Analysis of mutations of PARP1, RNF213, PAX8, KMT2C, MTRR in malignant mesothelioma of testicular tunica vaginalis testis

Introduction

We decided to present the case of malignant mesothelioma of tunica vaginalis testis (MMTVT) due to its rarity. Malignant mesothelioma of tunica vaginalis testis constitute less than 0,5% of the total of mesotheliomas of the human body [1, 2, 3]. Our case belongs to the category of mesotheliomas arising in coelomic cavities and such tumors of that counterpart of human body are truly rare with variety of histological patterns in the non-uniform group of neoplasms of that type in this anatomical setting [1, 2, 3]. Persistent hydrocele, herniorrhaphy, trauma, relapsing epididymitis are considered to be predisposing factors in the development of MMVT [4]. However, coexistence of these conditions could be more coincidence that real predisposition due to rarity of this tumor type in testicular region. Nevertheless, there is no doubt that the MMVTs are associated with exposure to asbestos (like in case pleural mesotheliomas) [5, 6]. The prognosis is dismal as – according to Recabal et al. – the tumor metastasize to lungs and groin with involvement of retroperitoneal and to lesser extend pelvic lymph nodes with deaths of five individuals from total studied group of 15 patients during 3.5-years-long follow-up [7]. There is little known about molecular biology of this tumor at the site of tunica vaginalis testis. However, with appliance of whole-genome sequencing method, Zhang et al. have recently reported on mutations in mesothelioma-associated genes such as KIF25, AHNAK, and PRDM2 and stated that MMVT harbored C-to-T and T-to-C mutations as well as it was characterized by amplification of copy number in its chromosomes 1 and 12 [8]. Our current report presents a case of malignant mesothelioma of tunica vaginalis testis (MMTVT) with detected for the first time mutations of PARP1, RNF213, PAX8, KMT2C, MTRR, in such a tumor in this location.

Material and methods

The postoperative specimen contained testicle with epididymis enveloped in testicular tunicas and spermatic duct of 81-year-old man. The postoperative specimen contained: the testis of dimensions: 6 × 3 × 2.5 cm without macroscopically visible focal lesions and 6 cm-long epididymis, mean up to 0.5 cm. The space was grossly enlarged between thickened visceral and parietal laminas of testicular tunica vaginalis with evident hydrocele. There were also numerous exophytic papillary excrescences with the largest diameter up to 3 cm that were mostly located in upper pole of the testicle on the visceral lamina of the tunica vaginalis. The papillary excrescences coalesced with one another to extend to greatest dimension to 6 cm in length, but this value was not diameter of the neoplasm as there was no one solid spherical tumor, but instead there was a multitude of papillary foci that studded both visceral and parietal lamina of tunica vaginalis testis. The neoplastic excrescences did not macroscopically show any deep invasion with mainly smooth interface with surrounding tissues. Representatives samples were taken from areas of tumor masses of testicular tunica vaginalis and underwent immunohistochemical staining with WT-1 (mouse monoclonal anti-human antibody, WT49, PA0562, dilution: 1:100), CK7 (NCL-CK7 OVTL dilution: 1:200), Calretinin (mouse monoclonal anti-human antibody PA0346, Clone CAL6, Novocastra Labs, Dilution: 1:40), CKAE1/AE3 (Monoclonal Mouse Anti-Human Cytokeratin, Clone: AE1/AE3 Isotype: IgG1, k. M3515, dilution:1:50 ), CK5 (mouse monoclonal anti-human antibody, NCL-CK5, Novocastra dilution: 1:100), PSA (mouse anti-human, monoclonal antibody, PA0431, Clone 35H9 Novocastra ready-to-use dilution to Bond system), TTF-1 (NCL-L-TTF-1, Clone SPT24 human thyroid transcription factor-1 (TTF-1), 1:50 dilution). Simultaneously gene profiling was performed. Status of hot spots found in 409 tumor genes were studied by means of next generation sequencing (NGS) technology (IonTorrent-Thermo Fisher Scientific, USA) using Ion AmpliSeq™ Comprehensive Cancer Panel. Detected mutations were annotated using COSMIC (https://cancer.sanger.ac.uk/cosmic), dbSNP database (https://www.ncbi.nlm.nih.gov/snp/) and Varsome database (https://varsome.com/).

Results

81-year-old man underwent total removal of testis, epididymis, testicular tunicas and part of spermatic cord due to clinically overt testicular hydrocele and later revealed papillary excrescences of tumor masses of vaginal tunica of the testis. The tumor was mainly composed of multifocal exophytic papillary-solid texture with necrosis fields among tightly packed areas of high cellularity with multiple mitoses, thus constituting a neoplasm of high grade malignancy. Actually, necrosis occupied 15% of tumor texture. Mitotic rate was 25 mitoses per 10 HPF on average. The cellular nuclei were predominantly oval, the chromatin was sparse in nuclei thus it was easy to notice that nuclei contained visible but not substantially enlarged nucleoli. The cytoplasm of neoplastic cells was rather eosinophilic with apparent shift toward amphophilic appearance in the highly cellular solid areas with high mitotic rate. The cells were cuboidal, oval and some of them a bit elongated. The cells were tightly packed in anastomosing rolls, rows and trabecules that streamed into more uniform solid areas without any discernible peculiar pattern, while on the periphery the cells were arranged in papillary-tubular glandular-like architecture. It occupied visceral lamina and to lesser extend parietal lamina of tunica vaginalis of testis with local invasion of rete testis without apparent extension beyond parietal lamina and without involvement of spermatic duct. Thus the tumor border and the interface of the tumor with surrounding tissue was rather smooth than infiltrative except from involvement of rete testis. The tumor was strongly positive for WT-1 (nuclear reaction) CKAE1/AE3 and calretinin (Fig. 1). It was also evidently positive for CK7 with negativity for CK5, PSA, TTF-1 as well as negative mucicarmine stain (Fig. 1). The tumor was diagnosed as malignant mesothelioma (epithelioid type) of the testicular vaginal tunica (pT2: 8th edition of pTNM staging) [9]. The use of next gene sequencing (NGS) enabled detection of six mutations in five genes (Table I). Namely, NGS revealed mutations in PARP1 (NM_001618: c.2285TG, p.K135R), MTRR (NM_024010: c.147A>G, p.I49M) and two sorts of mutations in structure of KMT2C gene (NM_170606: c.2447_2448insA (c.2447dupA), p.Y816fs and NM_170606: c.1042G>A, p.D348N) for the first time in this rare kind of tumor of genitourinary tract. In the analysis four mutations were classified as benign polymorphism (PARP1 p.V762A, RNF213 p.A1041T and MTRR pI49M, KMT2C p.Y816fs) and two as pathogenic mutations (PAX8 p.K135R and KMT2C p.D348N) (Fig. 2).

Discussion

In our opinion our tumor fits best to category of diffuse malignant mesothelioma with prominent papillary features in differential diagnosis of spectrum of borderline tumors between classic well differentiated papillary mesothelioma and diffuse malignant mesothelioma [10]. In regard to detected molecular gene abnormalities in mesothelioma, one should be aware of the fact that detected polymorphism (p.V762A) in the PARP1 encoding protein which is involved in the repair of DNA damage and cell proliferation and death, in a meta-analysis it was associated with an increased risk of cancer in Asian populations, but with a reduced risk for Caucasian populations [11]. Another meta-analysis also confirmed an increased risk of neoplastic incidence for gastric, cervical, and lung cancers as well as for gliomas in carriers of p.V762A in Asians [12]. The study conducted on the Polish population indicates this polymorphism as a risk factor for the development of cervical cancer [13]. The above data imply that the significance of polymorphism might be associated with the presence of other genetic factors, including hereditary polymorphisms and somatic changes in some cancers like malignant pleural mesotheliomas (MPM) [14]. p.A1041T polymorphism in the RNF213 (RNF213 possesses both ATPase activity and E3 ubiquitin-protein ligase activity) was detected in the present study. This polymorphism was previously described in the moyamoya population and control population [15]. The third of polymorphisms was detected in the MTRR gene, which codes for methionine synthase reductase, that is involved in the synthesis of methionine [16]. The polymorphism detected in the analyzed case p. I49M was previously described in the population with bone metastases of lung cancer [17]. Interestingly, deprivation of methionine is a potential strategy of oncological treatment analogously to arginine deprivation proposed for therapy of mesothelioma [18, 19, 20]. According to the bioinformatic analysis, the detected mutation in PAX8 is pathogenic. PAX8 is a transcription factor associated with embryogenic development, particularly in modeling of reproductive organs [21]. It is also used as a useful diagnostic marker of the differentiation of ovarian tumors [21]. On the other hand, this mutation (COSM6466208) has been flagged as a SNP and excluded from COSMIC database. It should be added that only focal and/or weak staining for PAX8 was described in pleural malignant mesothelioma [22]. Methylation, demethylation and acetylation of histone proteins have implications in cancerogenesis. Histone modifications are responsible for regulating the availability of transcription factors and other functional proteins to chromatin, thereby regulating transcription, translation, replication, DNA repair and recombination of DNA [23]. KMT2C (Lysine N-methyltransferase 2C 2-lysine methyltransferase)/MLL3 (myeloid/lymphoid or mixed myeloid/lymphoid or mixed-lineage leukemia) is regarded a significant tumor suppressor, due to its mutations that lead to the reading frame change were very often detected in colorectal cancer [24]. Recently, Wang et al. detected cancer mutational hotspot in MLL3 within the region that encodes its plant homeodomain (PHD) repeats to evidence involvement of this domain in interaction with the histone H2A deubiquitinase as well as tumor suppressor BAP1 with subsequent impairment of the interaction between MLL3 and BAP1 by mutations of MLL3 PHD repeats of poor prognosis for cancer patients [25]. In our current work, we have detected exactly mutations that change the reading frame (p.Y816fs) and point mutations (p.D348N) KMT2CA. Mutations p.Y816fs previously described in colon cancer [26] and pancreatic neuroendocrine tumors [27]. However, bioinformatic analysis classified this mutation as benign especially that this variant is very often detected in population (allelic frequency 0,48). In meanwhile, p.D348N mutations were detected in intestinal and pancreatic tumors, astrocytoma grade II and haemangioblastoma [27, 28, 29, 30]. To sum up, in the currently analyzed case, the molecular abnormalities significantly differ from the profile of mutated genes (CDKN2A, NF2, BAP1, EGFR, NRAS) described malignant pleural mesothelioma (MPM) [31, 32]. Although, we were not able to asses presence of homozygous deletion of CDKN2A with high confidence, looking at raw coverage data of the panel, we have noticed very low level of sequence depth (mostly less than 10 reads comparing to 137 reads [median]) of CDKN2A and CDKN2B locus. Considering 80% of tumor cells in the studied sample we suspect that both of CDKN2A alleles were lost in our case. Indeed, p16/CDKN2A is one of hallmark genes that get abrogated in diffuse malignant peritoneal mesothelioma (DMPM), so fluorescence in situ hybridization (FISH) detection of the homozygous deletion of p16/CDKN2A (p16) is an useful procedure for differential diagnosis of DMPM from reactive mesothelial hyperplasia (RMH), ovarian cancer and other malignancies that could spread to peritoneal surface [33].
Thus, the process of malignant transformation might develop in slightly different way and could be related to the anatomical location of the tumor. In spite of the numerous meticulous reports on morphology of malignant mesothelioma of testicular tunica vaginalis, there has been no molecular comprehensive research on gene profile in this rare malignancy so far [10, 34, 35]. Thus, our report certainly belongs to the first descriptions of the mutation profile of this category of tumors with some limitations as a single case study, as mesotheliomas present as truly heterogenous group of neoplasms in a variety of clinical settings [7, 36, 37, 38]. On the ground of our findings, development of MMTVT may be obscured by hereditary genetic predisposition in genes associated with DNA repair (PARP1), regulation of protein degradation (RNF213), surveillance of embryonic development (related to Müllerian duct origin) (PAX8), methionine synthesis (MTRR) and somatic changes linked to epigenetic regulation of gene expression (KMT2C). In our point of view, detection of mentioned mutations in MMTVT could be a ground for various, novel kinds of molecular targeted therapy, which have already been implemented in case of more frequent malignancies as colorectal cancer in case of anti-EGFR therapy that depends on KRAS and BRAF mutation status [39, 40, 41].
The authors declare no conflict of interest.

References

1. Shimada S, Ono K, Suzuki Y, Mori N. Malignant mesothelioma of the tunica vaginalis testis: a case with a predominant sarcomatous component. Pathol Int 2004; 54: 930-934.
2. Trenti E, Palermo SM, D’Elia C, et al. Malignant mesothelioma of tunica vaginalis testis: Report of a very rare case with review of the literature. Arch Ital Urol Androl 2018; 90: 212-214.
3. Mezei G, Chang ET, Mowat FS, Moolgavkar SH. Epidemiology of mesothelioma of the pericardium and tunica vaginalis testis. Ann Epidemiol 2017; 27: 348-359.e11.
4. Baqui AA, Boire NA, Baqui TT, Etwaru DJ. Malignant Mesothelioma of the Tunica Vaginalis Testis-A Malignancy Associated With Asbestos Exposure and Trauma: A Case Report and Literature Review. J Investig Med High Impact Case Rep 2019; 7: 2324709619827335.
5. Attanoos RL, Gibbs AR. Primary malignant gonadal mesotheliomas and asbestos. Histopathology 2000; 37:150-159.
6. Chekol SS, Sun CC. Malignant mesothelioma of the tunica vaginalis testis: diagnostic studies and differential diagnosis. Arch Pathol Lab Med 2012; 136:113-117.
7. Recabal P, Rosenzweig B, Bazzi WM, et al. Malignant Mesothelioma of the Tunica Vaginalis Testis: Outcomes Following Surgical Management Beyond Radical Orchiectomy. Urology 2017; 107: 166-170.
8. Zhang S, Zhang Q, Sun Q, et al. Genome Evolution Analysis of Recurrent Testicular Malignant Mesothelioma by Whole-Genome Sequencing. Cell Physiol Biochem 2018; 45: 163-174.
9. Brierley JD, Gospodarowicz MK, Wittekind C. TNM Classification of Malignant Tumours. 8th Edition. Wiley-Blackwell 2017; 1-272.
10. Brimo F, Illei PB, Epstein JI. Mesothelioma of the tunica vaginalis: a series of eight cases with uncertain malignant potential. Mod Pathol 2010; 23: 1165-1172.
11. Yu H, Ma H, Yin M, et al. Association between PARP-1 V762A polymorphism and cancer susceptibility: a meta-analysis. Genet Epidemiol 2012; 36: 56-65.
12. Qin Q, Lu J, Zhu H, et al. PARP-1 Val762Ala polymorphism and risk of cancer: a meta-analysis based on 39 case-control studies. PLoS One 2014; 9:e98022.
13. Roszak A, Lianeri M, Sowiñska A, et al. Involvement of PARP-1 Val762Ala polymorphism in the onset of cervical cancer in caucasian women. Mol Diagn Ther 2013; 17: 239-245.
14. Srinivasan G, Sidhu GS, Williamson EA, et al. Synthetic lethality in malignant pleural mesothelioma with PARP1 inhibition. Cancer Chemother Pharmacol 2017; 80: 861-867.
15. Zhang Q, Liu Y, Zhang D, et al. RNF213 as the major susceptibility gene for Chinese patients with moyamoya disease and its clinical relevance. J Neurosurg 2017; 126: 1106-1113.
16. https://www.snpedia.com/index.php/Rs1801394
17. Zhang K, Zhang M, Zhu J, et al. Screening of gene mutations associated with bone metastasis in nonsmall cell lung cancer. J Cancer Res Ther 2016; 12: C186-C190.
18. Cavuoto P, Fenech MF. A review of methionine dependency and the role of methionine restriction in cancer growth control and life-span extension. Cancer Treat Rev 2012; 38: 726-736.
19. Chaturvedi S, Hoffman RM, Bertino JR. Exploiting methionine restriction for cancer treatment. Biochem Pharmacol 2018; 154: 170-173.
20. Yap TA, Aerts JG, Popat S, et al. Novel insights into mesothelioma biology and implications for therapy. Nat Rev Cancer 2017; 17: 475-488.
21. Xiang L, Kong B. PAX8 is a novel marker for differentiating between various types of tumor, particularly ovarian epithelial carcinomas. Oncol Lett 2013; 5: 735-738.
22. Laury AR, Hornick JL, Perets R, et al. PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma. Am J Surg Pathol 2010; 34: 627-35.
23. Kouzarides T. Chromatin modifications and their function. Cell 2007; 128: 693-705.
24. Watanabe Y, Castoro RJ, Kim HS, et al. Frequent alteration of MLL3 frameshift mutations in microsatellite deficient colorectal cancer. PLoS One 2011; 6: e23320.
25. Wang L, Zhao Z, Ozark PA, et al. Resetting the epigenetic balance of Polycomb and COMPASS function at enhancers for cancer therapy. Nat Med 2018; 24: 758-769.
26. Lu YW, Zhang HF, Liang R, et al. Colorectal Cancer Genetic Heterogeneity Delineated by Multi-Region Sequencing. PLoS One 2016; 11: e0152673.
27. Ji S, Yang W, Liu J, et al. High throughput gene sequencing reveals altered landscape in DNA damage responses and chromatin remodeling in sporadic pancreatic neuroendocrine tumors. Pancreatology 2018; 18: 318-327.
28. Witkiewicz AK, McMillan EA, Balaji U, et al. Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets. Nat Commun 2015; 6: 6744.
29. Shankar GM, Lelic N, Gill CM, et al. BRAF alteration status and the histone H3F3A gene K27M mutation segregate spinal cord astrocytoma histology. Acta Neuropathol 2016; 131: 147-150.
30. Shankar GM, Taylor-Weiner A, Lelic N, et al. Sporadic hemangioblastomas are characterized by cryptic VHL inactivation. Acta Neuropathol Commun 2014; 2: 167.
31. Hylebos M, Van Camp G, van Meerbeeck JP, et al. The Genetic Landscape of Malignant Pleural Mesothelioma: Results from Massively Parallel Sequencing. J Thorac Oncol 2016; 11: 1615-1626.
32. Kim JE, Kim D, Hong YS, et al. Mutational Profiling of Malignant Mesothelioma Revealed Potential Therapeutic Targets in EGFR and NRAS. Transl Oncol 2018; 11: 268-274.
33. Ito T, Hamasaki M, Matsumoto S, et al. p16/CDKN2A FISH in Differentiation of Diffuse Malignant Peritoneal Mesothelioma From Mesothelial Hyperplasia and Epithelial Ovarian Cancer. Am J Clin Pathol 2015; 143: 830-838.
34. Chekol SS, Sun CC. Malignant mesothelioma of the tunica vaginalis testis: diagnostic studies and differential diagnosis. Arch Pathol Lab Med 2012; 136: 113-117.
35. Trpkov K, Barr R, Kulaga A, el al. Mesothelioma of tunica vaginalis of uncertain malignant potential – an evolving concept: case report and review of the literature. Diagn Pathol 2011; 6: 78.
36. Smith-Hannah A, Naous R. Primary peritoneal Epithelioid mesothelioma of clear cell type with a novel VHL gene mutation: a case report. Hum Pathol 2019; 83: 199-203.
37. Inaguma S, Lasota J, Wang Z, et al. Expression of ALCAM (CD166) and PD-L1 (CD274) independently predicts shorter survival in malignant pleural mesothelioma. Hum Pathol 2018; 71: 1-7.
38. Abello A, Steinkeler J, Das AK. A Bilateral Metachronous Mesothelioma of the Tunica Vaginalis. Urology 2018; 120: e1-e2.
39. Domaga³a P, Hybiak J, Sul¿yc-Bielicka V, et al. KRAS mutation testing in colorectal cancer as an example of the pathologist's role in personalized targeted therapy: a practical approach. Pol J Pathol 2012; 63: 145-164.
40. Wójcik P, Okoñ K, Osuch C, et al. BRAF mutations in sporadic colorectal carcinoma from polish patients. Pol J Pathol 2010; 61: 23-26.
41. Lasota J, Kowalik A, Wasag B, et al. Detection of the BRAF V600E mutation in colon carcinoma: critical evaluation of the imunohistochemical approach. Am J Surg Pathol 2014; 38: 1235-1241.