Clinicopathological characteristics of primary peritoneal epithelioid mesothelioma of clear cell type

Abstract Rationale: Primary peritoneal epithelioid mesothelioma of clear cell type is an extremely rare entity composed of clear cytoplasm. It is challenging to diagnose because of the morphological resemblance to clear cell tumor. Patient's concerns: A 69-year-old male patient had swollen lymph nodes in the right inguinal region for 7 months and was constipated for 1 month. Diagnosis: The patient was diagnosed as peritoneal epithelioid mesothelioma of clear cell type based on computed tomography scan, pathology, immunohistochemistry, special staining and whole-exome sequencing. This patient harbored VHL gene alteration in exon 1 and homologous recombination defect (with a score of 45). This finding indicated that this patient might be sensitive to platinum-based therapy and Poly ADP-ribose Polymerase (PARP) inhibitor. This patient carried no microsatellite instability, a low level of tumor mutation burden, and a high extent of intratumoral heterogeneity. Eighteen neoantigens were detected. Interventions: The patient received surgery-based multidisciplinary treatment by integrating cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC). HIPEC was administered with docetaxel 120 mg plus cisplatin 120 mg, at 43°C, for 60 minutes. After operation, the patient received intravenous (IV) chemotherapy with docetaxel 60 mg, pemetrexed 750 mg and cisplatin 100 mg, and then intraperitoneal (IP) chemotherapy with docetaxel 40 mg. The patient received interventional therapy of hepatic artery embolization for 5 times. Outcomes: Regular follow-up was performed until Oct 14, 2020. The patient died 31.6 months later owing to incomplete intestinal obstruction. Lessons: Primary peritoneal epithelioid mesothelioma of clear cell type needs to be differentiated from a variety of clear cell tumors. This disease is characterized by specific genetic alteration. Whole-exome sequencing contributes to guide individualized therapy. CRS-HIPEC helps achieve long-term overall survival.


Introduction
Malignant mesothelioma (MM) involves serosal surfaces, which originates from mesothelium comprising the pleura, peritoneum, pericardium and tunica vaginalis testis. Diffuse malignant peritoneal mesothelioma (DMPM) accounts for 7% to 30% of all cases. [1] Generally, DMPM is a rare and aggressive primary peritoneal malignancy, characterized by widespread multiple metastatic tumorous nodules originating from the peritoneum. The DMPM exhibits 3 major histologic subtypes, divided into epithelioid, sarcomatoid, or mixed (biphasic) categories in the updated 2015 World Health Organization classification. [2] The epithelioid type of mesothelioma frequently contains papillary or tubular components; however, clear cell variant is an extremely rare entity, which has only been described in a few case reports. [3,4] Diagnostic difficulties may be encountered because unusual clear cell morphological variants of mesothelioma can be confused with a variety of other clear cell tumors. We report on mesothelioma presenting as clear cell morphology, in attempt to explore differential diagnosis, molecular pathology and prognosis.

Case presentation
A 69-year-old male patient was diagnosed with primary peritoneal epithelioid mesothelioma of clear cell type. He had swollen lymph nodes in the right inguinal region for 7 months and dyssynergic defecation for 1 month, who presented to our hospital in October 2018. He had a history of asbestos exposure and a family history of cancer.

CT enhancement and 3D reconstruction imaging
The shape of the left kidney was normal. By contrast, multiple cystic low-density shadows were present in the right renal parenchyma, without obvious enhancement on enhanced scanning. Multiple nodules were identified in the abdominal cavity (Fig. 1A). A huge tumor was identified in the pelvic cavity ( Fig. 1B) with obvious inhomogeneous enhancement (Fig. 1C).

Intraoperative findings
Tumor nodules (with the maximum diameter of 3.5 cm) were scattered on the surface of small intestinal mesentery ( Fig. 2A). Tumor nodules were universally distributed on the surface of ligamentum teres and greater omentum, with the maximum diameter of 2 cm (Fig. 2B). Patchy tumor nodules were scattered in bilateral subphrenic peritoneum, with the largest size of 6 Â 6 Â 0.8 cm. Additionally, tumor nodules were present in the paracolonic sulcus on both sides, with maximum diameter of 3 cm on the left amd 2 cm on the right. The peritoneum of the left lower abdominal wall was adhered to a part of sigmoid colon, harboring scattered small tumor nodules with invasion to rectum and mesorectum. The small intestine, mesentery, ileocecal and pelvic masses (Fig. 2C, D) formed a dense adhesion at 400 cm from flexor ligament. The PCI (peritoneal cancer index) score was 24. The CCR (completeness of cytoreduction) was minimal residual disease.

Histopathological analysis
Removed surgical specimens were fixed in 10% phosphatebuffered, neutral formaldehyde solution at room temperature for 24 hours and dehydrated in an ascending series of ethanol. Samples were routinely embedded in paraffin, washed with xylene, rehydrated in a descending series of alcohol, washed with distilled water, and then stained with hematoxylin and eosin for 30 minutes at room temperature. Sections (4-mm thick) were observed under a light microscope with magnification of Â40, Â100, Â200, and Â400, respectively. Microscopically, most tumor cells were arranged in sheet-like structures without papillary patterns (Fig. 4A). Some tumor cells were arranged in tubular structures (Fig. 4B). Tumor cells had clear cytoplasm (Fig. 4C). The tumor was composed of large round to polygonal cells. Tumor cells displayed abundant clear cytoplasm with evident cytoplasmic membrane and eccentric small round nuclei (Fig. 4D). Mitotic figures were rare.

Immunohistochemical markers
Removed surgical specimens were fixed in 10% phosphatebuffered, neutral formaldehyde solution at room temperature for 24 hours. Tissue sections (4-mm thick) were deparaffinized, rehydrated and antigen retrieved with working solution of EnVision FLEX Target Retrieval solution High Ph (50 Â ) according to the manufacturer's protocol [EnVision FLEX+, Mouse, high Ph (Link) HRP; # K8002; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA] in PT Link (#PT100; Dako; Agilent Technologies, Inc.) at 95°C for 20 minutes, and washed in distilled water. [5] Endogenous peroxidase was blocked by DAKO EnVision FLEX peroxidase-blocking reagent for 10 minutes, and washed for 3 times in the PBS Wash buffer (Origene Technologies, Inc., Wuxi, China). The slides were incubated for 20 to 30 minutes at room temperature in humidity chamber with appropriate dilutions of primary antibodies (primary antibodies were detailed in Table 1) along with positive and negative controls. Immunohistochemistry was performed by automatic immunohistochemical instrument (intelliPATH FLX, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd). The sections (4-mm thick) were incubated with secondary antibody (MA-2000, Origene Technologies, Inc., Wuxi, China) for coupling reaction for 20 to 30 minutes at room temperature. The substrate (EnVision FLEX DAB+ Chromogen) was used to produce crisp brown color at the  site of target antigen. Then, hematoxylin (1-2 dips) was used as a counter stain. Sections were observed under a light microscope with magnification of Â40, Â100, Â200, and Â400, respectively. Immunohistochemical staining is depicted in Table 1. Tumor cells exhibited diffuse strong staining for cytokeratins (CKs) (

Special staining
Tissue sections (4-mm thick) were deparaffinized, washed with xylene, rehydrated in a descending series of alcohol, and washed with distilled water. The slides were oxidized for 10 minutes with periodic acid (batch #: C191201, Zhuhai Basso Biotechnology Co., Ltd), washed with distilled water; stained for 10 minutes with Schiff (batch #: C191201, Zhuhai Basso Biotechnology Co., Ltd), rinsed with running water; then stained for 3 minutes with hematoxylin and rinsed with running water. The slides were dehydrated, transparentized, and sealed with neutral gum. D-PAS was digested with diastase.

Whole-exome sequencing (WES) and somatic mutation calling
Tumor and matched normal DNA were extracted using GeneRead DNA FFPE Kit (Qiagen) from formalin-fixed paraffin-embedded (FFPE) tissues. Libraries were constructed by Agilent SureSelect v.4 Kit (Agilent) and sequenced with next-   The WES data were analyzed for mutations and human genome build hg19 was used as reference. Somatic SNVs and In/ Dels were analyzed with GATK MuTect2 (version 4.1). The sequenced reads were realigned to hg19 by Burrows-Wheeler Aligner BWA-MEM (http://biobwa.sourceforge.net/) to enhance valid SNVs.
Tumor mutation burden (TMB) represents the total number of somatic mutations in each MB base of exon coding region in a tumor sample. The calculation formula was as follows: TMB = total number of somatic mutations (including non-synonymous point mutations, insertions and deletions in exon coding region)/ target region size, with the unit of mutations/Mb. For this patient, TMB level was low (Table 5).
This experiment detected 18 kinds of neoantigens (Table 6). Twelve frameshift mutations had high affinity (IC50 < 150 nMol). Six were missense mutations. There was no in-frame deletion or in-frame insertion.
Detection of chemotherapy related genes was as follows. Compared with other genotypes, the subject might be sensitive to methotrexate, cyclophosphamide + epirubicin, fluorouracil or capecitabine based chemotherapy. Toxicity and side effects of cyclophosphamide, cyclophosphamide + doxorubicin, cyclophosphamide + epirubicin, irinotecan, fluorouracil/tegafur + folic acid, fluorouracil + folic acid + oxaliplatin might be relatively low. The subject might be resistant to paclitaxel, cisplatin + paclitaxel, cisplatin + cyclophosphamide, anthracycline, fluorouracil + oxaliplatin and gemcitabine. Toxicity and side effects of platinum compounds + paclitaxel, cisplatin + paclitaxel, cisplatin + cyclophosphamide, anthracycline and pemetrexed may be relatively high. This patient carried no reported genetic pathogenic variation.

Follow up
Before cytoreductive surgery (CRS) + hyperthermic intraperitoneal chemotherapy (HIPEC) operation, the patient received 2 cycles of intraperitoneal (IP) chemotherapy with cisplatin 120 mg  and intravenous (IV) chemotherapy with Preto 800 mg. After operation, the patient received IV chemotherapy with docetaxel 60 mg, pemetrexed 750 mg and cisplatin 100 mg, and IP chemotherapy with docetaxel 40 mg. The patient received interventional therapy of hepatic artery embolization for 5 times ( Table 7). The patient received no targeted therapy. The patient survived over 31+ months and died on October 14, 2020 owing to incomplete intestinal obstruction. This case report was approved by the ethics committee of our hospital. Informed consent was obtained from the patient's son.

Discussion
Clear cell mesothelioma, also called glycogen-rich or foamy mesothelioma, was considered a variant of epithelioid MM. As an extremely rare entity, it was first described by Ordoñez et al [7] in 1996. It can originate from pleura [3,4] or peritoneum. [3] Clinically, primary peritoneal epithelioid mesothelioma of clear cell type occurs mainly in aged people (Table 8). [3,[8][9][10][11] The majority of patients were males (male: female ratio of 6:2).  To diagnose clear cell peritoneal epithelioid mesothelioma can be challenging owing to a wide variety of tumors with similar morphologic features. Given extreme rarity of such an entity, diagnosis of clear cell mesothelioma could be made after excluding more common intra-abdominal tumors, including carcinomas arising from upper or lower gastrointestinal tract, pancreas, adrenal, kidney, liver and Müllerian system/primary HRD refers to patients with BRCA harmful mutations or suspected harmful mutations, or patients with genomic instability more than 6 months after the onset of the latest platinum chemotherapy. If there is a BRCA1/2 harmful mutation or suspected harmful mutation and/or a predefined HRD score more than 42, it is defined as HRD positive. If the HRD score is less than 42 and there is no BRCA1/2 harmful or suspected harmful mutation, it is defined as HRD negative. If the HRD analysis fails and the BRCA1/2 analysis is negative, the HRD status is unknown.   Table 6 Tumor neoantigens in this patient identified by whole-exome sequencing. peritoneum, or metastatic carcinomas from breast or lung origins. Other intra-abdominal tumors with clear cell morphology, within the scope of differential diagnosis, included myoepithelial carcinoma, epithelioid leiomyosarcoma, melanoma, perivascular epithelioid cell tumor, paraganglioma, gastrointestinal stromal tumor, epithelioid angiosarcoma, and epithelioid sarcoma. [11] Carcinomas in general were excluded by absent immunostaining for markers such as B72.3, Ber-EP4, and poly-CEA. [12,13] One of the most common genetic alterations in MM was homozygous deletion of 9p21 locus within a cluster of genes spanning cyclin-dependent kinase inhibitor (CDKN)-2A, CDKN2B, and methylthioadenosine phosphorylase. Deletion occurs in approximately 25% of peritoneal MM. [14] BAP1 (BRCA-associated protein 1) mutations or deletions, however, can occur in up to 80% of epithelioid mesothelioma cases. [15] Smith-Hannah et al reported VHL gene mutation in clear cell peritoneal epithelioid mesothelioma. [11] We also identified VHL gene alteration involving exon 1. The VHL, well-known as a tumor suppressor gene, was located in chromosome 3p25. Its protein product, pVHL, performs multiple biological functions. [16] Table 7 The procedure of treatment. Interventional therapy of hepatic artery embolization, liver protection therapy 2019-09-06
Most of homologous recombination defects (HRD) were caused by mutations/deletions in homologous recombination repair (HRR) genes. Since genomic damage could not be repaired in time, genomic defects would develop, characterized by chromosome breakage, loss of heterozygosity, and telomere instability. The HRD is evaluated by calculating loss of heterozygosity (hrdloh), large scale transitions (LST) and number of telomeric allelic imbalances (TAI). [22] This patient had a HRD score of 45, indicating sensitivity to platinum-based chemotherapy and PARP (Poly ADP-ribose Polymerase) inhibitor.
Neoplastic antigens are abnormal proteins, which could be produced by genetic mutations in cancer cells to activate immune system. Neoantigen can be an abnormal polypeptide produced by virus infection, genetic mutation or rearrangement during tumor development and progression. This neoantigen can be presented by major histocompatibility complex (MHC) molecules to activate antitumor immune response. Upon presented by MHC molecule, neoantigen can be recognized, processed and presented by antigen presenting cells (APC) to T cells through specifically binding to T cell receptor (TCR). This process leads to activation, proliferation and differentiation of T cells into cytotoxic effector cells, which initiates anti-tumor immune response. [23] Notably, neoantigens are only expressed on tumor cells with tumor-type specificity. Moreover, sequences of neoantigen peptides are different from normal proteins/peptides, which are not screened by thymus negative selection, so they harbor strong immunogenicity. [24] In addition, heterogeneity of neoantigen is an important factor for survival and prognosis of cancer patients. For example, patients with homogeneous tumors (ITH 1%) had a longer overall survival time than those with heterogeneous tumors (ITH > 1%). [25] All of these characteristics indicate that neoantigens could be used as a good target for immunotherapy. Neoantigen-based vaccines have provided a potential complementary therapeutic strategy by increasing immunogenic antigen load, which can enhance tumor-specific immune response. Further research is needed to explore this treatment option in mesothelioma and technological advances are required to translate this concept into clinical practice. [26] In conclusion, primary peritoneal epithelioid mesothelioma of clear cell type is an extremely rare entity. Tumor cells compose of abundant clear cytoplasm, evident cytoplasmic membranes and eccentric small round nuclei. It is necessary to be differentiated from a variety of clear cell tumors. Molecular testing identifies VHL gene alteration involving exon 1. Positive HRD indicates sensitivity to platinum-based chemotherapy and PARP inhibitor. Based on WES, 18 potential neoantigens are identified. This patient has a high level of intratumoral heterogeneity. Compared with other genotypes, this subject might be beneficial from methotrexate, cyclophosphamide + epirubicin, fluorouracil or capecitabine based treatment.