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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 1  |  Page : 64-72
Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas


1 Division of Molecular Pathology and Translational Medicine, Tata Memorial Centre, Mumbai, Maharashtra, India
2 Division of Molecular Pathology and Translational Medicine; Department of Surgical Pathology, Tata Memorial Centre, Mumbai, Maharashtra, India

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Date of Web Publication31-Jan-2020
 

   Abstract 


Background: Synovial sarcoma (SS) is an aggressive, but a relatively chemosensitive soft tissue sarcoma, characterized by a specific, t (X;18)(p11;q11) translocation, leading to formation of SS18–SSX chimeric transcript. This translocation can be detected by various techniques, such as fluorescence in-situ hybridization (FISH), reverse transcriptase PCR (RT-PCR) and fragment analysis. Objectives: To compare the results of detection of t (X;18)(p11;q11) translocation, across three different platforms, in order to determine the most optimal and sensitive technique. Methods: Formalin-fixed paraffin embedded (FFPE) tissue sections of 45 soft tissue sarcomas were analyzed, including 16 cases of SS confirmed by histopathology, immunohistochemistry and molecular technique (s)(Group 1); 13 cases, wherein SS was one of the differential diagnosis, preceding molecular testing (Group 2) and 16 cases of various other sarcomas (Group 3). Various immunohistochemical (IHC) markers studied, including INI1/SMARCB1. All cases were tested for t (X;18) translocation, by fragment Analysis, FISH and RT-PCR. Results: There were 23 cases of SS, including 16 of group 1 and 7 of group 2. By fragment analysis, t (X;18)(p11;q11) translocation was detected in 22/23 cases (95.6%). By FISH, SS18 gene rearrangement was detected in 18/22 cases (78.2%), whereas by RT-PCR, SS18-SSX transcripts were detected in 15/23 cases (65.2%). Immunohistochemically, a unique “weak to absent”/reduced INI1 immunostaining pattern was exclusively observed in 12/13 cases of SS (92.3%). Fragment analysis and FISH were relatively more sensitive techniques. Unique “weak to absent”INI1 immunoexpression significantly correlated with positive t (X;18) translocation results (P = 0.0001). Conclusion: The present study constitutes first such study from our subcontinent. Fragment analysis is a promising technique for detection of t (X;18)(p11;q11) translocation. FISH and INI1 immunostaining pattern were also relatively more sensitive, over RT-PCR.

Keywords: Fluorescence in-situ hybridization, fragment analysis, INI1/SMARCB1, molecular diagnosis in sarcomas, Reverse Transcriptase PCR, synovial sarcoma, t(X;18)(p11;q11)

How to cite this article:
Shetty O, Pai T, Gurav M, Rekhi B. Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas. Indian J Pathol Microbiol 2020;63:64-72

How to cite this URL:
Shetty O, Pai T, Gurav M, Rekhi B. Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Jul 5];63:64-72. Available from: http://www.ijpmonline.org/text.asp?2020/63/1/64/277430





   Introduction Top


Synovial sarcoma (SS) is a malignant mesenchymal tumor that displays a variable degree of epithelial differentiation, and is characterized by a specific t (X; 18)(p11;q11) SS18-SSX chromosomal translocation.[1],[2],[3] Clinically, SS is an aggressive sarcoma; accounts for 5–14% of soft tissue sarcomas and mostly occurs in the extremities of young adults. It is the most commonly diagnosed adult soft tissue sarcoma, especially in extremity sites and trunk, in Indian settings and is one of the few adult soft tissue sarcomas that is relatively chemosensitive.[4] Therefore, its exact diagnosis is crucial.

Despite availability of the various immunohistochemical (IHC) markers, including the relatively newly identified IHC maker, namely TLE1, presently, the diagnostic 'gold' standard of a SS is the demonstration of t (X; 18)(p11;q11) translocation.[1],[5],[6],[7] Molecular studies of the translocation breakpoints have unraveled that the proximal portion of the SS18(SYT) gene at 18q11 fuses with the distal portion of one of the SSX genes located on chromosome Xp11.[1] More than 95% of the SSs harbor these translocations, leading to the formation of specific chimeric transcripts, namely SS18/SSX1, SS18/SSX2, and SS18/SSX4. Some of these fusion transcripts have been observed to be associated with a specific histopathologic subtype and the clinical course of SS.[2],[3]

Multiple lines of evidence support SS18-SSX as the central genetic “driver” in this cancer. Foremost, its presence as the sole cytogenetic anomaly is seen in up to a third of cases. Secondly, there is low frequency of additional mutations in SSs. Finally, there is preservation of this specific underlying translocation in metastatic and advanced cases.[8],[9],[10]

Ideally, the diagnosis in case of sarcomas, characterized by recurrent genetic aberrations, should be confirmed by molecular test result, even if initially reported by a specialist pathologist.[11] However, cost and feasibility limit this practice in limited resource setting like ours. Nonetheless, molecular diagnosis is imperative in such tumors occurring at uncommon location and in cases with equivocal IHC results.

Since the first report by Kawai et al.[2], several techniques have been employed to demonstrate t (X; 18) translocation for detection of SS18-SSX fusion transcripts. Molecular analysis of SS18–SSX fusion transcripts by conventional reverse transcriptase polymerase chain reaction (RT–PCR) on archived formalin-fixed, paraffin wax embedded (FFPE) tumor specimens has been found as a feasible and a reliable technique for the molecular diagnosis of SS.[12] However, this technique has its limitations. Amplification of the fusion transcripts from the archived paraffin tissue blocks is difficult at times, as a result of inferior quality of the RNA, in case the tumor content is less, or the tissue (in certain referral cases, submitted to our centre) is suboptimally fixed.[13] This leads to a lower sensitivity. Quantitative Real-Time PCR (Q RT- PCR) is another sensitive technique. However, even this technique requires a reasonably good quality RNA.[3],[8] Fluorescence in-situ hybridization (FISH) is yet another technique for detecting SS18 gene rearrangement. Despite its reasonable sensitivity, this technique cannot differentiate between various molecular subtypes.[14] Lately, fragment size using capillary electrophoresis has been observed as a sensitive and rapid technique for detection chimeric fusion transcript amplicons. This is a multiplex assay comprising primers for general fusion transcript, as well as of specific genotype, which specifically bind to the chimeric fusion transcripts. The amplicons are resolved on the basis of differences in the fragment size. Till date, there has been only a single published study, based on this technique for molecular differentiation of SS, including its two molecular subtypes.[15] Although, there is available literature regarding comparison between interphase FISH, conventional RT-PCR and real time PCR, for detection of the chimeric fusion transcript in cases of SS, there is no documentation on comparison between results of fragment size analysis by capillary electrophoresis, FISH and conventional RT-PCR.

Presently, except 2 studies on immunohistochemical analysis, including TLE1 and INI1 immunoexpression, coupled with molecular results, there is no sizable published study related to molecular techniques for diagnosing SS, from our subcontinent.[5],[6] The present study was undertaken to compare results of detection of specific t (X;18) translocation, utilizing all the three platforms, in order to determine the most optimal, rapid, accurate technique. Furthermore, it was intended to detect all the 3 clinically relevant chimeric fusion transcript genotypes of SS, namely SS18-SSX1, SS18-SSX2 and SS18-SSX4 with the help of a single assay.


   Methods Top


Tumor samples and histopathologic evaluation

The present study included 45 cases of sarcomas, including SSs that were tested for the t(X;18)(p11;q11) translocation. It was performed on formalin-fixed paraffin embedded tissue sections (FFPE), which were retrieved from the Department of Pathology of our institution.

Clinical and demographic details of the patients were obtained from the electronic medical records (EMR). In all the cases, histopathologic sections, including hematoxylin and eosin (H&E) stained and IHC stained sections were reviewed by a senior pathologist (B.R.), routinely engaged in reporting bone and soft tumors, at our Institution. Diagnosis of SS was based on histopathologic features described in the World Health Organization (WHO) classification of tumors of soft tissues.[1] All cases of SS were further classified as monophasic (MP), biphasic (BP) and poorly differentiated (PD), wherever possible.

IHC staining was performed using the polymer technique (Dako REAL Envision detection system, Glostrup, Denmark) including peroxidase/3-3-diaminobenzidine tetrahydrochloride (DAB). Details of the various IHC antibody markers, utilized in cases of SS have been enlisted in [Table 1].
Table 1: List of the immunohistochemical antibody markers used in the present study

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There were 16 cases with an unequivocal diagnosis of SS, rendered on histopathology with immunohistochemical results and further confirmed by molecular testing; 13 cases, in which SS was one of the differential diagnosis, offered during histopathologic evaluation, preceding molecular testing and 16 cases of sarcomas, other than SSs. All these cases were placed in Group 1 (n = 16),

Group 2 (n = 13) and Group 3 (n = 16), respectively.

Paraffin blocks with more than 50% tumor tissue content were selected for molecular analysis.[15] FISH technique was considered as the diagnostic gold standard, against which RT-PCR, fragment analysis and IHC expression of INI1 were compared.

RNA extraction and cDNA synthesis

Total RNA was extracted from the representative FFPE samples. 4 sections, each of 10 μm thickness, were treated with limonene (Sigma Aldrich, USA) and alcohol followed by overnight digestion with proteinase K (Ambion, Thermo scientific, USA). Total RNA extraction was performed using RecoverAll total nucleic acid isolation kit (Ambion, Thermo scientific, USA). The quality of the extracted RNA was checked by measuring concentration and 260:280 ratio using Nanodrop (Thermo scientific, USA). First strand complementary DNA (cDNA) was synthesized from 100 ng of total RNA using RevertAid™ H MinusFirst Strand cDNA Synthesis Kit (Thermo Scientific, USA) as per the manufacturer's protocol.

Primers

All the RT-PCR primers and unlabeled primers for multiplex PCR were synthesized from Sigma genosys (Sigma Aldrich, USA). Fluorochrome labeled primer for fragment analysis platform were from Applied Biosystems (Thermo scientific, Foster city, USA). Sequences of all primers used in this study are shown in [Table 2].[14],[15]
Table 2: Primer Sequences,[13],[14]

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PCR for ACTNB

The integrity and the quality of the extracted RNA was assessed by performing PCR for beta actin (ACTB) housekeeping gene using cDNA as template. PCR comprised of primer pairs with product size of 323bp. cDNA samples amplifiable at this product size was selected for further analysis.

Conventional RT-PCR

All samples positive for ACTB PCR were analyzed for the presence of SS18-SSX (general), SS18-SSX1 and SS18-SSX2 fusion transcripts using specific primer sets designed, based on the known fusion genes and breakpoints. PCR amplification was carried out in a 20 μL reaction volume containing 2 μL of template cDNA, 1 μL of 10 pmol of each forward and reverse primer and 10 μL of 2 X PCR master mixes (Thermo Scientific, USA). The PCR conditions for the detection of SS18-SSX general were as follows: predenaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 30 seconds for 30 cycles, and a final extension of 10 minutes at 72°C. For SS18-SSX1 and SS18-SSX2, the conditions were 94°C for 3 minutes, followed by 35 cycles of 94°C for 45 seconds, 61°C for 1 minute and 72°C for 1 minute, and completed with an extension step at 72°C for 10 minutes. The PCR products were separated through a 10% polyacrylamide gel, stained with ethidium bromide and visualized under UV illumination (Alpha Imager, Bioscreen, USA).

Fluorescence In-Situ Hybridization (FISH)

FISH technique was performed on 2 μm paraffin sections of each sample using Zytolight SPEC SS18 Dual color break-apart probe (Zytovision GmBH, Germany). Sections were transferred to poly-l-lysine coated slides and baked overnight at 60°C. These sections were deparaffinized by xylene followed by dehydration with absolute alcohol. After pretreatment in citric acid buffer (pH 6.0) at 98°C for 15 minutes, slides are washed in 2X sodium chloride-sodium citrate buffer (SSC). Slides were incubated in 4 mg/ml of pepsin (in 10 mM HCL, pH 2.0) at 37°C for 25 minutes, dehydrated in increasing grades of alcohol (70°C, 85°C and 100°C for 1 minute each) and air dried completely. A 6-8 μL of SYT probe mixture was applied to the section and sealed under a coverslip with rubber cement. The slides were incubated at 75°C for 10 minutes to co-denature. Hybridization with the probe was done at 37°C for 16-20 hours. Following hybridization, the slides were washed with 2X SSC (pH 7.5) at 73°C for 4 minutes, and at room temperature for 2 minutes. The nuclei were counterstained with the 4′, 6 -diamidino-2-phenylindole (DAPI) in dark. The signals were viewed under an Olympus BX 53F upright fluorescence microscope equipped with filters specific for DAPI, TRITC/Spectrum Orange and FITC/Spectrum Green, QIcam Olympus camera and Q Capture pro 7.0 image analyzer software. For all cases, at least 100 non-overlapping tumour cell nuclei were counted. A probe was considered to be break-apart when a pair of orange and green signals was separated by a distance greater than the size of one signal. More than 20% tumor cells demonstrating split (break-apart) signals were classified as positive for SS18 gene rearrangement, while those with 'split' in less than 20% of cells were scored as negative.[3]

Fragment analysis by capillary electrophoresis

The analysis of the fusion transcript amplicons was performed by capillary electrophoresis. The assay was designed as per an earlier study, with some modifications.[14] Multiplex PCR comprising primers for SS18-SSX general primers and genotype specific viz; SS18-SSX 1, SS18-SSX2 and SS18-SSX4 was performed using fluorescence labeled SS18, the amplified PCR products were analyzed by capillary electrophoresis on ABI 3500 Genetic Analyzer (Thermoscientific, Foster city, CA, USA). Reaction comprised of 2X PCR master mixes, 150 nM of SS18 exon 10 labeled primers, unlabeled SSX 1/SSX 2/SSX 4 primers of 200 nM each and 1μL of template cDNA. Thermal cycling conditions were 95°C for 10 minutes, 40 cycles of 95°C for 20 seconds, 58°C for 30 seconds, 72°C for 1 minute. Following PCR amplification, 1 μl of amplified product was mixed with the 9 μl premix of HIDI formamide and internal size standard LIZ 500 (ThermoScientific, Foster city, USA) which was denatured at 95°C for 3 minutes, followed by immediate cooling on ice for 5 minutes. Samples were then run on an ABI 3500. Appropriate positive and negative controls were included in each run. Fragment analysis was performed using Gene Mapper ID Software v3.1. The assay was run in duplicates to confirm the results. The results were considered valid only when peaks for both general translocations as well genotype specific were present. One single peak depicting either general translocation or genotype specific was considered as uninterpretable and the assay was repeated.

Statistical analysis

The data was analyzed using SPSS 16.0 statistical software (IBM, USA). Concordance was calculated among results obtained from the 3 platforms, used for testing t (X; 18)(p11;q11) translocation. Clinicopathological correlation was performed using Fisher exact test. P value < 0.05 was considered as significant.


   Results Top


Histopathologic profile of 45 cases, including different groups along with t (X; 18) translocation results are enlisted in [Table 3].
Table 3: Histopathologic profile of 45 cases, including different groups, along with t(X;18) translocation results

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Out of 45 cases included in the study, 16 constituted as Group 1, 13 as Group 2, and remaining 16 cases as Group 3. The latter group included 4 cases of malignant peripheral nerve sheath tumors (MPNSTs), 2 of spindle cell sarcoma not otherwise specified (NOS); 3 of round cell sarcoma (NOS); 2 of embryonal rhabdomyosarcoma, and a single case, each, of Ewing sarcoma, small cell osteosarcoma, low-grade fibromyxoid sarcoma, solitary fibrous tumor; and a desmoplastic round cell tumor (DSRCT), respectively.

Final histopathologic diagnosis, after molecular testing, in 13 cases belonging to Group II was synovial sarcoma (n = 7), Ewing sarcoma (n = 1), adult fibrosarcoma (n = 1) round cell sarcoma, NOS (n = 3) and spindle cell sarcoma, NOS (n = -1).

Finally, there were 23 cases of SS, including 5 monophasic type, 10 poorly differentiated type, 3 biphasic SS and 5 cases of SS, not subtyped [Table 3]. 16 cases of SS were of group 1 and 7 of group 2.

IHC results of these cases are depicted in [Table 4]. Twelve out of 13 cases (92.3%) of SS displayed the characteristic “weak to absent”/reduced staining pattern for INI1 [Figure 1].
Table 4: Immunohistochemical results in 23 cases of synovial sarcomas

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Figure 1: Case 9. Poorly differentiated synovial sarcoma (SS). (a). Nests of tumor cells separated with 'slit-like' vasculature. Hematoxylin and Eosin (H and E, ×200). (b). Oval to short spindle-shaped cells with mitotic figures and interspersed 'slit-like' vasculature. H and E stain, ×400 (c). By immunohistochemistry (IHC), tumor cells show focal pan cytokeratin (AE1/AE3) positivity. (Diaminobenzidine, ×400). (d). By IHC, tumor cells show characteristic 'weak to absent' staining pattern for INI1. (DAB ×400)

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Detection of t(X;18)(p11;q11) translocation

Among 23 cases of SS, SS18-SSX translocation was detected in 22, 18 and 15 cases, using fragment analysis, FISH technique and RT-PCR, respectively. Furthermore, using fragment analysis, SS18-SSX1 genotype was observed in 14 cases and SS18-SSX2 in 8 cases, while using RT-PCR assay, SS18-SSX1 genotype was observed in 10 cases and SS18-SSX2 in 5 cases respectively [Figure 2], [Figure 3], [Figure 4].
Figure 2: Case 9. Positive results of t(X;18)(p11; q11) translocation with all the three techniques. (a) Fragment analysis. Positive for SS18-SSX general (97bp); SS18-SSX1 genotype positive (143bp), Gene scan LIZ 500 as size standard. (b) FISH test displaying SS18 rearrangement. Inset: 'split' red green signals, indicating SS18 rearrangement (c). Conventional RT-PCR for SS18-SSX1; RC Reagent control, S Sample, PC Positive control. The sample is positive for SS18-SSX1 (330bp). RT-PCR for SS18-SSX2; RC, S, PC. The sample is negative for SS18-SSX2 (330bp). Ladder: 100bp Molecular weight (wt.) marker

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Figure 3: Case 5. Poorly differentiated SS showing t(X;18) (p11; q11) translocation with all the three methods. (a) Fragment analysis. Positive for SS18-SSX general (97bp); SS18-SSX2 genotype positive (125bp), Gene scan LIZ 500 as size standard. (b) Interphase FISH displaying SS18 rearrangement. Inset: a single pair of fused and 'split' red green signals, indicative of SS18 rearrangement. (c). Conventional RT-PCR for SS18-SSX2; RC, S, PC. Positive for SS18-SSX2 (330bp). RT-PCR for SS18-SSX1; RC, S, PC. Negative for SS18-SSX1 (330bp). Ladder: 100bp Molecular weight (wt.) marker

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Figure 4: Case 13 showing differing t (X; 18)(p11; q11) translocation results across various techniques. (a) Fragment analysis. Positive for SS18-SSX general (97bp); SS18-SSX1 genotype positive (143bp), Gene scan LIZ 500 as size standard. (b) Interphase FISH test: Positive SS18 rearrangement. (c) Conventional RT-PCR for SS18-SSX1; RC, S, PC. Negative for SS18-SSX1 (330bp). RT-PCR for SS18-SSX2; RC, S, PCl. Negative for SS18-SSX2 (330bp). Ladder: 100bp Molecular weight (wt.) marker

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2 tumors revealed unusual complex pattern of signals, by FISH technique. Tumor sample of case no. 4 revealed significant number of red green 'split' signals, along with certain tumor cell nuclei displaying single fused signals. This tumor was positive for SS18-SSX1 transcript, by both, RT-PCR and fragment analysis. Another tumor (case no 27), diagnosed as an adult fibrosarcoma, although was negative for the t(X;18) (p11;q11) translocation, by fragment analysis and RT-PCR, revealed a complex pattern in form of unusual loss of 5′ end (loss of green signals) of SS18 gene with retained 3′ end of the SS18 gene (red signals) in nearly 90% cells, by FISH technique [Figure 5]).
Figure 5: Case 27 displaying complex signals on FISH. (a): Fragment analysis. Negative for SS18-SSX general (97bp); and for both the genotypes. (b) Complex pattern observed on Interphase FISH test with most tumor cells (approximately 90%) showing unusual loss of 5′ end (loss of green signals) of SS18 gene with retained 3′ end of the SS18 gene (red signals) Inset: two nuclei with single fused and red signals only. (c): Conventional RT-PCR for SS18-SSX1; RC, S, PC. Negative for SS18-SSX1 (330bp). RT-PCR for SS18-SSX2; RC, PC, S. Negative for SS18-SSX2 (330bp). Ladder: 100bp Molecular weight (wt.) marker

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Considering FISH technique as the present gold standard in our laboratory, for detecting SS18 gene rearrangement, the sensitivity of fragment analysis was 100% and for RT-PCR it was 81.8%.

Correlation between IHC markers and clinicopathologic features with molecular results

On correlation of IHC findings with molecular results among Group 1 and 2 it was observed that a unique “weak to absent”/reduced staining pattern of INI1 showed statistically significant association with the positive t (X; 18) (p11;q11) translocation results (P = 0.0001). All 11 cases showing this pattern demonstrated t(X;18)(p11;q11) translocations. 6 cases with its diffusely retained pattern were negative for the translocation (100% specificity).


   Discussion Top


It is an ideal and a desirable situation for a clinical laboratory to have an assay which can be utilized on fresh, as well as FFPE tissue samples; has a rapid turnaround time, and provides results which are accurately interpretable. The present study was mainly conducted to evaluate the comparison between three different testing platforms, to detect the t (X; 18)(p11;q11) specific translocation of SS, in order to identify the most optimal technique, especially in dilemmatic cases.

6 cases in group 2, including those with an equivocal diagnosis of SS, on histopathologic examination, including IHC results, were objectively recognized as SSs, utilizing various molecular techniques, in the present study. This reinforces the value of molecular techniques in diagnosing sarcomas, despite speciality reporting, at least in certain cases.[16]

There are recognizable strengths and limitations of the two techniques, namely RT-PCR and FISH for molecular diagnosis of sarcomas, including SS. The major limitation with RT-PCR is in obtaining intact amplifiable transcripts of a particular size nearly 300bp, whereas fragment analysis requires smaller product size with the provision for multiplex PCR. Various factors attribute towards obtaining intact transcripts for molecular assays, for example quality of the tissue, methods of fixation used, quality of the paraffin material used, as well as age of the FFPE block, that affects the yield of the RNA. All these factors have been very well documented in the literature, which in turn influence interpretation of results.[17]

In the present study, FISH technique could enable detection of SS18 gene rearrangement in 78.2% cases of SS, while RT-PCR was useful in detection of SS18-SSX transcripts in 65.2% cases, in contrast to fragment analysis.that was useful in detection of the same in 95.6% cases. A lower sensitivity of detection of the fusion transcript by RT-PCR is known, considering this is dependent on the quality of the amplifiable transcripts obtained from the fixed tissues.[18] Variations in tissue processing, as well as, usage of different fixative buffers significantly affects the quality and integrity of the tissues leading to poor quality of RNA and cDNA.[16] Although amplifiable cDNA is prepared form the degraded RNA, detecting the fusion transcripts of 330bp length becomes difficult which can lead to false negative result. On the other hand, FISH technique is relatively more sensitive and easily interpreted by the pathologists, as they are more familiar with analyzing tumor cells using a microscope.[8] Ideally, more than one technique should be employed for confirming the specific translocation results.[19]

SS18-SSX1 and SS18-SSX 2 are mutually exclusive variants of SS.[20] We observed SS18-SSX1 ( 14 cases, 63.6%) fusion transcript more frequent than SS18-SSX2 ( 8 cases, 36.3%), as reported in an earlier, including a study from India, but in contrast to a study from China, wherein the investigators observed SS18-SSX2 as the more frequent fusion.[5],[20],[21]

Fragment analysis using multiplex PCR was more sensitive in identification of the specific chimeric transcripts, than RT-PCR, in the present study. This technique was useful in confirming all 18 cases of SS, displaying SS18 rearrangement, by FISH. In addition, it uncovered positive translocation results in 4 cases of group 1, wherein FISH technique was uninterpretable. This assay is faster than conventional RT-PCR and FISH, economical and can simultaneously provide information regarding the translocations with the genotypes.[22],[23] Moreover, in case of real time PCR, several probes and paired primers are needed for translocations with more than one common breakpoint or for assays using small targets, such as fragmented templates extracted from FFPE tissues. Furthermore, the number of multiplexed probes is limited by the fluorochrome detection channels of the real-time machine. However, fragment analysis by capillary electrophoresis using two or three upstream primers for each potential breakpoint hot spot, to be paired simultaneously with a common, downstream primer can be designed in such a way that the presence of two peaks with the expected size difference (when analyzed by capillary electrophoresis) indicates the specific amplification of PCR products from the defined breakpoint locus. Considering only a single fluorochrome-labeled oligonucleotide is needed, the assays can be easily multiplexed to detect multiple breakpoints in a single PCR reaction.[24]

In this study, the assay was designed to detect all the three transcripts, SS18-SSX 1, SS18-SSX 2 and SS18-SSX 4. The primers were designed to get shorter fragments of size between 96 bp to 150 bp, to ensure that the relevant genotypes were not missed.

It is a multiplex assay, simultaneously detecting fusion transcripts along with genotypes thus, minimizing the chances of any false positive results. The reliability of this design for correctly genotyping tumor derived transcripts was confirmed by FISH and RT-PCR. The single tube format of this assay simplifies interpretation.

Apart from its highest sensitivity, followed by FISH, over RT-PCR, fragment analysis was found to be relatively economical, as compared to FISH and was less laborious, as compared to conventional RT-PCR in the present study. Current cost per test for RT-PCR and fragment analysis at our Institution is Indian rupees (INR) is Rs 7000, each, respectively, whereas for FISH is INR 10, 000. Immunohistochemical testing, inclusive of pathology consultation is offered for INR 3965, presently.

In 2 cases, wherein a rather unusual, complex signal pattern was observed by FISH, result with fragment analysis was in one, and a negative result in the other case. Previously, complex signals on FISH testing have been reported, apart from significant cells showing 'split' signals, in certain cases. Such cases have been interpreted as positive for translocation.[18]

A single case (case 23), despite negative results with RT-PCR, was retained with a diagnosis of SS, on histopathologic examination. This case did not yield interpretable results with fragment analysis and FISH. This underscores value of integrating molecular results with morphology. Lack of positive t(X;18) translocation result in any of the sarcomas other than SSs, with all 3 molecular techniques, indicates their 100% specificity. Among various IHC markers, this study further reinforced utility of characteristic “weak to absent”/reduced staining pattern of INI1 in the diagnosis of SS, as previously documented.[6],[7],[25],[26] This is especially useful in triaging cases for molecular diagnosis, in laboratories with limitations for molecular testing.

To conclude, this study reveals utility of fragment analysis as another robust technique, besides FISH, for detection of specific translocations, including genotyping in the diagnosis of sarcomas, characterized by a specific translocation, such as a SS. Among various immunohistochemical antibody results, unique 'weak to absent' immunostaining pattern of INI1 is fairly diagnostic of a SS, irrespective of its subtype. It also reinforces incorporation of molecular techniques (preferably more than one), in the diagnosis of sarcomas, including SS, especially in cases with equivocal histopathologic features and IHC results, despite speciality reporting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Bharat Rekhi
Department of Surgical Pathology, Room Number 818, 8th Floor, Annex Building, Tata Memorial Hospital, Dr E.B. Road, Parel, Mumbai - 400 012, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_851_18

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