Guidelines for Pathologic Diagnosis of Mesothelioma.

CONTEXT.­: Mesothelioma is an uncommon tumor that can be difficult to diagnose. OBJECTIVE.­: To provide updated, practical guidelines for the pathologic diagnosis of mesothelioma. DATA SOURCES.­: Pathologists involved in the International Mesothelioma Interest Group and others with expertise in mesothelioma contributed to this update. Reference material includes peer-reviewed publications and textbooks. CONCLUSIONS.­: There was consensus opinion regarding guidelines for (1) histomorphologic diagnosis of mesothelial tumors, including distinction of epithelioid, biphasic, and sarcomatoid mesothelioma; recognition of morphologic variants and patterns; and recognition of common morphologic pitfalls; (2) molecular pathogenesis of mesothelioma; (3) application of immunohistochemical markers to establish mesothelial lineage and distinguish mesothelioma from common morphologic differentials; (4) application of ancillary studies to distinguish benign from malignant mesothelial proliferations, including BAP1 and MTAP immunostains; novel immunomarkers such as Merlin and p53; fluorescence in situ hybridization (FISH) for homozygous deletion of CDKN2A; and novel molecular assays; (5) practical recommendations for routine reporting of mesothelioma, including grading epithelioid mesothelioma and other prognostic parameters; (6) diagnosis of mesothelioma in situ; (7) cytologic diagnosis of mesothelioma, including use of immunostains and molecular assays; and (8) features of nonmalignant peritoneal mesothelial lesions.

Detailed clinical course of a patient with rapidly progressing sarcomatoid pleural mesothelioma without p16 deletion with systemic haematogenous metastasis to soft tissues.

Sarcomatoid mesothelioma is difficult to differentiate from other mesotheliomas. Here, we describe the case of a man in his early 80s with sarcomatoid mesothelioma and a history of asbestos exposure. He initially presented with right-sided chest pain and was examined. Right-sided pleural effusion was detected; therefore, he was hospitalised. Based on the observed pleural effusion and biopsy result, the presence of a malignant tumour was excluded; hence, he was diagnosed with benign asbestos pleurisy. He subsequently developed left-sided pleural effusion, masses and lung nodules, and died 9.5 months after the initial examination. A definitive diagnosis of sarcomatoid mesothelioma with rapid systemic progression was established after detailed investigations using autopsy specimens. This rare case of mesothelioma-without p16 deletion (detected using fluorescence in situ hybridisation)-presented differently from the usual sarcomatoid mesothelioma.

Expression of laminin332 γ2 at the invasive front is associated with tumor budding and poor prognosis in cutaneous squamous cell carcinoma.

Laminin332 is a glycoprotein consisting of α3/ß3/γ2 chains, of which the γ2 chain (Ln-γ2) is expressed in tumor cells at the invasive front in many types of malignant tumors. We have previously reported that Ln-γ2 is associated with tumor invasion of cutaneous squamous cell carcinoma (cSCC) in vivo and in vitro. Recently, tumor budding (TB; invasion patterns in small clusters of less than five cancer cells in the stroma at the invasive front) has been reported to be a risk factor for lymph node metastasis in cSCC. Based on these findings, we speculated that expression of Ln-γ2 is related to TB in cSCC and would be an invasive factor that causes lymph node metastasis. In this study, we investigated the relationship between Ln-γ2 expression and clinicopathological findings, including TB, in 102 cases of cSCC using immunohistochemistry. The results showed that high expression of Ln-γ2 at the invasive front correlated with a high TB score. In addition, high Ln-γ2 expression at the invasive front was also associated with lymphatic invasion, lymph node metastasis, and poor prognosis (death or recurrence), as in TB. Furthermore, we showed a positive association between Ln-γ2 expression at the invasive front and Yes-associated protein (YAP) expression in the Hippo pathway. Our results suggest that Ln-γ2 expression at the invasive front may have a role in TB formation via YAP and contribute to prognosis by causing lymphatic invasion and lymph node metastasis. The expression of Ln-γ2 would be useful for risk assessment of lymph node metastasis and poor prognosis in routine practice of cSCC.

MMP-2 Regulation of Emmprin on Tumour Cells and CD73 on Fibroblasts During Tumour-Stromal Interaction.

BACKGROUND/AIM: We previously found that binding between CD73 and extracellular matrix metalloproteinase (MMP) inducer (emmprin) and suppression of CD73 in both tumour cells and fibroblasts suppressed MMP-2 production when co-cultured. However, the importance of CD73 expression in either fibroblasts or cancer cells for cancer invasion remains unknown. In this study, we used siRNA to separately down-regulate CD73 in individual cells, and then performed a 3D co-culture to investigate tumour invasion. MATERIALS AND METHODS: siRNA was used for suppression of CD73 in either fibroblasts (ST353i, HDF) or tumour cells (FU-EPS-1, A431, CRL-2095). Immunoblotting was performed for detecting MMP-2 production after CD73 suppression. 3D-co-cultures were performed for examining tumour invasion. RESULTS: CD73 suppression revealed that CD73 expression on fibroblasts and emmprin on tumour cells were important in regulating MMP-2 production, suggesting that emmprin on tumour cells does not bind CD73 at the cis-manner, but rather at the trans-manner to CD73 present on fibroblasts. CD73 suppression also reduced MMP-2 production at the transcription level and reduced tumour invasion. CONCLUSION: CD73 on fibroblasts acts as a receptor for emmprin, which forms a complex that increases MMP-2 production, possibly resulting in increased invasiveness.

Phosphaturic mesenchymal tumor: A chondromyxoid fibroma-like type.

Phosphaturic mesenchymal tumor (PMT) is a rare neoplasm that causes tumor-induced osteomalasia (TIO) in most affected patients, usually through the production of fibroblast growth factor 23 (FGF23). This tumor is often misdiagnosed due to its relative rarity and its widely varied histomorphologic spectrum. Here we describe a case of a 78-year-old woman who presented with a left middle tumor without symptoms of TIO. The histological features resembled chondromyxoid fibroma with smudgy calcification in the tumor matrix. In addition, we evaluated FGF23 expression through immunohistochemical study and reverse transcription polymerase chain reaction. PMT with chondromyxoid fibroma features are extremely rare. Examining the expression of FGF23 is useful in the diagnosis of PMT.

Usefulness of NF2 hemizygous loss detected by fluorescence in situ hybridization in diagnosing pleural mesothelioma in tissue and cytology material: A multi-institutional study.

OBJECTIVES: BAP1, CDKN2A, and NF2 are the most frequently altered genes in pleural mesotheliomas (PM). Discriminating PM from benign mesothelial proliferation (BMP) is sometimes challenging; it is well established that BAP1 loss, determined by immunohistochemistry (IHC), and CDKN2A homozygous deletion (HD), determined by fluorescence in situ hybridization (FISH), are useful. However, data regarding the diagnostic utility of NF2 FISH in PM is limited. Thus, we performed a multi-institutional study examining the utility of NF2 alterations determined by FISH for diagnosing PM in combination with BAP1 loss and CDKN2A HD. MATERIALS AND METHODS: Multi-institutional PM cases, including 106 surgical and 107 cell block samples as well as 37 tissue cases of benign mesothelial proliferation (BMP) and 31 cell block cases with reactive mesothelial cells (RMC), were collected and analyzed using IHC for BAP1 and FISH for CDKN2A and NF2. RESULTS: In PM, NF2 FISH revealed hemizygous loss (HL) in 54.7% of tissue cases (TC) and 49.5% of cell block cases (CBC), with about 90% of HL being monosomy. CDKN2A HD or BAP1 loss were detected in 75.5%/65.4% TC or 63.6%/60% CBC, respectively. BMP or RMC showed no BAP1 loss, CDKN2A HD, or NF2 HL. For discriminating PM from BMP, a combination of BAP1 loss, CDKN2A HD, and NF2 HL yielded enhanced sensitivity of 98.1% TC/94.4% CBC. BAP1 loss, CDKN2A HD, or NF2 HL were observed in 69%, 70%, or 58% of epithelioid PM, but in 9%, 91%, or 27% of sarcomatoid PM, respectively. Histotype, histological gradings, and CDKN2A deletion status showed significant differences in overall survival, while BAP1 loss and NF2 HL did not. CONCLUSION: NF2 HL, consisting predominantly of monosomy, can be detected by FISH in both TC and CBC of PM, and is effective for distinguishing PM from BMP, especially when combined with BAP1 loss and CDKN2A HD.

GLUT-1 expression is helpful to distinguish myxofibrosarcoma from nodular fasciitis.

Myxofibrosarcoma (MFS) is a fibroblastic/myofibroblastic neoplasm with a variably myxoid stroma. Histologically, MFS shows a wide spectrum of cellularity, pleomorphism and proliferative activity. Because of its variable morphology and lack of discriminatory markers, MFS can be difficult to distinguish from some benign soft-tissue tumors, especially nodular fasciitis (NF). Glucose transporter 1 (GLUT-1) is expressed in a variety of malignant mesenchymal tumors. In the current study, we evaluated GLUT-1 expression to determine its value in distinguishing MFS from NF. Tissue specimens from 14 MFS cases and 16 NF cases were sectioned and stained for GLUT-1 using immunohistochemistry. The percentage of GLUT-1-positive cells was scored as follows: 0 (no staining), 1+ (1-19%), 2+ (20-50%) and 3+ (>50%). Samples with a score of 1+ were defined as GLUT1-expressing samples. GLUT-1 expression was seen in all 14 MFS cases, whereas only 6 NF cases (37.5%) were positive for GLUT-1 and were scored 1+. Notably, 2-3+ GLUT-1 expression was found in 86% of MFS cases and 0% of NF cases. Our results indicate that GLUT-1 is a highly sensitive immunohistochemical marker for MFS and may be useful for the differential diagnosis of MFS and NF.

t(1;2)-Positive Localized Tenosynovial Giant Cell Tumor With Bone Invasion.

BACKGROUND: Localized tenosynovial giant cell tumor (LTGCT) is one of the most common benign soft-tissue tumors of the foot. Although pressure erosion in the adjacent bone may be seen, intraosseous invasion of LTGCT is extremely rare. Recent molecular studies have identified the presence of pathognomonic translocation involving the colony stimulating factor 1 (CSF1) gene at 1p13. CASE REPORT: We present an unusual case of LTGCT mimicking a malignant tumor on imaging. The patient was a 16-year-old woman with no history of trauma who presented with a 2-year history of a slow-growing, painless mass in the left fourth toe. Physical examination revealed a 2-cm, elastic hard, immobile, nontender mass. Plain radiograph showed a lytic lesion with a partially sclerotic rim in the proximal phalanx of the fourth toe. Computed tomography demonstrated an expansile lesion with plantar cortical destruction. Magnetic resonance imaging revealed a nodular mass with intermediate signal intensity on T1-weighted sequences and heterogeneous high signal intensity on T2-weighted sequences. The mass had intense contrast enhancement. Complete excision of the mass was performed, and the bone defect was repaired with calcium phosphate cement. Cytogenetic analysis revealed a t(1;2)(p13;q37) translocation as the sole anomaly. Fluorescence in situ hybridization demonstrated the presence of CSF1 rearrangements. CONCLUSION: Although extremely rare, LTGCT should be considered in the differential diagnosis of an intraosseous lesion near small joints, especially when seen in the toe.

Arthroscopic Excision of Intra-articular AHRR-NCOA2- positive Angiofibroma of Soft Tissue of the Knee: A Case Report.

BACKGROUND/AIM: Angiofibroma of soft tissue (AFST) is a rare benign soft-tissue tumor that most frequently occurs in the lower extremities. It has a characteristic genetic feature with a balanced chromosomal translocation t(5;8)(p15;q13), resulting in a fusion of aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2). CASE REPORT: A 55-year-old woman presented with a 2-year history of left knee pain and recently noticed the development of a palpable mass. Magnetic resonance imaging exhibited a well-defined intra-articular mass with iso-signal intensity relative to skeletal muscle on T1-weighted sequences, heterogeneous high signal intensity on T2-weighted sequences and avid, diffuse enhancement on contrast-enhanced fat-suppressed T1-weighted sequences. After an ultrasound-guided core needle biopsy, the lesion was successfully treated by arthroscopically-assisted complete excision. Histologically, the tumor was composed of uniform bland spindle cells in a myxoid to collagenous stroma with a prominent vascular network. Immunohistochemically, the spindle cells were diffusely positive for CD163 and CD68 and focally positive for estrogen receptor. Moreover, AHRR-NCOA2 fusion gene was detected by reverse transcription-polymerase chain reaction. There has been no clinical evidence of local recurrence at 1-year follow-up. CONCLUSION: This is the first report of the detection of an AHRR-NCOA2 gene fusion associated with intra-articular AFST. AFST should be included in the extended differential diagnosis of an intra-articular soft-tissue mass, particularly if the mass is vascular.

Improvement of vanishing bile duct syndrome with hyperlipidemia.

A 75-year-old man was admitted to our hospital with acute onset of marked jaundice, elevated liver enzymes, and hyperlipidemia. He had been taking clopidogrel and pemafibrate for 3 months. He tested negative for autoantibodies and hepatitis-causing viruses. Gadoxetate-enhanced magnetic resonance imaging showed diffusely hypointense liver parenchyma in the hepatobiliary phase, with no appreciable excretion of gadoxetate into the biliary system. Histological examination of a liver specimen revealed disappearance of the bile ducts in the portal area and decreased expression of organic transporting polypeptide 1B3 on immunostaining. The patient was diagnosed with drug-induced vanishing bile duct syndrome and treated with ursodeoxycholic acid. The signs of liver dysfunction shown on blood chemistry tests improved spontaneously. After the acute hepatitis and lipid abnormalities had improved, repeat liver biopsy and gadoxetate-enhanced magnetic resonance imaging revealed improvement of the vanishing bile duct syndrome and recovery of the expression of organic transporting polypeptide 1B3. The reduction of OATP1B3 expression might be involved in the development of vanishing bile duct syndrome.

Challenges and limitation of MTAP immunohistochemistry in diagnosing desmoplastic mesothelioma/sarcomatoid pleural mesothelioma with desmoplastic features.

AIM: Genomic-based ancillary assays including immunohistochemistry (IHC) for BRCA-1 associated protein-1 (BAP1) and methylthioadenosine phosphorylase (MTAP), and fluorescence in situ hybridization (FISH) for CDKN2A are effective for differentiating pleural mesothelioma (PM) from reactive mesothelial proliferations. We previously reported a combination of MTAP and BAP1 IHC effectively distinguishes sarcomatoid PM from fibrous pleuritis (FP). Nevertheless, cases of sarcomatoid PM with desmoplastic features (desmoPM) are encountered where the IHC assessment is unclear. METHODS AND RESULTS: We evaluated assessment of MTAP IHC, BAP1 IHC, and CDKN2A FISH in 20 desmoPM compared to 24 FP. MTAP and BAP1 IHC could not be assessed in 11 (55 %) and 10 (50 %) cases, respectively, due to loss or faint immunoreactivity of internal positive control cells, while CDKN2A FISH could be evaluated in all cases. The sensitivities for MTAP loss, BAP1 loss, and CDKN2A homozygous deletion in desmoPM were 40 %, 10 %, and 100 %. A combination of MTAP loss and BAP1 loss yielded 45 % of sensitivity. CONCLUSIONS: MTAP IHC is a useful surrogate diagnostic marker in differentiating ordinary sarcomatoid PM from FP, but its effectiveness is limited in desmoPM. CDKN2A FISH is the most effective diagnostic assays with 100 % sensitivity and specificity in discriminating desmoPM from FP in the facilities where the FISH assay is available.

Phase II study of S-1 plus cisplatin with concurrent radiotherapy for locally advanced thymic carcinoma: Results of the LOGIK1605/JART-1501 study.

BACKGROUND: Combination chemotherapy is used to treat advanced thymic carcinoma; however, the effects are insufficient. METHODS: Previously untreated patients with unresectable locally advanced thymic carcinoma received two cycles of 80 mg/m2 /day S-1 orally on days 1-14 plus 60 mg/m2 /day cisplatin intravenously on day 1, and concurrent radiotherapy (60 Gy). RESULTS: Three patients were enrolled into the study. Toxicity and survival were assessable in all patients, but the treatment response was only assessable in one patient. The study was terminated because of poor case recruitment. The patients' characteristics were as follows: male/female = 2/1; PS 0/1 = 2/1; median age (range) = 59 (55-72); and stage III/IV = 2/1. The patient in which the treatment response was assessed exhibited SD (response rate: 0%). In both nonevaluable cases, the second course of chemotherapy was judged to be post-protocol treatment because it was delayed by ≥14 days, but a CR and PR were achieved after the end of the study, respectively. G4 leukopenia/neutropenia and G3 febrile neutropenia occurred in one patient each (33%). The median time to tumor progression was 17.6 months, and the 1-, 2-, 3-, and 4-year survival rates were 67, 33, 33, and 33%, respectively. The median overall survival time was not reached, and the 1-, 2-, 3-, and 4-year survival rates were 100, 67, 67, and 67%, respectively. CONCLUSIONS: Although it was difficult to recruit patients, there was a long-term survivor >4 years who appeared to have achieved a CR, indicating that such chemoradiotherapy may be effective against locally advanced thymic carcinoma.

Update of pathological diagnosis of pleural mesothelioma using genomic-based morphological techniques, for both histological and cytological investigations.

As more than 80% of pleural mesothelioma (PM) cases start with pleural effusions, diagnosis with effusion smear cytology or pleural biopsy is important. For diagnosing PM, a three-step approach is used: (1) detecting atypical cells; (2) verifying their mesothelial origin using immunohistochemistry (IHC); and (3) discriminating PM from benign mesothelial proliferations (BMP). The third step is critical for diagnosing early lesions. In small biopsy or cytologic specimens in which tumor cell fat invasion cannot be assessed, genomic-based assays, including IHC-detected BAP1 loss and fluorescence in situ hybridization (FISH)-detected homozygous deletion (HD) of CDKN2A/p16, are effective for differentiation. Both BAP1 IHC and CDKN2A FISH can equally be applied to histologic and cytologic specimens, with 100% specificity in discriminating PM from BMP. We found that methylthioadenosine phosphorylase (MTAP) loss as detected by IHC could serve as a feasible alternative in tissue and cytologic preparations for CDKN2A FISH. However, a combination including FISH was still most effective: the addition of NF2 FISH to CDKN2A FISH and BAP1 IHC yielded a greater sensitivity of close to 100% in diagnosing PM tissues. Although IHC is more feasible than FISH, owing to remaining challenges in data interpretation, caution and familiarity are warranted when diagnosing PM.

A case of pituitary adenoma with infiltration into the sphenoid sinus accompanied by melanocyte proliferation.

A 71-year-old woman presenting with headache and nausea was admitted to hospital. Magnetic resonance imaging revealed a tumorous lesion that surrounded the sella turcica and infiltrated the sphenoid sinus with bone destruction. The tumor was removed by nasal endoscopy. The histology was consistent with pituitary adenoma; immunohistochemistry indicated silent corticotroph adenoma with melanocyte proliferation. The possibility that melanocytes were incorporated into the tumor mass in the sphenoid sinus and underwent proliferation was evaluated by investigating the mechanisms of melanocyte proliferation associated with basic fibroblast growth factor (bFGF) and α melanocyte-stimulating hormone (αMSH). In the normal tissue, the pars intermedia and adrenocorticotropic hormone (ACTH)-producing cells were positive for αMSH. None of the control adenoma tissues were positive for bFGF or αMSH by immunostaining. In the present case, bFGF-positive cells and αMSHpositive cells were observed, suggesting that both may have been involved in melanocyte proliferation. The expression of bFGF has been linked to aggressive disease. Pituitary adenoma with melanocyte proliferation has not been previously reported. Careful follow-up is deemed necessary in the future.

Combination of DYRK2 and TERT Expression Is a Powerful Predictive Marker for Early-stage Breast Cancer Recurrence.

BACKGROUND/AIM: DYRK2 is a dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase induces degradation of telomerase reverse transcriptase (TERT). The expression of both proteins in breast cancer were investigated as predictors of recurrence. PATIENTS AND METHODS: Two hundred and twenty-one patients with early breast cancer treated at our institute between 2000 and 2009, were included. We used immunohistochemical analyses to measure the expression of DYRK2 and TERT and correlated it with clinicopathological factors and survival. RESULTS: DYRK2 and TERT were positive in 58 (26%) and 86 (39%) of 221 patients, respectively. There was no correlation between DYRK2 and TERT expression and clinicopathological factors. Better disease-free survival was observed in the DYRK2-positive group (p=0.032), and poorer disease-free survival was noted in the TERT-positive group (p=0.023). The DYRK2-positive TERT-negative group exhibited significantly better disease-free survival than the other groups (p=0.006). CONCLUSION: The combination of DYRK2 and TERT may be a powerful tool to stratify breast cancer patients.

Angiofibroma of soft tissue: Current status of pathology and genetics.

Angiofibroma of soft tissue (AFST) is a new soft tissue tumor entity described in the 2020 World Health Organization Classification of Soft Tissue and Bone Tumors. It most often arises in the lower extremities of middle-aged adults and pursues a benign clinical course with a low rate of non-destructive local recurrence. Histologically, the lesion consists of uniform bland spindle cells in a fibromyxoid stroma with a prominent vascular network. The vascular component forms a complex arrangement of small, thin-walled branching blood vessels. By immunohistochemistry, AFST is variably positive for epithelial membrane antigen, desmin, smooth muscle actin, CD34, CD68, CD163 and estrogen receptor. The exact etiology of AFST remains unknown, but it appears genetically distinct, with a balanced t(5;8)(p15;q13) translocation resulting in a fusion of aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2). Knowledge of this recently described entity is important because it can mimic a variety of intermediate and malignant soft tissue tumors, including solitary fibrous tumor, low-grade fibromyxoid sarcoma, myxoid liposarcoma and low-grade myxofibrosarcoma. We review AFST, with an emphasis on the diagnostic spectrum, recent molecular genetic features and the differential diagnosis.

Functional Prognosis Following Temporal Bone Chondroblastoma Resection: A Systematic Review and Case Illustration.

BACKGROUND: Chondroblastomas are rare tumors that account for <1% of all bone tumors, and 5.7% of them occur in the skull. The aim of this study was to investigate factors related to their functional prognosis by conducting a systematic review, including our own case. METHODS: A systematic review was conducted of case reports that clearly stated postoperative symptoms in temporal chondroblastomas. Tumor localization was limited to cases of the temporal bone. Cases not described in English were excluded. RESULTS: We obtained 30 articles comprising 44 cases and included our own case for a total of 45 cases. Postoperative asymptomatic cases accounted for 53.3% (24/45), and symptomatic cases accounted for 46.7% (21/45). Complications were observed in 31.1% (14/45) of cases. The main complications were facial palsy (9 cases), occlusal disorders (4 cases), and hearing loss (4 cases). The occurrence of facial palsy as a complication was considered likely. Tumor size was confirmed in 36 cases. Cases with postoperative complications were more likely to involve tumors ≥5 cm in size (77.8%, 7/9) compared with cases without complications (40.7%, 11/27). There was a significant association between rate of postoperative complications and tumor size (P = 0.061). CONCLUSIONS: As temporal chondroblastoma poses a risk of residual postoperative symptoms when the tumor grows, aggressive surgical treatment should be considered even in asymptomatic or small tumors.

Eosinophilic Granuloma of the Liver Mimicking Metastatic Liver Tumor.

We herein report a case of coagulation necrosis with granulation and eosinophilic infiltration of the liver. A 37-year-old woman was diagnosed with a new mass lesion in the liver 1 month after breast cancer surgery and admitted for a further examination. Because the tumor occurred immediately after surgery, it was considered essential to determine whether or not it was a metastatic liver tumor from breast cancer. A percutaneous liver tumor biopsy revealed eosinophilic granuloma of the liver, which is considered to have a high possibility of visceral larva migrans with suspected gnathostomiasis infection. A detailed medical history and histological diagnosis are important for making a differential diagnosis.

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma.

Homozygous deletion (homo-d) of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene is frequently found in malignant pleural mesothelioma (MPM). Fluorescence in situ hybridization (FISH) is commonly used to detect chromosomal deletion, and sometimes reveals more frequent heterozygous deletion (hetero-d) compared with homo-d. In clinical practice, such CDKN2A FISH results belong to the 'borderline' homo-d rate, which makes it difficult to definitively diagnose MPM. Microdeletion, [<200 kilobase (kb)], can induce a 'pseudo' hetero-d signal in FISH assays with long probes owing to redundant probe reactivity. Thus, the present study hypothesized that shorter FISH probes can effectively detect the small deletion status of the CDKN2A gene and increase homo-d rate in MPM, which has high hetero-d and low homo-d status. The present study aimed to evaluate the effectiveness of a shorter CDKN2A FISH probe in diagnosing MPM. CDKN2A FISH with either a 222 kb long probe (L-probe) or a 57 kb short probe (S-probe) was performed in four MPM cases with high hetero-d and low homo-d patterns. Furthermore, immunohistochemistry for methylthioadenosine phosphorylase (MTAP) and quantitative (q)PCR analyses were performed to confirm the microdeletion of the 9p21 locus. The results demonstrated that all four MPM cases retained MTAP protein expression. CDKN2A FISH with L-probe revealed high hetero-d (cases 1-4; 73.3, 37.1, 59.2 and 64.8%, respectively) and low homo-d (cases 1-4; 12.1, 12.4, 25.4 and 22.2%, respectively). CDKN2A FISH with S-probe revealed high homo-d (cases 1-4; 96.8, 90.0, 87.5 and 82.6%, respectively), with low hetero-d (cases 1-4; 0.0, 1.2, 1.2 and 4.3%, respectively). qPCR analysis demonstrated no allele deletions of the MTAP gene and two-allele deletions of the CDKN2A gene in 3/4 cases. Taken together, these results suggest that the S-probe detects the short homo-d of the 9p21 locus more effectively than the L-probe in MPM. This can assist in solving diagnostic difficulties in cases involving high hetero-d with low homo-d.