A rare case of recurrent ovarian cancer with NTRK1-TPM3 gene rearrangement

NTRK gene fusion is rare in gynecologic cancer. Entrectinib is a novel targeted drug which is a potent inhibitor of TRK A, B and C. Here, we present a case of recurrent ovarian cancer with NTRK1-TPM3 rearrangement, which was detected by next-generation sequencing (NGS) and treated with entrectinib. Case with

immunohistochemistry with a pan-Trk monoclonal antibody was performed to determine the expression of NTRK. However, immunohistochemistry was negative for NTRK.

Conclusion:
We presented a rare case of recurrent ovarian cancer with NTRK1-TPM3 gene fusion, in which entrectinib was not effective. While NTRK gene fusion was detected by DNA-based NGS, immunohistochemistry was negative for NTRK. Immunohistchemitory should be performed for con rmation of NTRK protein expression before entrectinib administration.
Background NTRK gene fusions are consistently detected in rare types of cancers (secretory breast carcinoma, mammary analogue secretary carcinoma, congenital infantile brosarcoma, and congenital mesoblastic nephroma) and they are novel therapeutic targets across multiple tumor types. [1][2][3] On the other hand, these gene fusions are rare in common adult cancers. [1,2] In gynecologic oncology, NTRK gene fusion is also rare, although there are several reports of uterine sarcoma with this fusion gene. [4][5][6][7] A previous cohort study showed that NTRK1-TPM3 is most frequent in NTRK1 fusions across multiple histologies. [2] Immunohistochemistry (IHC) staining, uorescence in situ hybridization, reverse transcriptase polymerase chain reaction, DNA-based next-generation sequencing (NGS) and RNA-based NGS are used to identify patients with NTRK gene fusion cancer. Each method to detect NTRK gene fusion has its own characteristics. [3] Entrectinib is a potent inhibitor of TRKA, TRKB, TRKC, ROS1, and ALK, and is speci cally designed to have systemic activity. In gynecologic oncology, treatment using entrectinib is rare because of the low frequency of NTRK fusions. [1] Here, we report a case of recurrent ovarian cancer (OC) with NTRK1-TPM3 gene fusion and that was treated with entrectinib.

Case Presentation
In September 2013, a 56-year-old woman was referred to our hospital with bilateral ovarian tumor, multiple disseminations in the peritoneum, bilateral pleural effusion, and multiple swellings of the pelvic, and paraaortic lymph nodes. Her serum level of cancer antigen 125 (CA125) was elevated to 1740 U/ml. She was diagnosed as having stage OC according to the International Federation of Gynecology and Obstetrics (FIGO) 1988 because pleural effusion cytology was positive. Paclitaxel (175 mg/m 2 ) and carboplatin (area under the curve 6) were started as neoadjuvant chemotherapy. After four courses of chemotherapy, computed tomography (CT) revealed a reduction in tumor size. Interval debulking surgery including abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and pelvic and paraaortic lymphadenectomy, was performed. Histopathological diagnosis was high-grade serous carcinoma. Following this surgery, another three courses of the same regimen were administered, and the patient achieved clinical complete response.
Ten months after the last therapy, OC recurred. Because of the disease persistence, she was treated with nine regimens of chemotherapy combined with two surgeries for recurrent tumors; the rst was partial hepatectomy due to dissemination to the liver, and the second was enterectomy due to recurrence in the mesentery. Microsatellite stability was detected in specimens from the second recurrence resection.
Six years after initiation of therapy, FoundationOne® CDx (Foundation Medicine, Cambridge, MA), which is DNA based NGS and covers 324 genes, was performed based on the patient's archival tumor tissue from the second recurrence resection. This revealed a missense variant of TP53 (c.731G > A) and a rearrangement between exon 11 of NTRK1 (NM_002529) and somewhere around exon 2-3 of TPM3 (pos1="chr1:156844554-156844771", pos2="chr1:154155588-154155822"). Oral entrectinib (600mg/day) was started after discussing with experts. Six weeks after initiation of entrectinib, the patient's serum CA125 level elevated to 4360 U/ml, although it was 1712 U/ml before initiation of entrectinib and CT revealed progression of liver metastasis. (Fig. 1) Adverse events during entrectinib administration comprised grade 2 dysgeusia. One month after discontinuation of entrectinib, the patient died. (Fig. 2) After the patient's death, IHC staining with a pan-Trk monoclonal antibody (mAB) clone EPR17341 (Abcam, Cambridge, MA) was performed to assess for TRKA, TRKB, and TRKC expression as previously described.
[8] This mAB clone is most commonly used and has been investigated thoroughly. In addition, this mAB clone reacts with a conserved proprietary peptide from the C-terminus of TRKA, TRKB and TRKC, and is therefore reactive to any oncogenic NTRK fusion. [3] IHC was negative for all specimens from the primary site resection, as well as the rst and second recurrent site resections. (Fig. 3)

Discussion
Here we presented a case of recurrent OC with NTRK1-TPM3 rearrangement. Additionally, the present case demonstrates the discrepancy between gene rearrangement detected by NGS and protein expression. This discrepancy may be a biomarker for predicting the ineffectiveness of entrectinib for cancers with NTRK rearrangement detected by NGS.
In the current case, NGS revealed NTRK1-TPM3 rearrangement and a missense variant of TP53. There are few approved therapies for TP53, although almost all cases of ovarian high-grade serous carcinoma (95%) have somatic TP53 variants. [9] On the other hand, NTRK fusions are oncogenic drivers and novel targets. Doebele et al. reported the safety and activity of entrectinib in adult patients with advanced or metastatic NTRK fusion-positive cancer across three clinical trials (ALKA-372-001, STARTRK-1 and STARTRK-2). In these trials, only one ovarian cancer patient was included. They showed that the objective response rate, which included complete response and partial response, was 57% (95% CI 43.2-70.8). The median duration of response was 10 months (95% CI 7.1 to not estimable) and the percentage of progressive disease (PD) was only 7%. However, the characteristics of cases with PD remained unclear in their report. [1] In the present case, entrectinib was administered because NGS revealed NTRK1-TPM3 rearrangement and entrectinib was recommended after a discussion among experts. However, this novel target drug was ineffective. NTRK protein was not expressed, although IHC testing with a pan-Trk mAB clone [EPR17341] was performed. A previous study reported that gene fusions involving NTRK1, 2, and 3 and their partner genes result in a constitutive activation or overexpression of TRK receptors, potentially leading to oncogenesis. [10] Additionally, other reports have shown that pan-Trk IHC yielded a sensitivity of 75-95.2%, and a speci city of 92-100% and that the sensitivity of pan-Trk IHC for NTRK1 was 96.2%. [3,8,11,12] Pan-Trk IHC is a reliable screening method for the detection of NTRK gene fusions based on this date. Moreover, pan-Trk IHC can rapidly assess malignancies which may harbor possible NTRK fusions in order to determine eligibility of patients for targeted therapy with TRK inhibitors.
[8] However, it should be considered that there are NTRK rearrangements which are found to be negative by IHC, and can only be detected by NGS, such as in the present case.
Drilon et al. reported the e cacy of larotrectinib, which is a selective inhibitor of TRKA, TRKB and TRKC. In their study, six of an initial 55 patients showed primary resistance to larotrectinib. Three patients had tumor material available for central analysis, and in all three cases, pan-Trk IHC did not reveal the presence of TRK protein expression. This indicated that the rearrangements detected by NGS were false positives or that the identi ed fusion genes were not expressed at the protein level. [13] It is considered that entrectinib has the same characteristics as larotrectinib with regard to discrepancy between gene fusion and protein expression, as observed in the current case, and that this nding may be a key to predict the ineffectiveness of entrectinib for cancers with NTRK rearrangement detected by NGS.
To the best our knowledge, this is the rst case report of OC with NTRK rearrangement. It is known that a small percentage of common adult cancers carry fusions of NTRK genes. [2] A large cohort study revealed that the frequency of NTRK gene fusions was 0.25% of general cancers. [2,12] Therefore, physicians have few chances to experience this molecular characteristic. However, physicians should be aware of the pitfall that NTRK protein may not expression even if NGS reveals NTRK rearrangement.

Conclusion
In conclusion, we here presented a rare case of recurrent OC with NTRK1-TPM3 fusion. Physicians should be aware of the discrepancy of DNA rearrangement and protein expression, and IHC should be performed for con rmation of NTRK protein expression before entrectinib administration.

Consent for publication
Written informed consent was obtained from the patient for publication of this case report and the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Availability of data and materials
The data used or analyzed are all included in this published article

Competing interests
The authors declare that they have no competing interests.

Funding
We have no commercial or nancial incentives associated with the publication of this article.