The suitability of NONO-TFE3 dual-fusion FISH assay as a diagnostic tool for NONO-TFE3 renal cell carcinoma

NONO-TFE3 RCC is a subtype of Xp11.2 translocation renal cell carcinoma (RCC). So far, only a small amount of NONO-TFE3 RCC have been reported owing to lack of effective diagnosis methods. Utilizing the novel dual-fusion fluorescence in situ hybridization (FISH) probe reported here, 5 cases of NONO-TFE3 RCC were identified and were ultimately confirmed by RT-PCR. Histopathology, all 5 cases were consisted by sheets of epithelial cells and papillary architecture. The cytoplasm was abundantly clear, and nucleoli was not prominent. Besides, the nuclear palisading, subnuclear vacuoles and psammoma bodies were identified. The most distinctive features were strong positive TFE3 staining but equivocal split signals of the TFE3 probe, which might lead to the misdiagnosis of Xp11.2 translocation RCC. The median age and median tumor size of the five patients were 41.2 years and 3.6 cm, respectively. A median following follow-up of 27 months showed moderate disease progression and prognosis in NONO-TFE3 RCC patients. In conclusion, the present study demonstrates the effectiveness and reliability of the NONO-TFE3 dual-fusion FISH probe for diagnosing NONO-TFE3 RCC. Suspected cases of Xp11.2 translocation RCC showing biphasic pattern, strong positive TFE3 staining, and equivocal split signals in the TFE3 FISH assay indicated a possibility of NONO-TFE3 RCC.

www.nature.com/scientificreports/ in the TFE3 break-apart FISH assay, respectively 12 . However, TFE3 immunoreactivity only reflects the expression levels of TFE3 protein, and the FISH split signal merely indicates rearrangements in TFE3. Identification of fusion patterns still relies on fusion FISH assays 5,13 , reverse transcription-polymerase chain reactions (RT-PCR), cytogenetic karyotypic analysis 10 , or sequencing 14 . However, the fusion FISH assay represents the most inexpensive and accessible method, due to economic difficulties and complicated sample requirements of the other methods. NONO gene, a member of the Drosophila behavior/human splicing (DBHS) protein family, is located adjacent to TFE3. However, the adjacent location of NONO and TFE3 genes result in an equivocal split signal distance of TFE3, which leads to the misdiagnosis of NONO-TFE3 RCC as non-Xp11.2 translocation RCC 9,15 . Previously, Argani and Xia et al. 8,9 introduced their single-fusion NONO-TFE3 FISH assays. In the present study, we designed a novel NONO-TFE3 dual-fusion FISH assay and identified five cases of NONO-TFE3 RCC based on our FISH probe. Therefore, the present study was designed to determine the effectiveness of the novel assay and highlight the diagnostic potential of dual-fusion and single-fusion FISH assays. Fig. 1a, NONO gene was labeled with red fluorescence and TFE3 gene was green fluorescence by bacterial artificial chromosomes (BACs). In general, signals were considered to be fused when yellow and abutting (equal to or less than the one signals' diameters) green-red signals were observed. Given the small gap between gene specific probe (GSP) TFE3 and GSP NONO (nearly 21 Mb), controls might also show adjacent or partially overlapping signals. Therefore, patient gender and signal sizes needed to be taken into consideration for the evaluation of FISH results (Fig. 1b). For males, when the 2 fusion signals (2F) appeared simultaneously, the reciprocal translocation between the NONO and TFE3 genes could be confirmed (Fig. 2). For female cases, a pair of split signals (1G1R) were also required. In contrast, the presence of one fusion signal in males, and two fusion signals unaccompanied with two split signals in females, both represented adjacent TFE3 and NONO signals. By this method, the observed fusion and split signals could be accurately identified as NONO-TFE3 fusion genes. In addition, signal size was another factor used to identify the signal patterns. Fusion signals were consisted of two split signals, with sizes markedly smaller than integrated green or red signals. Therefore, there were two pairs of small split signals and one pair of large unsplit signal in females with the NONO-TFE3 gene fusion.

FISH analysis. As displayed in
In total, there were 5 cases were showed NONO gene and TFE3 gene fusion. All five cases showed equivocal split signals with distances of nearly one signal diameters, which might be analyzed as a false-negative diagnosis by an unexperienced pathologist. For males with NONO-TFE3 translocation (Fig. 3a,b), two fusion signals (2F) were observed in 14.1-35% of the tumor cells, and a pair of split signals as well as a fusion signal (1G1R) were observed in 13-25% cells. For NONO-TFE3 dual-fusion probe positive females (Fig. 3c,d), two fusion signals in addition to a pair of split signals (1G1R2F) were observed in 8-21% cells, and two pairs of split signals (2G2R) were observed in 19-27% cells. For negative cases (Fig. 4), such as ccRCC and pRCC, 2F and 1G1R signal patterns were observed in 1% and 22-88.35% of male tumor cells, respectively. Moreover, 1G1R2F and 2G2R signal patterns were observed in 3% and 4.71-67.3% of female tumor cells. In total, the rate of false positive detection of this fusion FISH probe was 1.8%, and the threshold value was set to 2%.
Adequate RNA was extracted from FFPE tissues from all 5 patients and subjected to RT-PCR analysis. ~ 200 bp bands extracted from two cases and a ~ 300 bp bands were reverse transcribed by the primer pairs NONO exon 9 (F) and TFE3 exon 7 (R). On the other hand, a ~ 300 bp bands and a ~ 500bps band from the remaining patient were reverse transcribed by the primer pairs NONO exon 7 (F) and TFE3 exon 8 (R). All PCR products were successfully extracted and further analyzed (Fig. 5). Finally, three fusion patterns were identified, including 3 cases (cases 1, 3 and 4) of a fusion point between exon 9 of NONO and exon 6 of TFE3, one case (cases 2) of a fusion point between exon 7 of NONO and exon 6 of TFE3, and one case (case 5) of a fusion point between exon 9 of NONO and exon 5 of TFE3 (Fig. 6).  Table 1. Of the 5 cases of NONO-TFE3 RCC, 4 cases were initially diagnosed as Xp11.2 translocation RCC and the other one was negative to the TFE3 break-apart probe. The mean age of these patients was 41.2 years and the mean tumor size was 3.6 cm. Four patients were treated with nephron-sparing surgery (NSS), with radical nephrectomy (RN) conducted on the remaining patient diagnosed with a tumor near the pelvis. According to the 2010 American Joint Committee on Cancer TNM staging system, 4 of these patients were classified as stage I and one as stage III. Follow-up studies were conducted on all five patients from 5 to 51 months (mean, 28.2 months; median, 27 months). Four patients were found to be alive with no evidence of disease, but a 42-year-old man died 28 months after the surgery.

Patients. Including 8 cases of
Morphology and IHC. All 5 cases displayed a distinctive biphasic pattern in papillary architecture accompanied with sheets of epithelial cells (Fig. 7). The predominantly papillary architecture was lined by columnar cells with clear cytoplasm (Fig. 7b). The nuclear palisading (nuclear palisading means a special pathological phenomenon that the nuclei are arranged as palisade) and subnuclear vacuoles were identified, and nucleoli was not prominent (WHO/ISUP grade 1 or 2; Fig. 7e). Psammoma bodies or calcification were observed in 4 cases (Fig. 7c). Three cases were associated with a predominantly papillary architecture, and the other two showed equal prevalence of papillary architecture and sheets of epithelial cells. Immunohistochemical assays showed that all 5 cases were immunoreactive for TFE3 (Fig. 7f) www.nature.com/scientificreports/ CA-IX (7 g), and CK7 (Fig. 7h). One out of the five cases had CD117-positive cells, with the other cases being completely negative (Supplementary Table 1).

Discussion
Xp11.2 translocation RCC is a rare tumor which mainly occurs in children and young adults. Xp11.2 translocation RCC has been reported to have an incidence of 30, 15, and 1.5% in pediatrics, young adults (younger than 45 years), and adults, respectively 16 . The first cases of Xp11.2 translocation RCC involved in t(X; 1) (p11; q21) were reported by de Jong et al. in 1987 17 . However, this rare but aggressive renal tumor did not attract much www.nature.com/scientificreports/ attention until 2004, when it was classified as a distinctive type of RCC by WHO 18 . In the following decades, distinctive features of Xp11.2 translocation RCC have been increasingly characterized. Nowadays, ASPSCR1, PRCC , and SFPQ are recognized as relatively common gene fusion partners associated with Xp11.2 translocation renal cell carcinoma 8,19 . However, the involvement of NONO, CLTC, LUC7L3, and RBM10 have only been discussed in case reports 8,[20][21][22] . FISH is currently the most convenient and effective method for identifying gene alteration by using custom BAC probes on FFPE tissue sections. Utilizing ASPSCR1-TFE3 and PRCC-TFE3 dual-fusion FISH assays   (Table 1).
In the present study, 5 cases of Xp11.2 translocation RCC were found to be positive to the NONO-TFE3 dualfusion FISH assay and the diagnosis was eventually confirmed by PCR. Gene sequencing of the PCR products revealed three NONO-TFE3 fusion points. These are exons 1-7 of NONO and exons 6-10 of TFE3, exons 1-9 of NONO and exons 5-10 of TFE3, and exons 1-9 of NONO and exons 6-10 of TFE3. The former two fusion  www.nature.com/scientificreports/ points have been reported earlier 9 . However, the present study is the first report of the last fusion point. Of the 5 NONO-TFE3 RCC cases, one (20%) was previously identified as ccRCC owing to equivocal split signals of the TFE3 gene. A possible remediation is that the possibility of Xp11.2 translocation RCC should not be eliminated if a case is moderate or strong positive to TFE3 stanning but negative to TFE3 break-apart assay, and other complementary approaches should be adopted to further clarify and confirm the diagnosis. In 2017, Xia et al. 9 introduced a single-fusion FISH assay for the identification of NONO-TFE3 translocation. Given the adjacent location of NONO and TFE3 genes, only two BAC clones, RP11-934E1 (labeled with 5-fluorescein-dUTP and located centromeric to the NONO gene locus) and RP11-416B14 (labeled with 5-ROX-dUTP and located telomeric to the TFE3 gene locus), were used in their study. Fusion of TFE3 and NONO genes led to overlap of the separated signals. The experimental design of the single-fusion assay ensures rapid analysis of signal patterns (fusion or split). However, other issues could not be suitably clarified by this assay. First, signals consisting of single BAC clones were weak and less obvious, which made data acquisition difficult and increased the possibility of a false-negative diagnosis. Second, it was hard to distinguish target signals from noises, especially for samples with dirty backgrounds, which also increased the difficulty in diagnosis. Third, although single-fusion events represent gene translocation, these may not always balance translocation, which was an important character of Xp11.2 translocation RCC. For example, the fusion of the ASPSCR1 and TFE3 genes could occur not only in ASPSCR1-TFE3 RCC, but also in alveolar soft part sarcoma (ASPS) 23,24 , a stromal tumor involving an unbalanced der (17) t(X; 17) (p11; q25) translocation. As a control, both TFE3 and NONO gene assays in our study consisted of more than 5 BAC clones, which improved signals due to increased signal to noise ratio (SNR). On the other hand, we modified the inter-spot spacing standard as one signal diameter, which was effective in distinguishing fusion signals from split signals. In addition, the dual-fusion assay includes both NONO-TFE3 fusion and TFE3-NONO fusion, and therefore, represents an intuitive and direct response to genetic changes in NONO-TFE3 RCC. Easy and accurate diagnosis by the dual-fusion assay required information on patient gender, the number of split and fusion signals, and signal size.
By utilizing the NONO-TFE3 dual-fusion FISH probe, 5 cases of NONO-TFE3 RCC were diagnosed, accounting for 17.2% of the total Xp11.2 translocation RCC population. In summary, the most distinctive morphological www.nature.com/scientificreports/ features of the 5 NONO-TFE3 RCC cases were sheets of epithelial cells and papillary architecture, which were consisted with previous report 8,9 . The cytoplasm was abundantly clear (or flocculent in eosinophilic cells); and the nuclei were rounded and uniform in shape and were oriented toward the luminal surface. Immunohistochemical assays on NONO-TFE3 RCC showed a strong immunoreactivity (3+++) to TFE3 antibody. According to Green et al. 6,25 , biphasic morphological pattern and strong TFE3 immunoreactivity were important features of NONO-TFE3 RCC. They proposed that NONO-TFE3 RCC should be taken into consideration for patients with biphasic morphological pattern and strong TFE3 immunoreactivity, combined with equivocal split signals. In our study, all of these features were verified in all 5 cases of NONO-TFE3 RCC. Taken together, IHC and break-apart FISH probe could serve as precursor methods, and suspected cases could be further evaluated by the NONO-TFE3 dual-fusion FISH for final confirmation. In general, Xp11.2 translocation RCC tends to occur in patients aged between 20 to 50 years, and Xp11.2 translocation RCC is believed to be 2 to 3 times more prevalent in females compared to males 2,8,9,19 . However, our data and previous reports seem to support male predominance in NONO-TFE3 RCC. Compared with ASPSCR1-TFE3 RCC and PRCC-TFE3 RCC (Supplementary Table 2), patients of NONO-TFE3 RCC were younger and had smaller tumor sizes. In terms of prognosis, NONO-TFE3 RCC were reported as one of the most moderate subtypes among all Xp11.2 translocation RCC patients 2,9 . Even our data showed that NONO-TFE3 RCC patients had a longer survival than ASPSCR1-TFE3 RCC and PRCC-TFE3 RCC in a short time follow-up study. However, there was a case at the pT1aM0N0 stage, where the patient was diagnosed with peritoneal metastasis at 10 months and dead at 28 months postoperation. The other 4 patients were alive with no evidence of disease progression after their initial resection with a median follow-up of 20.5 months. Given the low number of cases, it's hard to believe that any of these data are statistically significant. The physiological characteristics of NONO-TFE3 RCC need further investigation with a larger population and long-term follow-up.
In summary, we reported a newly designed NONO-TFE3 dual-fusion FISH assay and confirmed the accuracy of this novel probe for detecting NONO-TFE3 RCC. Five cases of NONO-TFE3 RCC were identified utilizing this probe and their clinicopathological characteristics were explored. The equivocal split signals make the diagnosis of NONO-TFE3 RCC difficult. However, this feature could be regarded as a diagnostic marker to provide additional insights to pathologists. In a short-term study, NONO-TFE3 RCC showed a moderate prognosis compared to that in ASPSCR1-TFE3 RCC and PRCC-TFE3 RCC. These findings enhance our understanding of NONO-TFE3 RCC. www.nature.com/scientificreports/ www.nature.com/scientificreports/
FISH experimental procedure. FISH were performed as previously described 9,11 . 3 mm thick sections from formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks were first heated overnight in an incubator maintained at 65 °C. Then, sections were deparaffinized by treatment with xylene for 30 min, followed by washing with absolute ethanol for 10 min. Sections were rehydrated in 100, 90, and 70% ethanol and double distilled water, in turn, for 3 min. This was followed by boiling these sections in sterilized purified water for 20 min and subsequent air drying. Sections were then digested with 10 μl pepsin (4 mg/mL, 0.02 M HCl; Sigma-Aldrich, Beijing, China) at 37.5 °C for 5 to 10 min and washed with 2X saline-sodium citrate (SSC) for 5 min. Subsequently, these sections were dehydrated in 70, 90, and 100% ethanol, in turn, for 3 min and air dried. Hybridization was performed with the newly designed FISH probe, denatured at 85 °C for 5 min, and incubated overnight (10-18 h) at 37 °C under lucifugal conditions. The sections were then immersed in 2X SSC for 10 min; and in 0.1% NP-40 and 2X SSC for 5 min at 37 °C. Then, the sections were immersed in 70% ethanol for 3 min and air dried. The nuclei were counterstained with 10 μl of 4′,6-diamidino-2-phenylindole (DAPI). After hybridization, all slides were stored in the dark.
FISH evaluation. Evaluation of FISH results was conducted with an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) fitted with 3 filters (DAPI/FITC/Texas Red) and the FISH analysis software (Imstar, Paris, France). The FISH assay is effective only when clear FISH signals are observed in > 100 nonoverlapping nuclei.