Assessment of clinical relevance of antigen improves diagnostic accuracy of hypersensitivity pneumonitis.

BACKGROUND: Exposure assessment is integral to the diagnosis of hypersensitivity pneumonitis (HP). Although the clinical relevance of exposed antigens is essential for the assessment, many of the previous guidelines or reports have only evaluated simple exposure histories or immunological tests. To overcome this problem, the Exposure Assessment Form (EAF) was developed as an assessment tool for classifying the exposure grade from G0 to G4. The EAF was modified from the description in the Japanese clinical practice guide 2022 for HP published by the Japanese Respiratory Society. METHODS: One hundred and seventy-two consecutive patients with interstitial lung disease who underwent multidisciplinary discussion (MDD) at our hospital were retrospectively examined. We assessed whether the use of the EAF improved the diagnostic performance of the international guideline of HP. We also evaluated whether the exposure grade affected the prognosis of HP. RESULTS: Even when a HP diagnosis was made with a confidence of 70% or higher according to the international guideline, less than half of these cases resulted in a final diagnosis of HP when the exposure grades were lower than G3. When the result of the EAF was integrated into the exposure definition of the international guideline, the specificity of the diagnostic performance improved, while sensitivity was maintained. Furthermore, HP patients with an exposure grade of G3 or higher showed a tendency to take a longer time to initiate medication. CONCLUSIONS: This is the first study to evaluate the clinical relevance of possible antigens using the EAF. Assessing the exposure grade prevents overdiagnosis and improves the diagnostic performance of the international guideline.

A Case of Solitary Pure Ground-glass Opacity Suspected of Being the Initial Presentation of Nonfibrotic Hypersensitivity Pneumonitis.

We herein report a rare case of hypersensitivity pneumonitis (HP) that was initially demonstrated as solitary pure ground-glass opacity (GGO) on chest computed tomography (CT). A 51-year-old woman with a history of breast cancer underwent follow-up CT, which revealed solitary pure GGO. The patient developed exertional dyspnea after two years, and CT revealed diffuse centrilobular nodules in addition to GGO, which had increased in size. An antigen avoidance test was performed to diagnose HP, leading to the resolution of CT abnormalities, including the GGO. Our findings suggested that nonfibrotic HP can present as solitary pure GGO.

Clinical Response of Primary Malignant Pericardial Mesothelioma with Peritoneal Dissemination to Nivolumab.

Malignant pericardial mesothelioma (MPM) is extremely rare, and peritoneal dissemination has not yet been reported. There is no consensus regarding appropriate pharmacological treatment for MPM, including immune checkpoint inhibitors (ICIs). We herein report a 36-year-old man with MPM diagnosed by peritoneal dissemination and treated with an ICI. Cytology of the ascites revealed malignant peritonitis, and a re-evaluation of a pericardial biopsy performed at the previous hospital led to a diagnosis of MPM. The patient was treated with nivolumab and showed a clinical response despite several complications, such as renal dysfunction and performance status deterioration. This case report provides suggestive information for the diagnosis and ICI therapy of a rare type of mesothelioma.

Case Report: The leopard sign as a potential characteristic of chronic granulomatous disease-associated colitis, unrelated to colitis severity.

Background: Chronic granulomatous disease (CGD) is an inborn immune disorder in which the phagocytic system cannot eradicate pathogens, and autoinflammation occurs. Approximately half of the patients have associated gastrointestinal symptoms. Although most cases with CGD-associated colitis present nonspecific histology, colonoscopy in some cases shows brownish dots over a yellowish oedematous mucosa, which is termed a "leopard sign". However, the significance of these signs remains unclear. Methods: We collected data from patients with CGD whose colonoscopic findings showed the leopard sign. Results: Three patients with CGD and leopard signs were enrolled in this study. One patient underwent colonoscopy for frequent diarrhoea and weight gain failure, and another for anal fistula. The third patient was without gastrointestinal symptoms and underwent colonoscopy as a screening test before allogeneic haematopoietic cell transplantation (HCT). Endoscopic findings showed a mild leopard sign in the first case; however, non-contiguous and diffuse aphthae were observed throughout the colon. The other two cases were unremarkable except for the leopard sign. All the patients achieved remission with oral prednisolone or HCT. One patient underwent colonoscopy after HCT; results revealed improvements in endoscopy (including the leopard sign) and histological findings. However, another patient underwent colonoscopy after prednisolone treatment; this revealed no change in the leopard sign. Conclusion: The leopard sign in the colon may be a characteristic endoscopic finding of CGD, even in patients who do not develop severe gastrointestinal symptoms; however, it does not reflect the severity of CGD-associated colitis.

High density and proximity of CD8+ T cells to tumor cells are correlated with better response to nivolumab treatment in metastatic pleural mesothelioma.

BACKGROUND: The efficacy of immune checkpoint inhibitors (ICIs) in pleural mesothelioma has recently been established. The response to ICIs can be predicted by quantitative analysis of cells and their spatial distribution in the tumor microenvironment (TME). However, the detailed composition of the TME in pleural mesothelioma has not been reported. We evaluated the association between the TME and response to ICIs in this cancer. METHODS: A retrospective analysis of 22 pleural mesothelioma patients treated with nivolumab in different centers was performed using surgical specimens. Four patients had a partial response to nivolumab (response group) and 18 patients had stable or progressive disease (nonresponse group). The number of CD4, CD8, FoxP3, CK, and PD-L1 positive cells, cell density, and cell-to-cell distance were analyzed by multiplex immunofluorescence. RESULTS: PD-L1 expression did not differ significantly between the response and nonresponse groups. The density of total T cells and of CD8+ T cells was significantly higher in the response than in the nonresponse group. CD8+ T cells were more clustered and located closer to tumor cells, whereas regulatory T cells were located further from tumor cells in the response than in the nonresponse group. CONCLUSIONS: High density and spatial proximity of CD8+ T cells to tumor cells were associated with better response to nivolumab, whereas the proximity of regulatory T cells to tumor cells was associated with worse response, suggesting that the distinct landscape of the TME could be a potential predictor of ICI efficacy in pleural mesothelioma.

[Malignant paraganglioma mimicking multiple myeloma].

We here present a 33-year-old woman who was referred to our hospital with a complaint of back pain and was found to have elevated IgG and hypercalcemia, as well as osteolytic lesions of pelvis and spines. 18F-FDG-PET/CT scan revealed numerous uptakes in the bones. An examination of the bone marrow revealed increased plasma cells (10.2%). Despite clinical similarities to multiple myeloma, any evidence of plasma cell clonal proliferation, including serum M-protein and light chain restriction, was not found. A reexamination of the bone marrow with a biopsy revealed the proliferation of abnormal cells with chromogranin A and synaptophysin expression but no expression of hematopoietic and epithelial cell markers. Based on these results together with extra-adrenal lesions, a diagnosis of malignant paraganglioma was made. Malignant paraganglioma is known to frequently cause bone metastasis and skeletal related events, whose clinical manifestations are similar to those of multiple myeloma. Since patients with osteolytic lesions, hypercalcemia, and hypergammaglobulinemia are likely to be referred to hematologists, malignant paraganglioma should be considered as a differential diagnosis.

PHOX2B is a Sensitive and Specific Marker for the Histopathological Diagnosis of Pheochromocytoma and Paraganglioma.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are non-epithelial neuroendocrine neoplasms originating from the adrenal medulla and paraganglion of the sympathetic and parasympathetic nervous system, respectively. PCCs and PGLs show histological similarities with other epithelial neuroendocrine neoplasms and olfactory neuroblastomas (ONBs), and the differential diagnosis of PGLs is particularly difficult. Therefore, we compared the sensitivity of PHOX2A, PHOX2B, and tyrosine hydroxylase (TH) in the histopathological diagnosis of PCCs and PGLs immunohistochemically using the tissue microarrays of 297 neoplasms including PCCs, PGLs, neuroblastomas, ganglioneuromas, epithelial neuroendocrine neoplasms, and ONBs. Using cutoff values of 25%, 5%, and 5% of tumor cells expressing PHOX2A, PHOX2B, and TH, respectively, as positive, 40 of 51 PCCs, 32 of 33 parasympathetic/head and neck PGLs (HNPGLs), 17 of 19 sympathetic/thoracoabdominal PGLs (TAPGLs), and 12 of 152 epithelial neuroendocrine neoplasms, including 123 well-differentiated and 29 poorly differentiated neuroendocrine neoplasms, were PHOX2A-positive. All 51 PCCs, 33 HNPGLs, and 19 TAPGLs were PHOX2B-positive, while all 152 epithelial neuroendocrine neoplasms were PHOX2B-negative. Moreover, 50 of 51 PCCs, 13 of 33 HNPGLs, all TAPGLs, and 12 of 152 epithelial neuroendocrine neoplasms were TH-positive. All ONBs were negative for PHOX2A, PHOX2B, and TH. PHOX2B was the most sensitive and specific diagnostic marker for PCCs and PGLs among PHOX2A, PHOX2B, and TH. PHOX2B can facilitate identification of PCCs and PGLs from epithelial neuroendocrine neoplasms and ONBs, especially in the case of HNPGLs, in which TH is often negative.

Overview of Ferroptosis and Synthetic Lethality Strategies.

Ferroptosis, a term first proposed in 2012, is iron-dependent, non-apoptotic regulatory cell death induced by erastin. Ferroptosis was originally discovered during synthetic lethal screening for drugs sensitive to RAS mutant cells, and is closely related to synthetic lethality. Ferroptosis sensitizes cancer stem cells and tumors that undergo epithelial-mesenchymal transition and are resistant to anticancer drugs or targeted therapy. Therefore, ferroptosis-inducing molecules are attractive new research targets. In contrast, synthetic lethal strategies approach mechanisms and genetic abnormalities that cannot be directly targeted by conventional therapeutic strategies, such as RAS mutations, hypoxia, and abnormalities in the metabolic environment. They also target the environment and conditions specific to malignant cells, have a low toxicity to normal cells, and can be used in combination with known drugs to produce new ones. However, the concept of synthetic lethality has not been widely adopted with ferroptosis. In this review, we surveyed the literature on ferroptosis-related factors and synthetic lethality to examine the potential therapeutic targets in ferroptosis-related molecules, focusing on factors related to synthetic lethality, discovery methods, clinical application stages, and issues in drug discovery.

SMARCA4-deficient thoracic tumor detected by [18F]FDG PET/CT.

BACKGROUND: SMARCA4-deficient thoracic tumor (SMARCA4-DTT) is a distinct entity of undifferentiated thoracic malignancies newly introduced in 2015. Due to its unique clinical characteristic with aggressive thoracic tumor mostly observed in heavy smoker man with emphysema, with poor prognosis, many physicians are becoming increasingly aware of the disease; however, reports on 2-deoxy-2-[18F] fluoroglucose positron emission tomography/computed tomography ([18F]FDG PET/CT) have been limited; thus, this disease is not yet widely known to nuclear medicine clinicians. As a first step in discussing the usefulness of [18F]FDG PET/CT for this disease, we present a case in which [18F]FDG PET/CT played a clinically important role. CASE: A 74-year-old heavy smoker man with an anamnesis of severe emphysema characterized by pleural thickening and abnormal enhancement in CT underwent 18F-FDG PET/CT for further examination. [18F]FDG-avid pleural nodules infiltrating into the chest wall were detected and pathologically diagnosed as SMARCA4-DTT with biopsy. CONCLUSION: SMARCA4-deficient thoracic tumor should be considered in a [18F]FDG-avid aggressive thoracic tumor in heavy smoker men with emphysema.

A case of vestibular schwannoma mimicking burning mouth syndrome.

BACKGROUND: An oral burning sensation with unidentified cause in patients with preexisting psychosocial conditions is usually diagnosed as burning mouth syndrome. However, unexpected organic lesions may be detected in rare cases. CASE PRESENTATION: A 35-year-old woman had chief complaints of a burning sensation and numbness of the right side of the lip and tongue, as well as a dry sensation of the mouth with a taste disturbance of the right side of the tongue. The symptoms were continuous and did not show any daily fluctuations. The symptoms started without any recognizable triggering factor six months before her first visit to our clinic,. No abnormality was detected in her mouth. MRI images revealed an approximately 30 × 30 mm well-defined mass localized in the right cerebropontine angle compressing the trigeminal nerve, which was diagnosed as schwannoma of the right auditory nerve. CONCLUSIONS: It is important for clinicians to consider the possibility of brain tumors in their differential diagnosis of BMS. Although it is not always easy to eliminate all diseases that may cause an oral burning sensation in patients with BMS-like symptoms, more attention and careful examination based on the patient's psychosomatic background features and other possible causes are needed to rule out organic diseases.

Schlafen 11 Is a Novel Target for Mucosal Regeneration in Ulcerative Colitis.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with an intractable course. Although the goal of UC therapy is to achieve mucosal healing, the pathogenesis of mucosal injury caused by chronic inflammation remains unknown. We therefore aim to elucidate molecular mechanisms of mucosal injury by establishing in vitro and in vivo humanised UC-mimicking models. METHODS: An in vitro model using human colon organoids was established by 60 weeks of inflammatory stimulation. The key gene for mucosal injury caused by long-term inflammation was identified by microarray analysis. An in vivo model was established by xenotransplantation of organoids into mouse colonic mucosa. RESULTS: An in vitro model demonstrated that long-term inflammation induced irrecoverable changes in organoids: inflammatory response and apoptosis with oxidative stress and suppression of cell viability. This model also mimicked organoids derived from patients with UC at the gene expression and phenotype levels. Microarray analysis revealed Schlafen11 [SLFN11] was irreversibly induced by long-term inflammation. Consistently, SLFN11 was highly expressed in UC mucosa but absent in normal mucosa. The knockdown of SLFN11 [SLFN11-KD] suppressed apoptosis of intestinal epithelial cells [IECs] induced by inflammation. Moreover, SLFN11-KD improved the take rates of xenotransplantation and induced the regenerative changes of crypts observed in patients with UC in remission. CONCLUSIONS: In vitro and in vivo UC-mimicking models were uniquely established using human colonic organoids. They revealed that SLFN11 is significant for mucosal injury in UC, and demonstrated its potential as a novel target for mucosal regeneration.

Long-term survival after radical cystectomy and mesenteric lymph node dissection for squamous cell carcinoma arising from augmented bladder with lymph node metastasis: a case report.

Surgical resection is commonly performed for augmented bladder cancer, yet an optimal treatment strategy for augmented bladder cancer with lymph node metastasis has not been established. Here, we report a case that achieved 7 years of survival after radical cystectomy and mesenteric lymph node dissection for squamous cell carcinoma arising from augmented bladder with lymph node metastasis. Extended surgery could be a useful treatment option for locally advanced augmented bladder cancer including mesenteric lymph node metastasis.

Adenocarcinoma arising in the multiple heterotopic submucosal glands of the intestine in a Satoyoshi syndrome patient: A case report.

Satoyoshi syndrome is a rare multisystemic disorder of unknown etiology characterized by progressive muscle spasms, alopecia and diarrhea. Multiple protruding lesions with cystic glands, namely gastroenterocolitis cystica polyposa, manifest in the gastrointestinal tract. Since the first report of these lesions in 1977, which was unique to Satoyoshi syndrome, few studies have focused on their role, and the associated clinicopathological features are not well understood. Here, we report a 64-year-old Japanese woman with Satoyoshi syndrome who presented with multiple polypoid lesions in the stomach, duodenum, jejunum, ileum and colon. Histologically, the polypoid lesions in the intestine comprised multiple heterotopic submucosal glands containing cystically dilated glands and smooth muscle fibers in the lamina propria mucosa and/or submucosa. Additionally, we observed stromal changes, such as fibrosis, discontinuous and thinning muscularis mucosae, and diffuse neural fiber proliferation in the entire intestinal tract. Furthermore, multiple foci of adenocarcinomas were identified within several heterotopic submucosal glands. We hypothesized that multiple heterotopic submucosal glands in the present case corresponded to previously reported gastroenterocolitis cystica polyposa, suggesting that these lesions are essential in the histopathology and are a unique manifestation of Satoyoshi syndrome.

Intestinal phenotype is maintained by Atoh1 in the cancer region of intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasm (IPMN) is a precancerous lesion of pancreatic cancer. Although there are 4 types of IPMN, among which intestinal-type IPMN is likely to progress into invasive cancer known as colloid carcinoma, no information regarding the involvement of the intestinal phenotype in the carcinogenesis of IPMN exists. The present study was conducted to explore how the intestinal differentiation system is maintained during the tumor progression of intestinal-type IPMN using surgical resection specimens. Results showed that Atoh1, a critical transcriptional factor for intestinal differentiation toward the secretory lineages of intestinal epithelial cells, was expressed in an invasive-grade IPMN. To determine the function of Atoh1 in pancreatic cancer, we generated a pancreatic ductal adenocarcinoma (PDAC) cell line overexpressing Atoh1. In a xenograft model, we successfully induced an IPMN phenotype in PDAC cells via Atoh1 induction. Finally, for the first time, we discovered that GPA33 is expressed in intestinal-type IPMN, thereby suggesting a novel target for cancer therapy. In conclusion, the intestinal differentiation system might be maintained during tumor progression of intestinal-type IPMN. Further analysis of the function of Atoh1 in IPMN might be useful for understanding the molecular mechanism underlying the malignant potential during the tumor progression of IPMN.

Frequent expression of conventional endothelial markers in pleural mesothelioma: usefulness of claudin-5 as well as combined traditional markers to distinguish mesothelioma from angiosarcoma.

OBJECTIVES: Distinguishing pleural sarcomatoid mesotheliomas from true sarcomas is challenging because the former does not always express the mesothelial markers, and diagnosis is often made on the basis of keratin expression. Consequently, sarcomas such as angiosarcomas that express keratin complicate the differential diagnosis. Furthermore, some mesotheliomas have been reported to express endothelial markers. The aim of this study is to identify useful markers for distinguishing pleural sarcomatoid mesothelioma from angiosarcoma. MATERIALS AND METHODS: This study enrolled 147 patients with pleural mesothelioma-93 with epithelioid, 25 with biphasic, and 29 with sarcomatoid subtypes-and 41 patients with angiosarcomas in various organs. The expression levels of cytokeratin, mesothelial, and endothelial markers were assayed in both groups to identify the markers that could assist in distinguishing mesothelioma from angiosarcoma. Cytokeratin (AE1/AE3, CAM 5.2), endothelial (CD31, CD34, ERG, factor VIII, and claudin-5), and mesothelial (calretinin, WT-1, podoplanin (D2-40), EMA, and CK5/6) markers were immunohistochemically assayed using tissue blocks. RESULTS: More than 90% of the mesotheliomas and less than 20% of the angiosarcomas expressed cytokeratin. Calretinin was expressed in 82% of all types of mesotheliomas but in only 48% of sarcomatoid mesotheliomas. Endothelial markers were expressed in mesothelioma tissues-CD31 in 10.3%, CD34 in 3.5%, ERG in 29%, and factor VIII in 3.4%-and the positivity was higher in sarcomatoid than in epithelioid and biphasic mesotheliomas. Claudin-5 was expressed in all the angiosarcomas, but not in any of the mesotheliomas. CONCLUSION: We found overlapping immunophenotypes in pleural mesotheliomas and angiosarcomas, but the sensitivity and specificity of claudin-5 expression were sufficient to distinguish between them. The differential diagnosis of mesothelioma should therefore include claudin-5 in a panel of immunohistochemical markers to distinguish mesothelioma from angiosarcoma.

Engrailed Homeobox 1 and Cytokeratin 19 Are Independent Diagnostic Markers of Eccrine Porocarcinoma and Distinguish It From Squamous Cell Carcinoma.

OBJECTIVES: The diagnostic utility of En1 in the histopathologic differentiation of eccrine porocarcinoma (EPC) from invasive squamous cell carcinoma (SCC) was investigated. METHODS: Expression of En1 and CK19 in 16 cases of EPC was immunohistochemically examined and compared with that in 32 cases of SCC. RESULTS: In all 16 EPCs, En1 was expressed in 3% to 100% of tumor cells. In 20 of the 32 SCCs, En1 was expressed in 3% to 90% of tumor cells. A total of 13 of the 16 EPCs and five of the 32 SCCs were judged as En1 positive, with a cutoff value of 25%. In addition, 11 of the 16 EPCs and four of the 32 SCCs were CK19 positive. The frequencies of En1- and CK19-positive cases were significantly higher in EPCs than in SCCs. In a logistic regression analysis for predicting EPC, En1 and CK19 were independent markers. When expression patterns of En1 and CK19 were combined, none of the 32 SCCs was both positive. In contrast, 15 of the 16 EPCs were positive for either En1 or CK19. CONCLUSIONS: A combination of En1 and CK19 expression can improve the accuracy of histologic diagnosis of EPC.

[A Case Report of P0CY1 Gastric Cancer Achieved Long-Term Survival after Treated with Ramucirumab Monotherapy].

The patient was a woman in her early 60s with type 4 advanced cancer which spread throughout the entire stomach. Total gastrectomy with regional lymphadenectomy was performed. She was diagnosed as Stage Ⅳ scirrhous gastric cancer with positive lavage cytology pathologically without any macroscopic peritoneal metastasis(P0CY1). S-1 plus cisplatin therapy was carried out as first-line therapy, but must be stopped after 2 courses because of appetite loss. As the second-line, ramucirumab monotherapy was administered, due to the patient's denial of alopecia and numbness as side effects of paclitaxel. Tumor marker value of CA19-9 remained high 24 months after ramucirumab chemotherapy, but gradually decreased near the normal level with no proof of distant metastasis or peritoneal dissemination. However, after 74 courses, CA19-9 value was elevated and peritoneal dissemination was detected from CT scan. Nivolumab therapy was started as third-line, but only for 5 courses because of indefinite complaints. Afterwards, no chemotherapy has been performed as the patient's request until almost 5 years after surgery. The prognosis of patients with P0CY1 gastric cancer is generally poor, but in our case long-term survival was obtained from ramucirumab therapy only. Recently, ramucirumab monotherapy is administered for advanced HCC patients and expect to be effective in AFP producing gastric cancer. There is an urgent need to elucidate potential predictive biomarkers of ramucirumab efficacy.

Pathological evaluation of the visceral pleura in the radical pleurectomy/decortication for malignant pleural mesothelioma patients.

BACKGROUND: Radical pleurectomy/decortication (P/D) is applied as a surgical treatment of resectable malignant pleural mesothelioma (MPM). Although P/D removed visceral pleura, dissection plain was not histologically explored previously. We examined a pathological evaluation of surgically removed visceral pleura in P/D. METHODS: Twenty-five patients with MPM who underwent P/D at the Tokyo Medical and Dental University Hospital between April 2010 and April 2018 were studied. The 25 cases included 20 with epithelioid tumors, 4 with a biphasic tumor and 1 with desmoplastic tumors. Nine, 1, 11 and 4 patients had mesotheliomas of stages I, II, III and IV, respectively. We analyzed the site of the visceral pleural lesions using the HE & Elastica van Gieson (EVG) staining. The tumor involvement of the pleura and the surgical dissection plane were defined using the depth criteria, D0-3. We added survival analyses according to the depth criteria. RESULTS: Ninety-nine lesions in total 45 lobes: 20 upper right, 14 middle, 20 lower, 22 upper left, and 23 lower were examined. Based on the depth D 0-3 criteria, there were 21 type D0, 18 type D1, 22 type D2 and 38 type D3 lesions. The growth of tumor cells in the pleura was partially diffuse or nodular in all cases. While 38 lesions which invaded the lung parenchyma were excised, another 61 lesions that reached within the pleura were dissected from lung parenchyma. Type D2&3 showed poor survivals than type D0&1. CONCLUSIONS: The lung parenchyma was always the dissection plane in P/D, regardless of tumor involvement in the visceral pleura. The depth criteria would help us in classifying pleural invasion histologically and possibly predicting the prognosis.

The quantitative analysis of the human papillomavirus DNA load in submandibular gland lesions with droplet digital polymerase chain reaction.

BACKGROUND: The relationship between infection with human papillomavirus (HPV) and tumorigenesis of salivary gland remains controversial. OBJECTIVES: This study explored the relationship between HPV and salivary gland lesions as well as that of the HPV infection status and p16INK4A immunoreactivity. The HPV DNA loads were also quantitatively evaluated. MATERIALS AND METHODS: Tissue samples from 31 submandibular gland lesions were evaluated. p16INK4A immunohistochemical (IHC) staining, nested polymerase chain reaction (PCR), DNA sequencing, and droplet digital PCR (ddPCR) were performed. RESULTS: Non-neoplastic lesion, benign tumors, and malignant tumors were noted in 9, 16, 6 cases, respectively. p16INK4A immunoreactivity was higher in malignant tumors than in benign tumors (50.0% vs. 6.3%). Single PCR with MY09/11 found that all samples were negative. Nevertheless, nested PCR revealed a high HPV-DNA positivity rate of 96.8%. No relationship between the HPV status and p16INK4A immunoreactivity was shown. HPV-18 was the only subtype identified in this study. ddPCR showed significantly lower HPV-18 DNA loads in submandibular gland lesions than in oropharyngeal cancers. CONCLUSIONS: HPV-DNA positivity and p16INK4A-immunoreactivity were not correlated in submandibular gland lesions. The loads of HPV DNA detected in this study were small. HPV positivity therefore may not be associated with tumorigenesis of the submandibular gland.