Imaging, pathological and molecular detection of malignant peritoneal mesothelioma (MPM): a case description

Introduction

Malignant peritoneal mesothelioma (MPM) is a very rare and highly aggressive malignancy originating from peritoneal mesothelial cells, accounting for 10–30% of malignant mesotheliomas. It is associated less frequently with asbestos exposure compared with pleural mesothelioma. The median age at diagnosis in MPM is higher than that in pleural mesothelioma (71 vs. 63 years) (1). The clinical symptoms of MPM are non-specific, characterized by abdominal distension and abdominal pain as the primary symptom. Other atypical manifestations include weight loss, anorexia, nausea, vomiting, diarrhea, ileus and perforation (2). Based on its variable clinical characteristics, MPM is usually discovered during accidental examination, such as abdominal computed tomography (CT), laparoscopy or abdominal surgery. The typical CT findings of MPM include ascites, abdominal mass and peritoneal thickening. Positron emission tomography-computed tomography (PET-CT) shows diffuse nodular thickening of the peritoneum or local mass, with an abnormal uptake in the lesions (3). The definitive diagnosis of MPM depends on peritoneal biopsy and immunohistochemistry findings. Tandon et al. showed that positive immunohistochemical markers of MPM included calretinin, WT1, CK5/6, mesothelin and D2-40 (4). Most patients are diagnosed at an advanced stage with a poor prognosis, resulting in an average survival time of only 8.4 months and a 5-year survival rate of approximately 50% after effective treatment (5). The accepted standard therapy is cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC). Metagenomic next-generation sequencing (mNGS) provides an unbiased and comprehensive approach to detect bacteria, fungi, viruses, parasites, and unknown pathogens by sequencing the nucleic acids in clinical specimen, which has been widely applied for the diagnosis of infectious disease (6). Some studies have shown that only 1–2% of the reads are from pathogens, while human derived sequences account for more than 90%, which can be used to analyze copy number variations (CNVs) to detect tumors (7). Here we report the first case of MPM auxiliary diagnosed by mNGS.

Case presentation

An 83-year-old male was admitted to Peking University First Hospital for “dry mouth and emaciation for 5 months, anorexia for 3 months”. Prior to hospitalization, his glycated hemoglobin value was 12.8% on physical examination, which suggested the diagnosis of diabetes mellitus. After administration of hypoglycemic therapy, the patient still had emaciation and anorexia. The blood routine test was normal, C-reactive protein (CRP) was 42.47 mg/L (reference range, 0–3 mg/L), erythrocyte sedimentation rate (ESR) was 88 mm/h (reference range, 0–15 mm/h). Tumor biomarkers showed no significant abnormality. Abdominopelvic enhanced CT showed punctate high-density shadow with a diffuse distribution in the greater omentum and multiple small lymph nodes in the hilum of the liver and retroperitoneal area (Figure 1A,1B). PET-CT showed peritoneal thickening, high glucose metabolism in the mesentery, omentum and local wall of the colon splenic flexure, several small lymph nodes in the mesentery and para-abdominal aorta (Figure 2). After admission, his temperature was monitored to fluctuate at 36.0–38.5 ℃, and the laboratory tests were as follows (Table 1): white blood cell count (WBC) 11.10×10⁹/L [reference range, (3.5–9.5)×10⁹/L], hemoglobin 83 g/L (reference range, 130–175 g/L), platelet 562×10⁹/L [reference range, (125–350)×10⁹/L], neutrophil 8.10×10⁹/L [reference range, (1.8–6.3)×10⁹/L], ESR 121 mm/h, interleukin-6 (IL-6) 157.67 pg/mL (reference range, <6.4 pg/mL), CRP 136.51 mg/L. Urine routine examination showed: heterogeneous red blood cells 100–120/HP. CYFRA21-1 was 12.88 ng/mL (reference range, <5 ng/mL). No abnormal findings were detected in immunoglobulin, complement C3, complement C4 and lymphocyte subsets. Autoantibody, immunofixation electrophoresis of blood and urine, sputum smear, T cell spot test tuberculosis (T-spot.TB), purified protein derivative (PPD) test, antigens and antibodies of respiratory pathogens, (1,3)-β-D-glucan and Galactomannan, Influenza A and B nucleic acids, and blood cultures were negative. Ultrasound of superficial lymph nodes and abdominal lymph nodes was normal. Chest CT and urinary magnetic resonance imaging (MRI) showed no active lesions. Bone marrow pathology showed no significant abnormalities. Abdominopelvic enhanced CT showed greater thickening of the peritoneum and slightly more abdominopelvic effusion compared to that prior to hospitalization (Figure 3A,3B).

Figure 1.

Abdominopelvic enhanced CT. (A,B) Axial CT images prior to hospitalization revealed punctate high-density shadow with a diffuse distribution in the greater omentum (white arrows) and small lymph nodes in the retroperitoneal area (red arrow). CT, computed tomography.

Figure 2.

PET-CT showed diffuse nodular thickening of the peritoneum with a high uptake (yellow arrows). PET-CT, positron emission tomography-computed tomography.

Table 1. Laboratory data.

Variable	Reference range	On admission
White cell count (109/L)	3.5–9.5	11.1
Hemoglobin (g/L)	130–175	83
Platelet count (109/L)	125–350	562
Neutrophil count (109/L)	1.8–6.3	8.1
Alanine aminotransferase (IU/L)	9–50	48
Aspartate aminotransferase (IU/L)	15–40	77
Albumin (g/L)	40–55	28.5
Creatinine (μmol/L)	44–133	83.5
Glucose (mmol/L)	3.61–6.11	9.18
Erythrocyte sedimentation rate (mm/h)	0–15	121
C-reactive protein (mg/L)	0–3	136.51
Interleukin-6 (pg/mL)	<6.4	157.67
CYFRA21-1 (ng/mL)	<5	12.88
Prothrombin time (sec)	10.1–12.6	12.7
Prothrombin-time international normalized ratio	0.88–1.1	1.1

Figure 3.

Abdominopelvic enhanced CT. (A,B) CT axial images after admission showed thickening of the greater omentum with soft-tissue density shadow (yellow arrows), a small volume of pelvic ascites (green arrow) and small lymph nodes in the retroperitoneal area (blue arrow). CT, computed tomography.

As a result, infection was primarily suspected and anti-infection treatment was given. The patient was first administered with meropenem. After treatment, the patient still had recurrent fever. The antibiotics were then adjusted to moxifloxacin, minocycline, fosfomycin, piperacillin tazobactam, linezolid. The patient’s symptoms and inflammatory indicators showed poor improvement. Due to poor effect of antibiotic therapy, the patient underwent laparoscopy, which showed ascites and multiple nodules on the peritoneum and omentum. The routine and biochemical results of ascites suggested exudate. But the conventional microbiological tests of ascites and peritoneal tissue were negative. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

mNGS testing

The peripheral blood, ascitic fluid and peritoneal tissue of the patient were sent for mNGS testing. The MD017 Reverse Transcription Module for NGS Library Preparation was performed using the MD001T Metagenomic DNA Library Preparation Kit (Hangzhou Matridx Biotechnology Co., Ltd., China). Gram-negative bacteria dominated by Pseudomonas aeruginosa were detected by mNGS of blood, which were not identified in other specimens (Table 2). mNGS of ascitic fluid and peritoneal tissues detected normal flora from human skin or environment source which were not regarded as pathogens. The CNV analysis of the chromosomes by mNGS of ascitic fluid and peritoneal tissue showed significant alterations in chromosomes 7, 11, 16, 17, which suggested the presence of tumor (Figure 4).

Table 2. The microorganisms detected by mNGS in the peripheral blood, ascitic fluid and peritoneal tissue of the case.

Sample	Genus			Species
	Name	Sequence number†		Name	Sequence number†
Peripheral blood
Plasma	Stenotrophomonas	15		Stenotrophomonas acidaminiphila	12
	Pseudomonas	4		Pseudomonas aeruginosa	3
Blood corpuscle	Pseudomonas	165		Pseudomonas aeruginosa	150
	Citrobacter	126		Citrobacter freundii complex	116
	Stenotrophomonas	40		Stenotrophomonas acidaminiphila	36
Ascitic fluid	Mycoplasma	1		Mycoplasma pneumoniae	1
	Moraxella	1		Moraxella osloensis	1
	Malassezia	2		Malassezia globosa	1
	Saccharomyces	1		Saccharomyces cerevisiae	1
Peritoneal tissue	Cutibacterium	1		Cutibacterium acnes	1

^†, the sequence number of the microorganism detected at the level of the genus/species. mNGS, metagenomic next-generation sequencing.

Figure 4.

CNV analysis of the patient’s chromosomes. Both ascites and peritoneal tissue showed large CNVs on chromosomes 7, 11, 16 and 17. CNV, copy number variation.

Pathology and immunohistochemistry findings

Peritoneal tissues cytology exhibited large areas of epithelioid cells with moderate atypia in a diffuse growth pattern between adipose lobes, accompanied by mononucleated and multinucleated giant tumor cells. Immunohistochemistry staining showed that cells were strongly positive for CKPan (AE1/AE3), CK7, calretinin, CK5/6, p53, focally positive for vimentin, WT1, D2-40, CK19, negative for CK20, CDX-2, p40, Syn, TTF-1, TG, CEA, CA199, PSA, GATA3, hepatocyte, and Ki67 index was approximately 30% for tumor cells (Figure 5). Hence, the initial diagnosis was epithelioid malignant mesothelioma.

Figure 5.

Peritoneal tissues cytology and immunohistochemistry. (A) Epithelioid cells with moderate atypia in a diffuse growth pattern between adipose lobes HE staining (200×). (B) Cells positive for CK7 staining (100×). (C) Cells positive for Calretinin staining (100×). (D) Cells positive for Ki67 staining (100×).

Clinical outcomes

The patient was diagnosed as epithelioid malignant mesothelioma. Consultation from the oncology and chemotherapy department determined that the chemotherapy was not suitable for the patient due to his poor physical condition and supportive care was suggested. After a discussion with his family, his family elected to pursue supportive treatment.

Discussion

MPM is a very rare tumor arising from peritoneal mesothelial cells with a dismal prognosis. Due to the non-specific symptoms and signs of MPM, it is usually discovered during accidental examination and diagnosed at an advanced stage. In comparison to other abdominal tumors, the imaging findings of MPM are non-specific, including ascites, peritoneal thickening, peritoneal nodules and masses (8). PET-CT has a significant value in differentiating between malignant and benign lesions (9) The definitive diagnosis of MPM relies on peritoneal biopsy and immunohistochemistry. The most sensitive immunohistochemical markers include calretinin (100%), WT1 (94%) and CK5/6 (89%) (1). CRS combined with HIPEC is considered the standard therapy. However, the majority of patients are diagnosed at an advanced stage, resulting in a poor prognosis after effective treatment.

In this case, the patient presented with dry mouth, emaciation and anorexia. Abdominal enhanced CT and PET-CT showed ascites, peritoneal thickening and abdominal lymph node enlargement. Infection could not be excluded combined with elevated temperature and inflammatory markers but anti-infection treatment was ineffective. Fever of unknown cause can be attributed to many factors, such as infection, tumor, autoimmune disease (10). No obvious abnormalities were found by conventional microbiological testing, tumor biomarkers, autoantibody, bone marrow biopsy of the patient. No pathogens were found in ascites and peritoneal tissue by culture. mNGS of blood identified Gram-negative bacteria, suggesting that infection could not be completely excluded. mNGS of ascites and peritoneal tissue showed significant CNVs of chromosomes, which suggested the presence of tumor and was diagnosed by peritoneal tissue biopsy.

mNGS can simultaneously provide detection of pathogens and cancers through analysis of nucleic acids. CNV refers to structural variations greater than 1 KB with an alteration of copy number between two or more genomes, such as deletions, duplications, insertions of genomic regions (11), which can cause multilocus alterations in the genome and are involved in the development and progression of various cancer types, including breast, ovarian, pancreas, skin and brain (12). CNV analysis of human derived sequences has been recently used in the auxiliary diagnosis of tumors, including lymphoma, colorectal cancer and central nervous system (CNS) tumors (13-15). Gu et al. found that CNV detection in cerebrospinal fluid by mNGS had 55% sensitivity and 100% specificity for detecting CNS malignant neoplasms (15).

Tissue biopsy and pathological staining are the gold standard for cancer diagnosis, which require an invasive and time-consuming procedure. Due to concerns about the harm of invasive procedures, patients may refuse or delay the examination, thereby delaying diagnosis and treatment. However, mNGS can be conducted on various clinical samples and supply results within hours. mNGS can simultaneously provide detection of pathogens and cancers through analysis of nucleic acids for patients with unknown etiologies, thereby optimizing diagnosis, treatment and prognosis.

In summary, we report the first case of MPM auxiliary diagnosed by mNGS. mNGS is useful in pathogen identification and auxiliary diagnosis of cancers by CNV analysis.

Supplementary

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Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.