 71°C ± 2°C. Melting temperatures were calculated using a biocalculator (Metabion International AG). Primer walking PCR used GoTaq
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Green Master Mix (by Promega, Madison, Wisconsin, USA) according to protocol, annealing temperatures were programmed at 68°C to ensure high specificity. Breakpoints were narrowed down to 200-500 base pairs before performing a deletion spanning PCR. Deletion spanning PCR was performed using DreamTaq DNA Polymerase (by Thermo Scientific, Waltham, Massachusetts, USA) as well as Taq DNA Polymerase (by Invitrogen, Karlsruhe, Germany). The distinct band in the agarose gel (1.2 % + 1µl Ethidium bromide/100ml) was cut out and sequenced. The sequence was analyzed using the Basic Local Alignment Search Tool (NCBI Nucleotide BLAST release 2.2.28).
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ACCEPTED MANUSCRIPT FISH FISH was applied on paraffin-embedded tissues using two assays with the differentially labelled BAC clones RP11-1112A23 (labelled in Spectrum Orange) and RP11-76E8 (labelled in Spectrum Green) in Assay 1 andRP11-
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71G19 (labelled in Spectrum Orange) and RP11-911F12 (labelled in Spectrum Green) in Assay 2. A commercial probe for the centromeric region of chromosome 6 (CEP6, labelled in Spectrum Blue, Abbott, Wiesbaden,
Germany) served as internal hybridization control in both assays. Labelling of
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probes, pretreatment of FFPE-sections of tumor tissues, hybridization, washing and evaluation were performed in accordance with recently
described protocols (Ventura et al., 2006). Slides were evaluated by two
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observers using Zeiss fluorescence microscopes equipped with appropriate filter sets and documented using the ISIS software (MetaSystems, Altlussheim, Germany).
Sequencing
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PCR-gel-products were sequenced on an ABI-Prism 3130 Genetic Analyzer using ABI Prism BIG DYE Terminator Cycle Kit according to protocol.
Fragment Analysis
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The actual mechanism of loss of heterozygosity causing homozygosity for the deletion of 5006 base pairs was further analyzed using fragment analysis and
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a total of sixteen 5’-FAM-marked primer pairs for single nucleotide polymorphism (SNPs) distributed across chromosome 22, flanking the SMARCB1 region.
Primer design for residual tumor cell search Once the mutations had been identified in the tumor samples, multiple tumor specific primers were designed and subsequently tested for highest specificity. The published FASTA-formatted sequence of Chromosome 22, NCBI Reference Sequence NT_011520.12 was loaded onto Lasergene 8 SeqBuilder Program (by DNAstar, Madison, Wisconsin, USA). The program allows DNA sequence alteration and markings on the sequence, as well as p6
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ACCEPTED MANUSCRIPT primer design. The program was also used to keep an overview of all the primers used along the sequence. When two mutations were found, primers for both mutations were designed and tested for highest specificity. The mutation with better results in primer specificity was amplified during tumor cell search.
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Real-Time PCR for residual tumor cell search
Real-time PCR for residual tumor cell search was performed using Quantifast SYBR Green I (by Qiagen, Hilden, Germany) as well as the FastStart DNA MasterPLUS HybProbe PCR (by Roche Applied Sciences, Mannheim,
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Germany) based on the TaqMan-Principle with a dual-marked probe. Mutation specific primers and - for enhanced specificity - a dual-marked probe located
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between the forward and reverse primers labeled with a 5’6-FAM (Reporter) 3’BHQ-1 (Quencher) were designed. Residual tumor cell search real-time PCRs were performed on a Light Cycler Instrument (by Roche Applied Sciences, Mannheim, Germany). A series of primer pairs were designed and the primer pair with highest specificity was used for residual tumor cell detection.
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The extracted tumor cell DNA was serially diluted in wild type DNA, in order to determine the detection limit. The initial concentration for serial dilution was predetermined by the concentration of the tumor samples provided. The serial
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dilution was adjusted to the given initial concentration, causing varying serial dilution protocols among the patients. The lowest concentration of tumor cell DNA in a background of wild type DNA that could still be detected was
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determined as the detection limit.
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Results Mutation Identification In none of the five available CSF samples tumor cells were microscopically detected (s. Table 1).
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We excluded germline mutations in all seven patients and detected bi-allelic mutations in the tumor material using direct sequencing, MLPA and FISH. (s. Table 2).
In the tumor DNA of patient 1 we identified two different but overlapping large
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heterozygous deletions including SMARCB1. In the tumor DNA of patient 5, a large homozygous deletion ranging from the GNAZ locus to Exon 9 of SMARCB1 was present. However, the deletion breakpoints of patients 1 and
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5 could not be identified. Failure of the primer walking strategy in these cases can be due to more complex DNA rearrangement that we have experienced also in other genes, like discontinuous deletions, deletion/insertions or gene inversions.
In the tumor DNA of patient 2 we identified a homozygous deletion of
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SMARCB1 exon 1 (Figure 1) by MLPA mapping which was further characterized as a 5006 base pair deletion (Figure 2). In tumor samples of patient 3 two different heterozygous duplications were identified within SMARCB1 exon 5: one duplication of thirteen base pairs, and on the other
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allele a combined duplication of ten base pairs and an insertion of a single base pair at the fusion point. Tumor DNA of patient 4 revealed a homozygous
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deletion of two base pairs within Exon 6 of SMARCB1, which causes a frameshift and an early translational termination. In patient 6 a seemingly homozygous duplication of 43 base pairs in exon 5 of SMARCB1 was found. However, by FISH a large heterozygous deletion was visualized, suggesting compound heterozygosity for the duplication and the deletion. In the tumor DNA of patient 7 we identified by MLPA a very large heterozygous deletion (≈ 10 MB) ranging from TBX1 to the NIPSNAP1 locus of SMARCB1 and a duplication of two base pairs at positions 3.526.414/5 in SMARCB1 exon 3 on the other allele, also resulting in a bi-allelic alteration of SMARCB1. In contrast, FISH did not detect this large deletion. This can probably be
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ACCEPTED MANUSCRIPT explained by the different tumor material provided. Whereas the DNA for MLPA and sequencing was derived from fresh frozen tumor, only FFPE material was available for FISH. The identified large deletions are mapped and presented in Figure 3. Breakpoint Identification of Patient No° 2
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Deletion spanning PCR revealed a PCR product of approximately 600 base
pairs that did not appear in the wild type controls. The band was cut out and sequenced. The sequence contained the fusion point at position 3.515.705 and 3.520.710 on chromosome 22, where 5006 base pairs were deleted,
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including Exon 1 of SMARCB1 (Figure 2). Exon 1 of SMARCB1 is 289 base pairs long, located at positions 3.519.926 to 3.520.018 and thus within the
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deleted region, whereas Exon 2 is not affected by the deletion, since it is located 3801 base pairs further downstream. This deletion was discovered when comparing the sequence to the wild type reference sequence (GenBank Number NT_01152.12). The sequence was analyzed using the RepeatMasker program. No long interspersed nucleotide elements (LINEs), flanking the deleted sequence which could indicate homologous recombination were
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found. However, the deleted sequence was flanked by GC dinucleotides socalled mini-direct repeats (MDRs). Deletions between such micro homology sequences are regarded as being the result of DNA double-strand-breaks
nucleotides.
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followed by a non-homologous-end-joining repair mechanism with the loss of
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Mechanisms of Loss-Of-Heterozygosity (LOH) Patient’s heterozygous SNPs were analyzed in the tumor DNA: While D22S425 was in the heterozygous state like in constitutive DNA the germline heterozygous markers D22S1174 and D22S1169 were homozygous in the tumor DNA, indicating partial uniparental isodisomy of SMARCB1.Therefore, a second event, independent of the first, has probably caused partial uniparental isodisomy which resulted in the duplication of the deletion on the 2nd chromosome and thereby loss of heterozygosity (LOH).
Residual tumor cell search
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ACCEPTED MANUSCRIPT Overall the tumor samples were heterogeneous in quality and quantity, causing considerable variation of results during residual tumor cell search, Tumor cell search was performed for patients 2, 3, 4, 6 and 7 (s. Table 1). The detection limit for patient 2 was 3 ng tumor cell DNA in 30 ng whole DNA (10%). For patient 3, the detection limit was 0.35 ng tumor cell DNA in 1.38 ng
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whole DNA (18%). For patient 4 the detection limit was 0.038 ng tumor cell DNA in 3.8 ng whole DNA (1%). For patient 6 a reliable detection limit could not be obtained. For patient 7 the detection limit was 0.08 ng tumor DNA in
Discussion
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Breakpoint identification and mapping
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1.25 ng whole DNA (6%).
SMARCB1 is located in a particular liable region of chromosome 22, which seems to be prone to DNA double strand breaks followed by deficient repair mechanism pathways, such as non-homologous end-joining, as was observed in patient 2. Jackson et al. (2009) studied rhabdoid tumor mutations in this
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region. Multimodal mutation search using SNP-based oligonucleotide arrays, MLPA, fluorescence in situ hybridization as well as coding sequence analysis was required to cover the wide spectrum of SMARCB1 mutation mechanisms found in these tumors. They found the majority of breakpoints were located
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between low copy number repeat (LCR) regions rather than within LCR regions.[19]. For clinical diagnostics this effort may be rather costly and for
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residual tumor cell search the mutation mechanism must not only be identified but precise knowledge of the mutation sequence is required. A standard procedure, possibly involving next generation sequencing in the future might allow efficient further identification of deletion breakpoints of SMARCB1 in the future. Compiling them in a single map could reveal possible breakpoint accumulations and reasons for this liability.
Primer walking PCR setback The breakpoints for patients 1 and 5 could not be further identified using primer walking PCR. General possible reasons are the lack of quality of the
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ACCEPTED MANUSCRIPT sample material (highly fragmented material will not yield proper deletion spanning PCR products) and complex mutation mechanisms, e.g. large deletion/insertion mutations, discontinuous deletions and inversions. As for patient 1 in particular, the challenge was the heterozygous deletion that made a definite negative PCR signal impossible. Therefore, in order to distinguish a
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fifty-percent signal, found when only one allele was deleted, and a hundredpercent signal, found when no allele was deleted; real-time PCR was used for primer walking. This was an experimental first attempt at PCR signal
quantification for breakpoint identification using tumor samples of this kind.
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The real-time PCRs did not reveal any clearly distinguishable results,
probably due to an indefinite amount of tumor cells in the sample material. Further optimization of the mapping strategy may further enhance the range
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of mutations suitable for this method in the future. Residual tumor cell search in PBL and CSF
The evolution of cancer cells frequently leads to the formation of multiple clones due to the inherent genetic instability of most cancer types. These clones may often escape detection or targeted treatment. Under these
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premises any genetic molecular marker would eventually become useless. However, Lee and colleagues [20] have found bi-allelic losses of SMARCB1 are genetically stable, which not only affirms the high specificity of the gene
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but also makes it an ideal molecular marker. The detection limit for residual tumor cell search varied (1-18%) depending on quality and quantity of the amount of initial template tumor DNA in the reaction vessel. Gormally and
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colleagues [17] reported of similar results, “when mutant and wild-type DNA are mixed together prior to PCR, an experimental condition, which reproduces the analysis of actual biological specimens”, sensitivities of 1-6% are reached.” No residual tumor cells were detected, which overall coincides with the metastatic status of our patients at the time of sample-taking. Table 1 summarizes the follow-up data that we received from the EU-RHAB Register. Our calculations are only valid under the assumption that the tumor material consisted purely of tumor DNA. This ideal was not met in reality, as extracted DNA from solid tumors is often contaminated with normal cell DNA deriving from blood vessels nurturing the tumor mass and normal cells surrounding the
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ACCEPTED MANUSCRIPT tumor. Furthermore, FFPE tumor sample material is known to amplify less efficiently during PCR. Thus, it can be assumed that the amount of tumor cells in the reaction vessels was lower than indicated and that the sensitivity of the method could actually be higher than primarily being assumed on the grounds of these findings.
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The challenge in detecting residual solid tumor cells in liquid biopsies
The challenge in detecting residual tumor cells deriving from solid tumors in
the blood stream or CSF is the lack of understanding the actual mechanisms behind the release of tumor cells into these body fluids. In contrast to
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leukemia, where blood or bone marrow primarily presents itself filled with malignant cells as a consequence of the disease, solid tumors must yet
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release the tumor cells or mutant DNA, into the blood stream and/or into CSF in the case of brain tumors. Principally, the source of mutant DNA could therefore be either viable or apoptotic tumor cells or free mutant DNA released by the tumor, since it has been observed that fragments of tumor DNA circulate within blood [21].The clinical relevance of free tumor DNA fragments in body fluids, however, is currently not yet clear and does not
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necessarily indicate the presence of viable metastatic cells. In AT/RT, tumor cells maybe released into the cerebrospinal fluid. The current treatment protocol recommended by the EU-RHAB registry group includes
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frequent intra-thecal therapy. Therefore the CSF punctures at the respective time points would offer an ideal opportunity for tumor DNA monitoring and
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correlation with the clinical course and finally, outcome.
Sample quantity and quality One of the obstacles in our study was the varying sample quality. We realized that this issue is the limiting factor for the identification of suitable targets and the sensitivity and validity of our results. Since downstream analysis can nowadays be regarded as rather reliable, future organization of respective studies should focus on quality and quantity of tumor material by ensuring comprehensive sample asservation (fresh frozen, FFPE, PBL, CSF, touch prints),uniform DNA extraction methods and tumor material with minimal contribution of normal cells. Furthermore, a sufficient quantity of tumor sample
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ACCEPTED MANUSCRIPT material must be provided. Target identification and mutation specific primer testing may require multiple attempts, depending on the mutation type.
Suitable mutations for this method Though small deletions, small insertions and point mutations are very
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easy to identify and do contribute to half of the molecular defects in our database of 150 samples (Figure 4), they are not a good target due to limited specificity at a low tumor cell number. In contrast, large
deletions which make up another half of mutations are an ideal target for
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residual tumor cell detection, and are superior in specificity to single
base mutations. However, the identification of the deletion breakpoints is not very much straightforward as we experienced with tumor samples
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of patient 1 and 5: despite having narrowed down the breakpoints using primer walking PCR, finally no deletion spanning PCR could be established. Conclusively, due to lack of specificity single base pair mutations are not an ideal target and breakpoint analysis of very large deletions may be challenging and costly. This situation requires
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additional strategies: i) to optimize specificity for single base mutations as targets and ii) to narrow the candidate gene region of the deletion breakpoints by screening technologies like e.g. SNP arrays or next
Conclusions
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generation sequencing.
The contemporary model for the treatment of cancer underlies a continuous
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cycle of early detection, multimodal treatment and again early detection by closely monitoring the patients during follow-up. Therefore, there is much effort being put into the development of methods that could sensitively predict the presence of cancer cells [21]. Stable molecular markers could enable highly specific and sensitive tumor DNA detection after successful treatment for follow up monitoring remission or relapse as has been successfully applied in hematologic malignancies. Our results of residual AT/RT marker detection provide evidence that it is principally feasible to use mutation specific primers for detecting Rhabdoid tumor cells in peripheral blood or CSF of the patient and that SMARCB1 mutations are suitable molecular markers. The method is
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ACCEPTED MANUSCRIPT specific for each individual patient’s tumor cells and can be optimized to enhance sensitivity. A definitive proposition concerning the detection limit, i.e. sensitivity of the method, however, is currently not possible. This can further be analyzed on the grounds of these findings.
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Competing interests The authors declare no competing interests.
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Acknowledgements
This study was made possible by financial support provided by the Fördergemeinschaft Kinderkrebszentrum Hamburg e.V. to Reinhard
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Schneppenheim and was conducted in accordance with the EU-RHAB Register Study. Martin Hasselblatt is supported by IZKF Münster (Ha3/016/11) Kornelius Kerl was supported by the Sonja-Wasowicz-Stiftung. The European Registry for Rhabdoid Tumors (EU-RHAB) is supported by the “Deutsche Kinderkrebsstiftung”, the “Gesellschaft für Kinderkrebsforschung”
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and the “Parent Organization Horizonte”, Weseke, Germany. The research of Reiner Siebert was supported by the “Kinderkrebs-Initiative”
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Buchholz/Holm-Seppensen.
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Hasselblatt M, Gesk S, Oyen F, Rossi S, Viscardi E, Giangaspero F, Giannini C, Judkins AR, Fruhwald MC, Obser T et al: Nonsense mutation and inactivation of SMARCA4 (BRG1) in an atypical teratoid/rhabdoid tumor showing retained SMARCB1 (INI1) expression. The American journal of surgical pathology 2011, 35(6):933-935. Lee RS, Roberts CW: Rhabdoid tumors: an initial clue to the role of chromatin remodeling in cancer. Brain pathology 2013, 23(2):200-205. Hasselblatt M, Isken S, Linge A, Eikmeier K, Jeibmann A, Oyen F, Nagel I, Richter J, Bartelheim K, Kordes U et al: High-resolution genomic analysis suggests the absence of recurrent genomic alterations other than SMARCB1 aberrations in atypical teratoid/rhabdoid tumors. Genes, chromosomes & cancer 2013, 52(2):185-190. Kieran MW, Roberts CW, Chi SN, Ligon KL, Rich BE, Macconaill LE, Garraway LA, Biegel JA: Absence of oncogenic canonical pathway mutations in aggressive pediatric rhabdoid tumors. Pediatric blood & cancer 2012, 59(7):1155-1157. Lee RS, Stewart C, Carter SL, Ambrogio L, Cibulskis K, Sougnez C, Lawrence MS, Auclair D, Mora J, Golub TR et al: A remarkably simple genome underlies highly malignant pediatric rhabdoid cancers. The Journal of clinical investigation 2012, 122(8):2983-2988. Eaton KW, Tooke LS, Wainwright LM, Judkins AR, Biegel JA: Spectrum of SMARCB1/INI1 mutations in familial and sporadic rhabdoid tumors. Pediatric blood & cancer 2011, 56(1):7-15. NCBI Nucleotide Database. In. Jackson EM, Sievert AJ, Gai X, Hakonarson H, Judkins AR, Tooke L, Perin JC, Xie H, Shaikh TH, Biegel JA: Genomic analysis using high-density single nucleotide polymorphism-based oligonucleotide arrays and multiplex ligation-dependent probe amplification provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors. Clinical cancer research : an official journal of the American Association for Cancer Research 2009, 15(6):1923-1930. Gormally E, Caboux E, Vineis P, Hainaut P: Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutation research 2007, 635(2-3):105-117.
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Table 1: Metastatic status at the time of sample-taking and patient follow-up data concerning the disease progression Table 2: Patients' data and material. PN = Patient No., A@D = age at diagnosis, ff = fresh frozen, FFPE = formalin fixed paraffin embedded, CSF = availability of cerebro-spinal fluid, lod = limit of detection given in ng and in percentage in relation to whole DNA. *Compound heterozygosity for the two mutations confirmed by cloning.
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Figure legends Figure 1: MLPA of tumor DNA from patient 2. Compared to the reference bars (grey) the bars representing particular SMARCB1 exons (light grey) are comparable in height, except those representing exon 1, which are missing (E01), suggesting a homozygous deletion of
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exon 1. The graph below represents an approximation of the particular gene dosage which is reduced below 25 % (dark grey bars) and indicative for a homozygous deletion.
Figure 2: Map of the chromosomal region of 22q with the genes included in the
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MLPA assay and the identified large deletions including SMARCB1. Grey bars represent the deletions and their extension. Distances between the different genes fit
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roughly to scale.
Figure 3: Map and breakpoints of the deleted SMARCB1 sequence in patient 2. 5006 base pairs are missing at the fusion site of SMARCB1 exon 1. GC dinucleotide mini direct repeats are flanking the deleted region.
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Figure 4: Distribution of mutation types among 150 SMARCB1-deficient tumors.
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The majority of mutations are large deletions, nonsense mutations and small deletions.
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ACCEPTED MANUSCRIPT Table 1: Metastatic status at the time of sample-taking and patient follow-up data concerning the disease progression Status
Validation
Follow-up
1
M0
MRT/CSF Cytology
remission
2
M2b/M+
MRT/CSF Cytology
deceased
3
M0
MRT
deceased
4
M0
MRT/CSF Cytology
intracranial metastasis
5
M0
MRT/CSF Cytology
massive progression
6
n/a
n/a
n/a
7
M0
MRT/CSF Cytology
remission
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Patient No°
PN A@D
Mutation 1
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Table 2: Patients' data and material. PN = Patient No., A@D = age at diagnosis, ff = fresh frozen, FFPE = formalin fixed paraffin embedded, CSF = availability of cerebro-spinal fluid, lod = limit of detection given in ng and in percentage in relation to whole DNA. *Compound heterozygosity for the two mutations confirmed by cloning. Mutation 2
Tumor CSF
lod (ng)
lod (%)
24
delSNAP29_SEZ6L
delGNAZ_SMARCB1
ff
no
no
no
2
13
delExon1
delExon1
FFPE
yes
3
10
3*
10
c.550_559dup10 / c.559_560insG
c.570_582dup13
FFPE
no
0,35
25
c.726_727delAC
FFPE
no
0,038
1
FFPE
no
no
no
delSMARCB1
FFPE
no
no
no
delTBX1_NIPSNAP1
ff
yes
0.085
7
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1
34 63
c.726_727delAC
delGNAZ_SMARCB1 delGNAZ_SMARCB1
6
56
c.543_585dup43
7
23
c.330_331dupGT
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4 5
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Vu-Han et al. Figure 1: MLPA of tumor DNA from patient 2 displaying a homozygous deletion of SMARCB1 exon 1.
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TBX1
DGCR8
LZTR1 PPIL2
SNRPD3
SMARCB1
SEZ6L
NIPSNAP1
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SNAP29
GNAZ
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Vu-Han et al. Figure 2: Map of the chromosomal region of 22q with the genes included in the MLPA assay and the identified large deletions including SMARCB1. Grey bars represent the deletions and their extension. Distances between the different genes fit almost to scale.
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cen
Patient 1
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Patient 2 (homozygous)
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RP11-76E8 RP11-911F12 RP11-71G19 RP11-1112A23
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Patient 5 (homozygous)
Patient 6 (compound-heterozygous)
+c.543_585dup43
Patient 7 (compound-heterozygous + c.330_331dupGT)
≈ 10 MB
tel FISH probes
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Vuhan et al. Figure 3: Map and breakpoints of the deleted SMARCB1 sequence in patient 2. 5006 base pairs are missing at the fusion site of SMARCB1 exon 1. GC dinucleotide mini direct repeats are flanking the deleted region.
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S2210-7762(14)00094-5
DOI:
10.1016/j.cancergen.2014.05.008
Reference:
CGEN 291
To appear in:
Cancer Genetics
Received Date: 1 March 2014 Revised Date:
12 May 2014
Accepted Date: 14 May 2014
Please cite this article as: Vu-Han T-L, Frühwald MC, Hasselblatt M, Kerl K, Nagel I, Obser T, Oyen F, Siebert R, Schneppenheim R, Identifying molecular markers for the sensitive detection of residual AT/RT cells, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MRD in AT/RT
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ACCEPTED MANUSCRIPT Identifying molecular markers for the sensitive detection of residual AT/RT cells
Tu-Lan Vu-Hana, Michael C. Frühwaldb, Martin Hasselblattc, KorneliusKerld, Inga Nagele, Tobias Obsera, Florian Oyena, Reiner Sieberte, Reinhard
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Schneppenheima*
a) University Medical Center Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology, Hamburg, Germany
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b) Children’s Hospital Augsburg, Swabian Children’s Cancer Center Augsburg, Germany
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c) Institute of Neuropathology, University Hospital Münster Muenster, Germany
d) University Children's Hospital Münster, Department of Pediatric Hematology and Oncology, Münster, Germany
e) Christian-Albrechts-University Kiel, Institute for Human Genetics, Kiel,
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Germany
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Corresponding author Reinhard Schneppenheim, MD, PhD University Medical Center Hamburg-Eppendorf, Department of Pediatric Hematology and Oncology Martinistr. 52 20246 Hamburg, Germany phone: +49 40 7410 54270 fax: +49 40 7410 54601 email: [email protected]
running title: MRD in AT/RT Key words: AT/RT, Rhabdoid tumor, SMARCB1 Words 3585 Characters 23807
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Abstract Introduction Atypical teratoid rhabdoid tumors (AT/RT), a rare and highly malignant tumor entity of the central nervous system in early childhood, have a poor prognosis.
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They are characterized by biallelic inactivating mutations of the gene SMARCB1 in 98% of the patients which may serve as molecular markers for residual tumor cell detection in liquid biopsies. We developed a markerspecific method to detect residual AT/RT cells.
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Methods
Seven of 150 patient samples were selected, each with a histological and genetically ascertained diagnosis of AT/RT. Tumor tissue was either formalin
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fixed or fresh frozen. DNA was extracted from the patients’ peripheral blood leukocytes (PBL) and cerebro-spinal fluid (CSF). MLPA, DNA sequencing and FISH were used to characterize the tumors’ mutations. Residual tumor cell detection used mutation specific primers and real-time PCR. Results
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The detection limit for residual tumor cell search ranged from 1-18 % depending on the quality of the template provided. Residual tumor cell search in PBL and CSF was negative for all seven patients. Conclusion
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The SMARCB1 region of chromosome 22 is liable to DNA double strand breaks. The individual breakpoints and breakpoint specific PCR, offer the
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option to detect minimal residual tumor cells in CSF or blood. Even if we did not detect minimal residual tumor cells in the investigated material, proof of principle for this method was confirmed.
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Introduction Atypical teratoid rhabdoid tumor (AT/RT) is a rare and highly malignant tumor of the central nervous system, which was first described as a distinct entity in 1996 [1, 2]. Currently, AT/RT are WHO-classified under embryonal tumors
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9508/3 and received a WHO-Grade IV [3]. AT/RT account for approximately 1-2% of all tumors of the central nervous system during childhood. However, data from institutional reviews and institutional cancer registries encourage
the supposition that AT/RT constitute about 50% of all malignant brain tumors
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in children below the age of one [4]. AT/RT have an unfavorable prognosis
and the actual number of cases has likely been underestimated in the past [5], due to its close resemblance to the more prevalent childhood tumor
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medulloblastoma [6]. Rhabdoid tumors may also occur at other sites of the body like the kidney, the liver and in soft tissues [7]. One unique feature that the majority of AT/RT and other Rhabdoid tumors have in common is a biallelic alteration of the gene SMARCB1, a tumor suppressor gene located on chromosome 22q11.2 corresponding with Knudson’s two-hit recessive model
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of oncogenesis [8-10]. About 20% of the patients carry heterozygous germline mutations of SMARCB1 which defines the Rhabdoid Tumor Predisposition Syndrome type 1 (RTPS1, OMIM #609322) characterized by very early onset of tumor manifestation.
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SMARCB1/INI1 is a subunit of the SWI/SNF chromatin remodeling complex, an evolutionarily conserved multi-subunit chromatin remodeling complex,
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which uses the energy of ATP hydrolysis to mobilize nucleosomes and remodel chromatin and thereby regulate transcription of target genes [11]. Recently, loss of function mutations of the ATPase subunit BRG1/SMARCA4 of the SWI/SNF chromatin remodeling complex have also been implicated in the pathogenesis of AT/RT and other Rhabdoid Tumors [12, 13]. Constitutional inactivating mutations in SMARCA4 define Rhabdoid Tumor Predisposition Syndrome type 2 (RTPS2, OMIM# 613325) [12], further underlining the importance of this complex to suppress the generation of tumors. An inactivation or loss of the SWI/SNF complex core proteins leads to increased transcription of many genes, promoting tumor genesis [8]. These
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ACCEPTED MANUSCRIPT findings indeed provided the first link between chromatin remodeling complexes and tumor suppression [14]. AT/RT have since obtained a special position among the different tumor entities because of its distinct pathogenesis, its association with SMARCB1 and its seemingly independence from further accumulation of genetic alterations, as Hasselblatt et al. [15] and
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Kieran et al. [16] have screened AT/RT for genetic alterations other than SMARCB1 and detected none. The thus suggested genetic stability [17],
makes the gene SMARCB1 an ideal molecular marker because its clones
rarely escape detection or targeted treatment by change of genetic make-up
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[17]. The most common germline mutations have been found to be nonsense and frameshift mutations that lead to a premature truncation of the protein most preferentially in the region of exons 4 and 5 [18]. Intragenic deletions are
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likewise distributed among both somatic as well as germline abnormalities. They may involve single exons or the complete SMARCB1gene and even other flanking genes. The deletions and their corresponding breakpoints, however, have not yet been examined extensively. Thus, one research objective was to identify the breakpoints of SMARCB1 deletions as the region
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is apparently liable to DNA double strand breaks. The final objective was a proof-of-principle study whether SMARCB1-mutations may serve as molecular markers for the sensitive detection of residual tumor cells by means of real-time PCR of DNA from liquid biopsies, such as peripheral blood or
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CSF. Since the risk for an early relapse of these tumors is very high, a specific and sensitive method for early detection of tumor recurrence would be
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highly desirable.
Materials and Methods Patient-Sample Selection Seven individuals out of a cohort of 150 consecutive patients were studied. The diagnosis of AT/RT was based on the histopathology and SMARCB1/INI1 staining, which was negative in all cases. The diagnosis was also confirmed by the molecular identification of biallelic SMARCB1 alterations. Patients with germline mutations were excluded since their diagnostic targets were not specific for tumor cells. Tumor material was provided by the EU-RHAB
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ACCEPTED MANUSCRIPT neuropathology reference center (Institute of Neuropathology, University Hospital Münster), or by the Department of Pediatric Hematology and Oncology at the University Medical Center Hamburg-Eppendorf. This scientific study was covered by approval of the ethics committee and informed consent by the patients’ parents.
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DNA Extraction
Genomic DNA from either formalin-fixed paraffin-embedded (FFPE) tumor or from fresh frozen (ff) tumor tissue, as well as peripheral blood leukocytes
(PBL) and cerebrospinal fluid (CSF) from the patients was extracted by the
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QIAamp DNA Mini Kit for Tissue and Blood, according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Negative wild type control DNA was
Breakpoint Localization MLPA
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extracted from peripheral blood leukocytes of tumor-negative humans.
The breakpoints of the SMARCB1 deletions were roughly localized using multiplex-ligand-dependent probe amplification (MLPA) by the SALSA MLPA
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kit P250 DiGeorge and SALSA MLPA kit P258 SMARCB1 (both by MRCHolland, Amsterdam, Netherlands) and further narrowed down using primer walking PCR. All primers were designed using the NCBI reference Sequence NT_011520.12 of chromosome 22. Primers were designed with melting
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temperatures Tm at 71°C ± 2°C. Melting temperatures were calculated using a biocalculator (Metabion International AG). Primer walking PCR used GoTaq
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Green Master Mix (by Promega, Madison, Wisconsin, USA) according to protocol, annealing temperatures were programmed at 68°C to ensure high specificity. Breakpoints were narrowed down to 200-500 base pairs before performing a deletion spanning PCR. Deletion spanning PCR was performed using DreamTaq DNA Polymerase (by Thermo Scientific, Waltham, Massachusetts, USA) as well as Taq DNA Polymerase (by Invitrogen, Karlsruhe, Germany). The distinct band in the agarose gel (1.2 % + 1µl Ethidium bromide/100ml) was cut out and sequenced. The sequence was analyzed using the Basic Local Alignment Search Tool (NCBI Nucleotide BLAST release 2.2.28).
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ACCEPTED MANUSCRIPT FISH FISH was applied on paraffin-embedded tissues using two assays with the differentially labelled BAC clones RP11-1112A23 (labelled in Spectrum Orange) and RP11-76E8 (labelled in Spectrum Green) in Assay 1 andRP11-
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71G19 (labelled in Spectrum Orange) and RP11-911F12 (labelled in Spectrum Green) in Assay 2. A commercial probe for the centromeric region of chromosome 6 (CEP6, labelled in Spectrum Blue, Abbott, Wiesbaden,
Germany) served as internal hybridization control in both assays. Labelling of
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probes, pretreatment of FFPE-sections of tumor tissues, hybridization, washing and evaluation were performed in accordance with recently
described protocols (Ventura et al., 2006). Slides were evaluated by two
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observers using Zeiss fluorescence microscopes equipped with appropriate filter sets and documented using the ISIS software (MetaSystems, Altlussheim, Germany).
Sequencing
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PCR-gel-products were sequenced on an ABI-Prism 3130 Genetic Analyzer using ABI Prism BIG DYE Terminator Cycle Kit according to protocol.
Fragment Analysis
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The actual mechanism of loss of heterozygosity causing homozygosity for the deletion of 5006 base pairs was further analyzed using fragment analysis and
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a total of sixteen 5’-FAM-marked primer pairs for single nucleotide polymorphism (SNPs) distributed across chromosome 22, flanking the SMARCB1 region.
Primer design for residual tumor cell search Once the mutations had been identified in the tumor samples, multiple tumor specific primers were designed and subsequently tested for highest specificity. The published FASTA-formatted sequence of Chromosome 22, NCBI Reference Sequence NT_011520.12 was loaded onto Lasergene 8 SeqBuilder Program (by DNAstar, Madison, Wisconsin, USA). The program allows DNA sequence alteration and markings on the sequence, as well as p6
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ACCEPTED MANUSCRIPT primer design. The program was also used to keep an overview of all the primers used along the sequence. When two mutations were found, primers for both mutations were designed and tested for highest specificity. The mutation with better results in primer specificity was amplified during tumor cell search.
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Real-Time PCR for residual tumor cell search
Real-time PCR for residual tumor cell search was performed using Quantifast SYBR Green I (by Qiagen, Hilden, Germany) as well as the FastStart DNA MasterPLUS HybProbe PCR (by Roche Applied Sciences, Mannheim,
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Germany) based on the TaqMan-Principle with a dual-marked probe. Mutation specific primers and - for enhanced specificity - a dual-marked probe located
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between the forward and reverse primers labeled with a 5’6-FAM (Reporter) 3’BHQ-1 (Quencher) were designed. Residual tumor cell search real-time PCRs were performed on a Light Cycler Instrument (by Roche Applied Sciences, Mannheim, Germany). A series of primer pairs were designed and the primer pair with highest specificity was used for residual tumor cell detection.
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The extracted tumor cell DNA was serially diluted in wild type DNA, in order to determine the detection limit. The initial concentration for serial dilution was predetermined by the concentration of the tumor samples provided. The serial
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dilution was adjusted to the given initial concentration, causing varying serial dilution protocols among the patients. The lowest concentration of tumor cell DNA in a background of wild type DNA that could still be detected was
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determined as the detection limit.
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Results Mutation Identification In none of the five available CSF samples tumor cells were microscopically detected (s. Table 1).
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We excluded germline mutations in all seven patients and detected bi-allelic mutations in the tumor material using direct sequencing, MLPA and FISH. (s. Table 2).
In the tumor DNA of patient 1 we identified two different but overlapping large
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heterozygous deletions including SMARCB1. In the tumor DNA of patient 5, a large homozygous deletion ranging from the GNAZ locus to Exon 9 of SMARCB1 was present. However, the deletion breakpoints of patients 1 and
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5 could not be identified. Failure of the primer walking strategy in these cases can be due to more complex DNA rearrangement that we have experienced also in other genes, like discontinuous deletions, deletion/insertions or gene inversions.
In the tumor DNA of patient 2 we identified a homozygous deletion of
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SMARCB1 exon 1 (Figure 1) by MLPA mapping which was further characterized as a 5006 base pair deletion (Figure 2). In tumor samples of patient 3 two different heterozygous duplications were identified within SMARCB1 exon 5: one duplication of thirteen base pairs, and on the other
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allele a combined duplication of ten base pairs and an insertion of a single base pair at the fusion point. Tumor DNA of patient 4 revealed a homozygous
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deletion of two base pairs within Exon 6 of SMARCB1, which causes a frameshift and an early translational termination. In patient 6 a seemingly homozygous duplication of 43 base pairs in exon 5 of SMARCB1 was found. However, by FISH a large heterozygous deletion was visualized, suggesting compound heterozygosity for the duplication and the deletion. In the tumor DNA of patient 7 we identified by MLPA a very large heterozygous deletion (≈ 10 MB) ranging from TBX1 to the NIPSNAP1 locus of SMARCB1 and a duplication of two base pairs at positions 3.526.414/5 in SMARCB1 exon 3 on the other allele, also resulting in a bi-allelic alteration of SMARCB1. In contrast, FISH did not detect this large deletion. This can probably be
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ACCEPTED MANUSCRIPT explained by the different tumor material provided. Whereas the DNA for MLPA and sequencing was derived from fresh frozen tumor, only FFPE material was available for FISH. The identified large deletions are mapped and presented in Figure 3. Breakpoint Identification of Patient No° 2
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Deletion spanning PCR revealed a PCR product of approximately 600 base
pairs that did not appear in the wild type controls. The band was cut out and sequenced. The sequence contained the fusion point at position 3.515.705 and 3.520.710 on chromosome 22, where 5006 base pairs were deleted,
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including Exon 1 of SMARCB1 (Figure 2). Exon 1 of SMARCB1 is 289 base pairs long, located at positions 3.519.926 to 3.520.018 and thus within the
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deleted region, whereas Exon 2 is not affected by the deletion, since it is located 3801 base pairs further downstream. This deletion was discovered when comparing the sequence to the wild type reference sequence (GenBank Number NT_01152.12). The sequence was analyzed using the RepeatMasker program. No long interspersed nucleotide elements (LINEs), flanking the deleted sequence which could indicate homologous recombination were
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found. However, the deleted sequence was flanked by GC dinucleotides socalled mini-direct repeats (MDRs). Deletions between such micro homology sequences are regarded as being the result of DNA double-strand-breaks
nucleotides.
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followed by a non-homologous-end-joining repair mechanism with the loss of
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Mechanisms of Loss-Of-Heterozygosity (LOH) Patient’s heterozygous SNPs were analyzed in the tumor DNA: While D22S425 was in the heterozygous state like in constitutive DNA the germline heterozygous markers D22S1174 and D22S1169 were homozygous in the tumor DNA, indicating partial uniparental isodisomy of SMARCB1.Therefore, a second event, independent of the first, has probably caused partial uniparental isodisomy which resulted in the duplication of the deletion on the 2nd chromosome and thereby loss of heterozygosity (LOH).
Residual tumor cell search
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ACCEPTED MANUSCRIPT Overall the tumor samples were heterogeneous in quality and quantity, causing considerable variation of results during residual tumor cell search, Tumor cell search was performed for patients 2, 3, 4, 6 and 7 (s. Table 1). The detection limit for patient 2 was 3 ng tumor cell DNA in 30 ng whole DNA (10%). For patient 3, the detection limit was 0.35 ng tumor cell DNA in 1.38 ng
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whole DNA (18%). For patient 4 the detection limit was 0.038 ng tumor cell DNA in 3.8 ng whole DNA (1%). For patient 6 a reliable detection limit could not be obtained. For patient 7 the detection limit was 0.08 ng tumor DNA in
Discussion
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Breakpoint identification and mapping
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1.25 ng whole DNA (6%).
SMARCB1 is located in a particular liable region of chromosome 22, which seems to be prone to DNA double strand breaks followed by deficient repair mechanism pathways, such as non-homologous end-joining, as was observed in patient 2. Jackson et al. (2009) studied rhabdoid tumor mutations in this
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region. Multimodal mutation search using SNP-based oligonucleotide arrays, MLPA, fluorescence in situ hybridization as well as coding sequence analysis was required to cover the wide spectrum of SMARCB1 mutation mechanisms found in these tumors. They found the majority of breakpoints were located
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between low copy number repeat (LCR) regions rather than within LCR regions.[19]. For clinical diagnostics this effort may be rather costly and for
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residual tumor cell search the mutation mechanism must not only be identified but precise knowledge of the mutation sequence is required. A standard procedure, possibly involving next generation sequencing in the future might allow efficient further identification of deletion breakpoints of SMARCB1 in the future. Compiling them in a single map could reveal possible breakpoint accumulations and reasons for this liability.
Primer walking PCR setback The breakpoints for patients 1 and 5 could not be further identified using primer walking PCR. General possible reasons are the lack of quality of the
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ACCEPTED MANUSCRIPT sample material (highly fragmented material will not yield proper deletion spanning PCR products) and complex mutation mechanisms, e.g. large deletion/insertion mutations, discontinuous deletions and inversions. As for patient 1 in particular, the challenge was the heterozygous deletion that made a definite negative PCR signal impossible. Therefore, in order to distinguish a
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fifty-percent signal, found when only one allele was deleted, and a hundredpercent signal, found when no allele was deleted; real-time PCR was used for primer walking. This was an experimental first attempt at PCR signal
quantification for breakpoint identification using tumor samples of this kind.
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The real-time PCRs did not reveal any clearly distinguishable results,
probably due to an indefinite amount of tumor cells in the sample material. Further optimization of the mapping strategy may further enhance the range
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of mutations suitable for this method in the future. Residual tumor cell search in PBL and CSF
The evolution of cancer cells frequently leads to the formation of multiple clones due to the inherent genetic instability of most cancer types. These clones may often escape detection or targeted treatment. Under these
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premises any genetic molecular marker would eventually become useless. However, Lee and colleagues [20] have found bi-allelic losses of SMARCB1 are genetically stable, which not only affirms the high specificity of the gene
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but also makes it an ideal molecular marker. The detection limit for residual tumor cell search varied (1-18%) depending on quality and quantity of the amount of initial template tumor DNA in the reaction vessel. Gormally and
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colleagues [17] reported of similar results, “when mutant and wild-type DNA are mixed together prior to PCR, an experimental condition, which reproduces the analysis of actual biological specimens”, sensitivities of 1-6% are reached.” No residual tumor cells were detected, which overall coincides with the metastatic status of our patients at the time of sample-taking. Table 1 summarizes the follow-up data that we received from the EU-RHAB Register. Our calculations are only valid under the assumption that the tumor material consisted purely of tumor DNA. This ideal was not met in reality, as extracted DNA from solid tumors is often contaminated with normal cell DNA deriving from blood vessels nurturing the tumor mass and normal cells surrounding the
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ACCEPTED MANUSCRIPT tumor. Furthermore, FFPE tumor sample material is known to amplify less efficiently during PCR. Thus, it can be assumed that the amount of tumor cells in the reaction vessels was lower than indicated and that the sensitivity of the method could actually be higher than primarily being assumed on the grounds of these findings.
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The challenge in detecting residual solid tumor cells in liquid biopsies
The challenge in detecting residual tumor cells deriving from solid tumors in
the blood stream or CSF is the lack of understanding the actual mechanisms behind the release of tumor cells into these body fluids. In contrast to
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leukemia, where blood or bone marrow primarily presents itself filled with malignant cells as a consequence of the disease, solid tumors must yet
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release the tumor cells or mutant DNA, into the blood stream and/or into CSF in the case of brain tumors. Principally, the source of mutant DNA could therefore be either viable or apoptotic tumor cells or free mutant DNA released by the tumor, since it has been observed that fragments of tumor DNA circulate within blood [21].The clinical relevance of free tumor DNA fragments in body fluids, however, is currently not yet clear and does not
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necessarily indicate the presence of viable metastatic cells. In AT/RT, tumor cells maybe released into the cerebrospinal fluid. The current treatment protocol recommended by the EU-RHAB registry group includes
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frequent intra-thecal therapy. Therefore the CSF punctures at the respective time points would offer an ideal opportunity for tumor DNA monitoring and
.
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correlation with the clinical course and finally, outcome.
Sample quantity and quality One of the obstacles in our study was the varying sample quality. We realized that this issue is the limiting factor for the identification of suitable targets and the sensitivity and validity of our results. Since downstream analysis can nowadays be regarded as rather reliable, future organization of respective studies should focus on quality and quantity of tumor material by ensuring comprehensive sample asservation (fresh frozen, FFPE, PBL, CSF, touch prints),uniform DNA extraction methods and tumor material with minimal contribution of normal cells. Furthermore, a sufficient quantity of tumor sample
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ACCEPTED MANUSCRIPT material must be provided. Target identification and mutation specific primer testing may require multiple attempts, depending on the mutation type.
Suitable mutations for this method Though small deletions, small insertions and point mutations are very
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easy to identify and do contribute to half of the molecular defects in our database of 150 samples (Figure 4), they are not a good target due to limited specificity at a low tumor cell number. In contrast, large
deletions which make up another half of mutations are an ideal target for
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residual tumor cell detection, and are superior in specificity to single
base mutations. However, the identification of the deletion breakpoints is not very much straightforward as we experienced with tumor samples
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of patient 1 and 5: despite having narrowed down the breakpoints using primer walking PCR, finally no deletion spanning PCR could be established. Conclusively, due to lack of specificity single base pair mutations are not an ideal target and breakpoint analysis of very large deletions may be challenging and costly. This situation requires
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additional strategies: i) to optimize specificity for single base mutations as targets and ii) to narrow the candidate gene region of the deletion breakpoints by screening technologies like e.g. SNP arrays or next
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generation sequencing.
The contemporary model for the treatment of cancer underlies a continuous
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cycle of early detection, multimodal treatment and again early detection by closely monitoring the patients during follow-up. Therefore, there is much effort being put into the development of methods that could sensitively predict the presence of cancer cells [21]. Stable molecular markers could enable highly specific and sensitive tumor DNA detection after successful treatment for follow up monitoring remission or relapse as has been successfully applied in hematologic malignancies. Our results of residual AT/RT marker detection provide evidence that it is principally feasible to use mutation specific primers for detecting Rhabdoid tumor cells in peripheral blood or CSF of the patient and that SMARCB1 mutations are suitable molecular markers. The method is
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Competing interests The authors declare no competing interests.
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Acknowledgements
This study was made possible by financial support provided by the Fördergemeinschaft Kinderkrebszentrum Hamburg e.V. to Reinhard
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Schneppenheim and was conducted in accordance with the EU-RHAB Register Study. Martin Hasselblatt is supported by IZKF Münster (Ha3/016/11) Kornelius Kerl was supported by the Sonja-Wasowicz-Stiftung. The European Registry for Rhabdoid Tumors (EU-RHAB) is supported by the “Deutsche Kinderkrebsstiftung”, the “Gesellschaft für Kinderkrebsforschung”
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and the “Parent Organization Horizonte”, Weseke, Germany. The research of Reiner Siebert was supported by the “Kinderkrebs-Initiative”
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Buchholz/Holm-Seppensen.
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Table 1: Metastatic status at the time of sample-taking and patient follow-up data concerning the disease progression Table 2: Patients' data and material. PN = Patient No., A@D = age at diagnosis, ff = fresh frozen, FFPE = formalin fixed paraffin embedded, CSF = availability of cerebro-spinal fluid, lod = limit of detection given in ng and in percentage in relation to whole DNA. *Compound heterozygosity for the two mutations confirmed by cloning.
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Figure legends Figure 1: MLPA of tumor DNA from patient 2. Compared to the reference bars (grey) the bars representing particular SMARCB1 exons (light grey) are comparable in height, except those representing exon 1, which are missing (E01), suggesting a homozygous deletion of
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exon 1. The graph below represents an approximation of the particular gene dosage which is reduced below 25 % (dark grey bars) and indicative for a homozygous deletion.
Figure 2: Map of the chromosomal region of 22q with the genes included in the
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MLPA assay and the identified large deletions including SMARCB1. Grey bars represent the deletions and their extension. Distances between the different genes fit
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roughly to scale.
Figure 3: Map and breakpoints of the deleted SMARCB1 sequence in patient 2. 5006 base pairs are missing at the fusion site of SMARCB1 exon 1. GC dinucleotide mini direct repeats are flanking the deleted region.
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Figure 4: Distribution of mutation types among 150 SMARCB1-deficient tumors.
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The majority of mutations are large deletions, nonsense mutations and small deletions.
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Validation
Follow-up
1
M0
MRT/CSF Cytology
remission
2
M2b/M+
MRT/CSF Cytology
deceased
3
M0
MRT
deceased
4
M0
MRT/CSF Cytology
intracranial metastasis
5
M0
MRT/CSF Cytology
massive progression
6
n/a
n/a
n/a
7
M0
MRT/CSF Cytology
remission
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Patient No°
PN A@D
Mutation 1
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Table 2: Patients' data and material. PN = Patient No., A@D = age at diagnosis, ff = fresh frozen, FFPE = formalin fixed paraffin embedded, CSF = availability of cerebro-spinal fluid, lod = limit of detection given in ng and in percentage in relation to whole DNA. *Compound heterozygosity for the two mutations confirmed by cloning. Mutation 2
Tumor CSF
lod (ng)
lod (%)
24
delSNAP29_SEZ6L
delGNAZ_SMARCB1
ff
no
no
no
2
13
delExon1
delExon1
FFPE
yes
3
10
3*
10
c.550_559dup10 / c.559_560insG
c.570_582dup13
FFPE
no
0,35
25
c.726_727delAC
FFPE
no
0,038
1
FFPE
no
no
no
delSMARCB1
FFPE
no
no
no
delTBX1_NIPSNAP1
ff
yes
0.085
7
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1
34 63
c.726_727delAC
delGNAZ_SMARCB1 delGNAZ_SMARCB1
6
56
c.543_585dup43
7
23
c.330_331dupGT
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Vu-Han et al. Figure 1: MLPA of tumor DNA from patient 2 displaying a homozygous deletion of SMARCB1 exon 1.
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TBX1
DGCR8
LZTR1 PPIL2
SNRPD3
SMARCB1
SEZ6L
NIPSNAP1
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GNAZ
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Vu-Han et al. Figure 2: Map of the chromosomal region of 22q with the genes included in the MLPA assay and the identified large deletions including SMARCB1. Grey bars represent the deletions and their extension. Distances between the different genes fit almost to scale.
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Patient 1
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Patient 2 (homozygous)
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RP11-76E8 RP11-911F12 RP11-71G19 RP11-1112A23
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Patient 5 (homozygous)
Patient 6 (compound-heterozygous)
+c.543_585dup43
Patient 7 (compound-heterozygous + c.330_331dupGT)
≈ 10 MB
tel FISH probes
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Vuhan et al. Figure 3: Map and breakpoints of the deleted SMARCB1 sequence in patient 2. 5006 base pairs are missing at the fusion site of SMARCB1 exon 1. GC dinucleotide mini direct repeats are flanking the deleted region.
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