Detection of FOXO1 (FKHR) Gene Break-apart by Fluorescence In Situ Hybridization in Formalin-fixed, Paraffin-embedded Alveolar Rhabdomyosarcomas and Its Clinicopathologic Correlation : Diagnostic Molecular Pathology

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00019606-200803000-00003ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008 p 14-20Detection of FOXO1 (FKHR) Gene Break-apart by Fluorescence In Situ Hybridization in Formalin-fixed, Paraffin-embedded Alveolar Rhabdomyosarcomas and Its Clinicopathologic CorrelationOriginal ArticlesMehra, Shveta MD; de la Roza, Gustavo MD; Tull, Jamie BS; Shrimpton, Antony PhD; Valente, Alfredo MD; Zhang, Shengle MDDepartment of Pathology, SUNY Upstate Medical University, Syracuse, NYReprints: Shengle Zhang, MD, Department of Pathology, SUNY Upstate Medical University, 750 East Adams Street, WH 2319, Syracuse, NY 13210 (e-mail: [email protected]).AbstractChromosomal translocations of t(2;13)(q35;q14) and t(1;13)(p36;q14), resulting in PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR) gene fusions, have been found to be specific molecular markers for alveolar rhabdomyosarcomas (ARMS) and can be identified in approximately 80% cases. As the prognosis of ARMS is worse than that of embryonal rhabdomyosarcomas (ERMS), it is important to accurately distinguish between these 2 subtypes. This distinction may be difficult on the basis of morphology alone. To detect the genetic alterations, reverse transcriptase polymerase chain reaction (RT-PCR) or dual-color dual-fusion fluorescence in situ hybridization (FISH) have been used in most studies so far. In this study, we used FOXO1 (FKHR) gene break-apart FISH probe, which can detect both of the translocations involving the FOXO1 gene, and tested 20 cases of rhabdomyosarcoma (RMS) including 6 cases of ARMS, 8 ERMS, 1 pleomorphic type, 5 not otherwise specified (RMS-NOS), and 10 non-RMS sarcomas. A home-brew RT-PCR that could detect both PAX3-FOXO1 and PAX7-FOXO1 was also performed. Four pathologists independently reviewed all RMS and a consensus diagnosis was also reached in discrepant cases. Histologic and molecular findings were correlated with clinical outcomes with an average of a 49-month follow-up. FOXO1 break-apart by FISH was positive in 4 of 6 (66%) ARMS and 2 of 5 (40%) RMS-NOS cases. All other cases, including all ERMS, were negative. RT-PCR assay confirmed all FISH results. While 2 of 6 (33%) RMS patients with a FOXO1 break-apart died of the disease, there were no deaths among the patients with negative result. The FOXO1 gene break-apart FISH probe is a simple and accurate tool to detect the translocations associated with ARMS. As characteristic genetic alterations of ARMS can be identified in 40% of RMS-NOS cases in our study, the FISH assay would provide an additional useful tool in the diagnosis and prognosis of ARMS, and an alternative to RT-PCR.Rhabdomyosarcoma (RMS) is the most common childhood malignancy, accounting for approximately 8% of all pediatric cancers, and its subtypes include alveolar (ARMS), embryonal (ERMS) and, rarely, pleomorphic (PRMS) variants. ARMS has a worse prognosis than ERMS; therefore, it is important to differentiate it from ERMS for appropriate patient management.1Histologically, ARMS is characteristically composed of ill-defined aggregates of poorly differentiated round to oval cells with central discohesion, which create spaces with free floating cells that impart an alveolar pattern. These aggregates are separated by dense and vascularized fibrous septa. Some cases, however, are composed of densely packed round tumor cells that lack the alveolar pattern altogether and are difficult to distinguish from the undifferentiated form of ERMS.2 Although the fibrovascular network is still present in this “solid” form of ARMS, the histologic diagnosis of the solid forms of ARMS rests mostly on individual cell morphology. Tumor cells in both solid and alveolar areas of ARMS have round or oval hyperchromatic nuclei with scant amounts of indistinct cytoplasm.3 In keeping with this difficulty in the histologic subclassification of RMS, up to 25% of RMS cases in our practice are signed out as unclassified RMS or RMS not otherwise specified (RMS-NOS).Molecular studies have demonstrated the presence of characteristic chromosomal translocation t(2,13)(q35;q14) and t(1;13)(p36;q14), rendering PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR), respectively, in ARMS.4–6 These genetic alterations, resulting in fusion proteins with abnormal transcriptional regulatory activity, are considered tumorigenic and specific genetic markers for ARMS.5 To detect these genetic alterations, home-brew reverse transcriptase polymerase chain reaction (RT-PCR) probes, primarily on t(2;13)(q35;q14) and dual-color dual-fusion fluorescence in situ hybridization (FISH) have been used in most studies so far.6–10In this study, we evaluated FISH assay using commercial FOXO1 (FKHR) gene break-apart probe, which can detect both translocations involving FOXO1 gene, on 20 cases of RMS and 10 other sarcomas on formalin-fixed, paraffin-embedded tissue. The samples were also analyzed by a home-brew RT-PCR probe that could detect both translocations and, if positive, by DNA sequencing. Histologic diagnosis made by 4 separate pathologists and molecular findings were correlated with clinical outcomes on follow-up.MATERIALS AND METHODSCase SelectionAfter approval by the Institutional Review Board of the SUNY-Upstate Medical University, Syracuse, New York, paraffin blocks and histologic slides from 35 cases of RMS were retrieved from archives of the Department of Pathology. For patients with multiple specimens, only 1 specimen was selected for FISH and PCR study, depending on the amount of tumor present on the sample and availability of the tissue. Twenty cases of RMS, which based on the original diagnosis on file included 9 ARMS, 4 ERMS, 6 RMS-NOS, and 1 PRMS, were selected for the study on the basis of tissue availability and at least 12 months of clinical follow-up. The average age of the patients is 16 years (range, 1 to 62 y) with 13 male and 7 female patients (Table 1). Ten cases of different types of sarcomas (1 osteosarcoma, 4 Ewing/primitive neuroectodermal tumor sarcomas, 2 undifferentiated pleomorphic sarcomas, 1 low-grade fibromyxoid sarcoma, and 2 unclassified spindle cell sarcomas) were also included as negative controls. Four pathologists independently reviewed all cases and the results were compared with the original diagnosis. If discrepancies were identified, cases were resolved by consensus diagnosis at a multiheaded microscope by 3 of the pathologists. The patient's medical records were reviewed and all relevant clinical and follow-up information was recorded.JOURNAL/dimp/04.03/00019606-200803000-00003/table1-3/v/2021-02-17T195944Z/r/image-tiff Pathologic Diagnosis and Clinical Data on Patients With RMSFISHFormalin-fixed, paraffin-embedded tissue specimens were cut in 3 to 5-μm sections on a microtome. The sections were then placed on positive charged slides, dewaxed in xylene 3 times (5 min each), in ethanol 2 times (1 min each), and then dried. They were then pretreated with boiled citrate buffer (pH 6.0) for 20 minutes followed by protease digestion for another 20 minutes. LSI FKHR (13q14) Dual Color, Break Apart Rearrangement Probe (Vysis Inc, Des Plaines, IL) was then applied before putting a coverslip. The slides were placed on a HYBrite at 75°C for 5 minutes (denaturing DNA) and then at 37°C overnight (probe hybridizing). After hybridization, the unbound probe was removed by washing and the nuclei then counterstained with DAPI, a DNA-specific stain that fluoresces blue. The slides were examined using a fluorescence microscope with appropriate excitation and emission filters that allow visualization of orange, green, and blue fluorescent signals. Hybridization signals were counted in at least 20 to 40 morphologically intact, nonoverlapping nuclei. The probes flank the FOXO1 gene located on chromosome 13. In normal cells with lack of 13q14 rearrangement in the FOXO1 gene, a 2-fusion signal (yellow) pattern was observed, which reflects 2 intact copies of FOXO1. In abnormal cells with FOXO1 break-apart showed 1 fusion (red and green overlapping), 1 green, and 1 orange signal. A break-apart signal to intact signals ratio was obtained. A ratio greater than 0.10 was considered positive for FOXO1 gene break-apart, indicating the presence of a translocation.RNA ExtractionRNA was extracted from paraffin-embedded sections using Optimum FFPE RNA Isolation kit (Ambion Diagnostics, Austin, TX). Upon pathologic examination, tumor areas were outlined and extracted after deparaffinizing. Samples were digested overnight at 37°C and then applied to filter cartridge, which binds to total RNA. After 3 washes, RNA was eluted from column, and used in RT-PCR without further purification.RT-PCRConsensus primers were designed by us to target both PAX3-FOXO1 and PAX7-FOXO1 gene fusions with amplicon of 143 bp and 155 bp, respectively. The primer sequences were 5′-CMGACASCAGCTCTGCCTAC-3′ (PAX-7M, sense) and 5′-GGACAGATTATGACGAATTGAA-3′ (FKHR-2M, antisense) (Fig. 4A). Primers for the housekeeping gene glucose 6 phosphate dehydroglucose (G6PD) were used as an internal control. They are 5′-TTGGGATCATCCGGGACGTG-3′ (sense) and 5′-GACCACATTGTTGGCCTGCA-3′ (antisense) and were designed to produce an amplicon of 151 bp. All sequences were synthesized by Sigma Genosys (The Woodlands, TX).RT-PCR was performed using Qiagen One-Step RT-PCR kit (Qiagen, Valencia, CA) with a 50 μL of reaction mixture containing 0.6 μM of all primers on Gene Amp PCR System 9700. The instrument was set up as follows: 30 minutes at 50°C for reverse transcription, 15 minutes initial PCR activation step at 95°C, followed by 40 cycles of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, 1 minute of extension at 72°C, and a final extension of 10 minutes at 72°C. The PCR products were electrophoresed on 10% polyacrylamide 1× TBE gel, stained in ethidium bromide, and visualized under a UV lamp.DNA SequencingThe Qiagen Gel Extraction kit (Qiagen, Valencia, CA) was used for DNA amplicon purification. The purified DNA was sequenced using the BigDye Terminator v3.1 kit (Applied Biosystems, Foster City, CA) according to manufacturer's protocol and run on an ABI 3100 Avant genetic analyzer (Applied Biosystems, Foster City, CA).Cell LinesCell lines SJRH30 (Douglass EC) (ATCC, Manassas, VA) and CW9019 (gift from Dr B Hall of Columbus Children's Research Institute, Columbus, OH) were used as positive controls for the PAX3/FOXO1 and PAX7/FOXO1 gene fusion, respectively. The cell lines were cultured in RPMI 1640 medium with 2 mM of L-glutamine, 1.5 g/L of sodium bicarbonate, 4.5 g/L of glucose, 10 mM of HEPES, 1.0 mM of sodium pyruvate, and 10% of fetal bovine serum. RNA was extracted with QIAamp RNA Blood Mini kit (Qiagen, Valencia, CA) according to manufacturer's instructions.RESULTSMost RMS arose from extremities and head/neck region. Seven of 16 RMS (44%) and 1 of 4 ERMS (25%) show tumor metastasis at or after the diagnosis. Of 20 RMS patients, 7 patients were lost to follow-up. Two of 6 ARMS died at 2 and 48 months after the diagnosis, whereas none of 8 ERMS or 5 RMS-NOS were dead after an average of 30 and 60-month follow-ups, respectively (Table 1).After histologic review by all 4 different pathologists and consensus diagnosis of cases with discrepant diagnosis by 3 pathologists (G.D.R., A.L.V., and S.Z.), the 20 selected cases of RMS were reclassified as: ARMS-6 cases (including 2 solid variant), ERMS-8 cases, PRMS-1 case, and RMS-NOS-5 cases (Table 2). Among RMS patients with available immunostain results, all the cases except 1 (case 5) show at least 1 positive muscular marker (myo-D1, myogenin, or desmin). There is no significant difference of immunostaining pattern between ARMS and ERMS. Four of 6 cases (67%) of ARMS and 2 of 5 (40%) RMS-NOS were positive for FOXO1 break-apart by FISH, whereas all 8 ERMS and 1 PRMS were negative (Figs. 1–3). The 2 ARMS patients (cases 2 and 9) who died of disease ARMS were positive for the FOXO1 break-apart. Two cases of ARMS without a detectable FOXO1 genetic alteration (cases 5 and 6) were alive after 10 and 6-year follow-ups. The 10 cases of non-RMS sarcoma, as expected, were all negative by FISH (Table 3).JOURNAL/dimp/04.03/00019606-200803000-00003/table2-3/v/2021-02-17T195944Z/r/image-tiff FOXO1 (FKHR) Gene Break-apart, PAX-FOXO1 Gene Fusion and Muscular Markers on RMSJOURNAL/dimp/04.03/00019606-200803000-00003/table3-3/v/2021-02-17T195944Z/r/image-tiff FOXO1 (FKHR) Gene Break-apart and PAX/FOXO1 Gene Fusion on Non-RMSJOURNAL/dimp/04.03/00019606-200803000-00003/figure1-3/v/2021-02-17T195944Z/r/image-jpeg FOXO1 (FKHR) gene break-apart FISH probes on ARMS (case 3). Pattern of 1 red, 1 green, and 1 yellow (positive) in ARMS cells (A), and pattern of 2 yellow signals (negative) in normal stroma cells (B).JOURNAL/dimp/04.03/00019606-200803000-00003/figure2-3/v/2021-02-17T195944Z/r/image-jpeg FOXO1 (FKHR) gene break-apart FISH probes on ERMS and non-RMS. Pattern of 2 yellow signals in ERMS (A, case 18) and Ewing tumor (B, case 23).JOURNAL/dimp/04.03/00019606-200803000-00003/figure3-3/v/2021-02-17T195944Z/r/image-jpeg Two RMS-NOS, (A) (case 4) and (B) (case 15), with positive FOXO1 (FKHR) gene break-apart, show no “classic” histologic features of ARMS.All the above cases, except for cases 8, 19, and 20 due to insufficient tissue, were tested by RT-PCR targeting PAX3-FOXO1 and PAX7-FOXO1. All the cases with positive FKHR gene break-apart were also positive for PAX-FOXO1 gene fusion by RT-PCR (Table 2). The cases with positive gene fusion by RT-PCR were all PAX3-FOXO1 fusion type by direct DNA sequencing (Figs. 4A–C). All the non-RMS sarcomas were negative for PAX-FOXO1 fusion by RT-PCR (Table 3).JOURNAL/dimp/04.03/00019606-200803000-00003/figure4-3/v/2021-02-17T195944Z/r/image-jpeg A, Forward (PAX7M-FWD1) and reverse (FKHR2M-REV1) primers targeting PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR) gene. B, MW-PhiX174 Hinf 1 Marker, 1—CW9019 (PAX7-FOXO1), 2—CRL2601 (PAX3- FOXO1), 3—case 1 (PAX3- FOXO1), 4—negative. C, PAX3-FOXO1 (FKHR) fusion point of case 1 by DNA sequencing.All RMS cases were reviewed by 4 pathologists. In 7 of 20 cases, there was a discrepancy between the original diagnosis and the consensus diagnosis of the authors. Among the authors, there was a complete agreement in the histopathologic classification of 5 of 20 RMS cases, and in the diagnosis of 2 of 6 ARMS (Table 4).JOURNAL/dimp/04.03/00019606-200803000-00003/table4-3/v/2021-02-17T195944Z/r/image-tiff Histologic Diagnosis of RMS by Pathologists*DISCUSSIONDetection of FOXO1 gene break-apart by FISH is a simple and reliable assay, which can be consistently performed on small, formalin-fixed, and paraffin-embedded tissue samples. The FOXO1 gene break-apart rearrangements of RMS by FISH correlated well with PAX-FOXO1 gene fusion by RT-PCR with 100% consistence. All the gene fusions by RT-PCR were PAX3-FOXO1, the most common ARMS subtype.5 The FISH assay targeting FKHR gene break-apart is highly specific for ARMS. FOXO1 gene break-apart by FISH was positive in 4 of 6 ARMS (67%) and negative in all ERMS (8 cases) and non-RMS sarcomas (10 cases). Previous studies have shown PAX-FOXO1 gene fusion in approximately 80% of ARMS.4,6Only 2 of 20 patients died of disease after 2 and 48 months of diagnosis. These 2 patients were histologically classified as ARMS and were positive for FOXO1 gene break-apart by FISH. On the other hand, the 2 cases of ARMS that were negative for FOXO1 break-apart by FISH were alive after follow-up for 6 and 10 years. Interestingly, cases 4 and 15 of RMS-NOS, lack of classic ARMS histologic features, with positive FOXO1 genetic alteration showed bone marrow and lung metastasis, respectively, at the time of diagnosis. Although this represents a small number of cases, these findings are consistent with previous studies, suggesting that FOXO1 gene fusion may represent adverse prognostic factor for ARMS.11,12 As it seems that ARMS without FOXO1 alterations often behave more like ERMS than ARMS, molecular classification of RMS based on status of FOXO1 gene fusion may be a better tool than morphology in terms of predicting clinical behavior and prognosis.As Table 4 illustrates, the diagnosis of ARMS could be proven difficult when it is based on morphology alone, particularly when the characteristic alveolar features are not evident. Relatively recent studies have shown that myogenin and myo-D1, extremely sensitive and specific markers for RMS, could be useful in the subclassification of these tumors, given the significantly greater extent of expression of these markers in ARMS than in ERMS, but their results have shown significant overlap among the different tumor subtypes.2,13 Our immunostain data also show no significant different expression of muscular markers between ARMS and ERMS.ARMS with PAX3-FOXO1 showed worse prognosis than that with PAX7-FOXO1.5,7 The subtypes of PAX-FOXO1 can be identified by PCR-based assay but not by FISH with FOXO1 break-apart probe. However, clinical utility of subtyping PAX-FOXO1 remains indeterminate in the management of patients with ARMS.In summary, detection of FOXO1 gene break-apart by FISH is a simple and reliable assay, which can be consistently performed on small, formalin-fixed, and paraffin-embedded tissue samples. 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Link]1664520600019606-200803000-0000300002366_1995_12_186_biegel_rhabdomyosarcoma_|00019606-200803000-00003#xpointer(id(citation_FROM_JRF_ID_d458e688_citationRF_FLOATING))|11065213||ovftdb|SL0000236619951218611065213citation_FROM_JRF_ID_d458e688_citationRF_FLOATING[CrossRef]10.1002%2Fgcc.287012030500019606-200803000-0000300002366_1995_12_186_biegel_rhabdomyosarcoma_|00019606-200803000-00003#xpointer(id(citation_FROM_JRF_ID_d458e688_citationRF_FLOATING))|11065405||ovftdb|SL0000236619951218611065405citation_FROM_JRF_ID_d458e688_citationRF_FLOATING[Medline Link]753645700019606-200803000-0000300005180_1996_178_410_mcmanus_rhabdomyosarcoma_|00019606-200803000-00003#xpointer(id(citation_FROM_JRF_ID_d458e728_citationRF_FLOATING))|11065213||ovftdb|SL00005180199617841011065213citation_FROM_JRF_ID_d458e728_citationRF_FLOATING[CrossRef]10.1002%2F%28SICI%291096-9896%28199604%29178%3A4%3C410%3A%3AAID-PATH508%3E3.0.CO%3B2-A00019606-200803000-0000300005180_1996_178_410_mcmanus_rhabdomyosarcoma_|00019606-200803000-00003#xpointer(id(citation_FROM_JRF_ID_d458e728_citationRF_FLOATING))|11065405||ovftdb|SL00005180199617841011065405citation_FROM_JRF_ID_d458e728_citationRF_FLOATING[Medline Link]8691319 Pathologic Diagnosis and Clinical Data on Patients With RMS FOXO1 (FKHR) Gene Break-apart, PAX-FOXO1 Gene Fusion and Muscular Markers on RMS FOXO1 (FKHR) Gene Break-apart and PAX/FOXO1 Gene Fusion on Non-RMS FOXO1 (FKHR) gene break-apart FISH probes on ARMS (case 3). Pattern of 1 red, 1 green, and 1 yellow (positive) in ARMS cells (A), and pattern of 2 yellow signals (negative) in normal stroma cells (B). FOXO1 (FKHR) gene break-apart FISH probes on ERMS and non-RMS. Pattern of 2 yellow signals in ERMS (A, case 18) and Ewing tumor (B, case 23). Two RMS-NOS, (A) (case 4) and (B) (case 15), with positive FOXO1 (FKHR) gene break-apart, show no “classic” histologic features of ARMS. A, Forward (PAX7M-FWD1) and reverse (FKHR2M-REV1) primers targeting PAX3-FOXO1 (FKHR) and PAX7-FOXO1 (FKHR) gene. B, MW-PhiX174 Hinf 1 Marker, 1—CW9019 (PAX7-FOXO1), 2—CRL2601 (PAX3- FOXO1), 3—case 1 (PAX3- FOXO1), 4—negative. C, PAX3-FOXO1 (FKHR) fusion point of case 1 by DNA sequencing. Histologic Diagnosis of RMS by Pathologists*Detection of FOXO1 (FKHR) Gene Break-apart by Fluorescence In Situ Hybridization in Formalin-fixed, Paraffin-embedded Alveolar Rhabdomyosarcomas and Its Clinicopathologic CorrelationMehra Shveta MD; de la Roza, Gustavo MD; Tull, Jamie BS; Shrimpton, Antony PhD; Valente, Alfredo MD; Zhang, Shengle MDOriginal ArticlesOriginal Articles117p 14-20

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