Identification of highly sensitive biomarkers that can aid the early detection of pancreatic cancer using GC/MS/MS-based targeted metabolomics.

BACKGROUND: To improve prognosis of pancreatic cancer (PC) patients, the discovery of more reliable biomarkers for the early detection is desired. METHODS: Blood samples were collected by 2 independent groups. The 1st set was included 55 early PC and 58 healthy volunteers (HV), and the 2nd set was included 16 PC and 16HV. The 16 targeted metabolites were quantitatively analyzed by gas chromatography/tandem mass spectrometry together with their corresponding stable isotopes. In the 1st set, the levels of these metabolites were evaluated, and diagnostic models were constructed via multivariate logistic regression analysis, leading to validation using the 2nd set. RESULTS: In the 1st set, model X consisting of 4 candidates based on our previous report possessed higher sensitivity (74.1%) than carbohydrate antigen 19-9 (CA19-9). Model Y, consisting of 2 metabolites newly selected from 16 metabolites via stepwise method possessed higher sensitivity (70.4%) than CA19-9. Furthermore, combining model Y with CA19-9 increased its sensitivity (90.7%) and specificity (89.5%). In the 2nd set, combining model Y with CA19-9 displayed high sensitivity (81.3%) and specificity (93.8%). In particular, it displayed very high sensitivity (100%) for resectable PC. CONCLUSIONS: Quantitative analysis confirmed that metabolomics-based diagnostic methods are useful for detecting PC early.

Identification of IGFBP2 and IGFBP3 As Compensatory Biomarkers for CA19-9 in Early-Stage Pancreatic Cancer Using a Combination of Antibody-Based and LC-MS/MS-Based Proteomics.

Pancreatic cancer is one of the most lethal tumors, and reliable detection of early-stage pancreatic cancer and risk diseases for pancreatic cancer is essential to improve the prognosis. As 260 genes were previously reported to be upregulated in invasive ductal adenocarcinoma of pancreas (IDACP) cells, quantification of the corresponding proteins in plasma might be useful for IDACP diagnosis. Therefore, the purpose of the present study was to identify plasma biomarkers for early detection of IDACP by using two proteomics strategies: antibody-based proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Among the 260 genes, we focused on 130 encoded proteins with known function for which antibodies were available. Twenty-three proteins showed values of the area under the curve (AUC) of more than 0.8 in receiver operating characteristic (ROC) analysis of reverse-phase protein array (RPPA) data of IDACP patients compared with healthy controls, and these proteins were selected as biomarker candidates. We then used our high-throughput selected reaction monitoring or multiple reaction monitoring (SRM/MRM) methodology, together with an automated sample preparation system, micro LC and auto analysis system, to quantify these candidate proteins in plasma from healthy controls and IDACP patients on a large scale. The results revealed that insulin-like growth factor-binding protein (IGFBP)2 and IGFBP3 have the ability to discriminate IDACP patients at an early stage from healthy controls, and IGFBP2 appeared to be increased in risk diseases of pancreatic malignancy, such as intraductal papillary mucinous neoplasms (IPMNs). Furthermore, diagnosis of IDACP using the combination of carbohydrate antigen 19-9 (CA19-9), IGFBP2 and IGFBP3 is significantly more effective than CA19-9 alone. This suggests that IGFBP2 and IGFBP3 may serve as compensatory biomarkers for CA19-9. Early diagnosis with this marker combination may improve the prognosis of IDACP patients.

Plasma biomarker for detection of early stage pancreatic cancer and risk factors for pancreatic malignancy using antibodies for apolipoprotein-AII isoforms.

We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19-9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words).

Altered plasma apolipoprotein modifications in patients with pancreatic cancer: protein characterization and multi-institutional validation.

BACKGROUND: Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection. METHODS: We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system. RESULTS: We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10(-21), P = 4.35×10(-14), and P = 1.83×10(-24) (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany. CONCLUSIONS: We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer.

Carcinoma ex pleomorphic adenoma of the sublingual gland: a case report.

We report a case of carcinoma ex pleomorphic adenoma of a sublingual gland in a 70-year-old man. Under a clinical diagnosis of benign salivary gland tumor, excision of the mass with the sublingual salivary gland in an en bloc fashion via an intraoral approach was performed. Histopathologically, there was a rupture of the fibrous capsule and diffuse cell-rich sheets composed of myoepithelial cells with round nuclei were also seen. Immunohistochemically, the cells that composed of cell rich sheets were positive to smooth muscle actin. Final diagnosis of myoepithelial carcinoma ex pleomorphic adenoma was made.

Nicorandil-induced tongue ulceration with or without fungal infection.

Oral ulceration is one of the common adverse effects of nicorandil in European countries. In Japan, however, only 9 cases of nicorandil-induced oral ulceration have been reported. Here, we report 3 cases of nicorandil-induced oral ulceration, one of which exhibited a unique clinical course associated with Candida infection. In this case, the initial discontinuation of nicorandil failed to ameliorate the lesion. However, the second discontinuation of the drug after the control of the Candida infection overlying the surface of the ulcer produced a favorable effect. This patient was diagnosed with nicorandil-induced tongue ulceration with Candida infection.

Identification of adipophilin as a potential plasma biomarker for colorectal cancer using label-free quantitative mass spectrometry and protein microarray.

BACKGROUND: The aim of this study was to identify a new plasma biomarker for use in early detection of colorectal cancer. METHODS: Using the combination of hollow fiber membrane (HFM)-based low-molecular weight protein enrichment and two-dimensional image converted analysis of liquid chromatography and mass spectrometry (2DICAL), we compared the plasma proteome of 22 colorectal cancer patients with those of 21 healthy controls. An identified biomarker candidate was then validated in two larger cohorts [validation-1 (n = 210) and validation-2 (n = 113)] using a high-density reverse-phase protein microarray. RESULTS: From a total of 53,009 mass peaks, we identified 103 with an area under curve (AUC) value of 0.80 or higher that could distinguish cancer patients from healthy controls. A peak that increased in colorectal cancer patients, with an AUC of 0.81 and P value of 0.0004 (Mann-Whitney U test), was identified as a product of the PLIN2 gene [also known as perilipin-2, adipose differentiation-related protein (ADRP), or adipophilin]. An increase in plasma adipophilin was consistently observed in colorectal cancer patients, including those with stage I or stage II disease (P < 0.0001, Welch's t test). Immunohistochemical analysis revealed that adipophilin is expressed primarily in the basal sides of colorectal cancer cells forming polarized tubular structures, and that it is absent from adjacent normal intestinal mucosae. CONCLUSIONS: Adipophilin is a plasma biomarker potentially useful for the detection of early-stage colorectal cancer. IMPACT: The combination of HFM and 2DICAL enables the comprehensive analysis of plasma proteins and is ideal for use in all biomarker discovery studies.

Plasma biomarker discovery and validation for colorectal cancer by quantitative shotgun mass spectrometry and protein microarray.

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting-edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94,803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex-matched healthy controls using 2D image-converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex-matched healthy controls) using high-density reverse-phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94×10(-4) and 1.43×10(-12) (Student's t-test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage-I and -II diseases (P=3.01×10(-3) and P=1.13×10(-5) , respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer.

Fluorine-18-labeled boronophenylalanine positron emission tomography for oral cancers: Qualitative and quantitative analyses of malignant tumors and normal structures in oral and maxillofacial regions.

The present study aimed to demonstrate the features of fluorine-18-labeled boronophenylalanine positron emission tomography ((18)F-BPA-PET) to reveal oral cancer, as well as normal structures in the oral and maxillofacial regions. We analyzed (18)F-BPA-PET findings from 8 patients with histologically confirmed recurrent and/or advanced oral cancer scheduled for boron neutron capture therapy. The capacity of (18)F-BPA-PET to delineate tumor and normal structures was assessed qualitatively and quantitatively. Tumors were easily identified as high uptake areas in all cases. Although the eyes, which were depicted as a low uptake area, and tongue musculature were readily identified, major vessels were not noted in any of the cases. Areas corresponding to the surface of the dorsum tongue to middle pharynx were expressed as high uptake areas in all of the cases. Quantitatively, tumors were expressed as the highest uptake area in 6 of the 8 cases, while the dorsum tongue had the highest uptake area in the remaining 2 cases. (18)F-BPA-PET is useful in demonstrating the presence of a tumor. Thus, it is crucial to note the presence of a high uptake area corresponding to the dorsum area of the tongue when diagnosing a tumor using this technique.

Reduced plasma level of CXC chemokine ligand 7 in patients with pancreatic cancer.

BACKGROUND: Early detection is essential to improve the outcome of patients with pancreatic cancer. A noninvasive and cost-effective diagnostic test using plasma/serum biomarkers would facilitate the detection of pancreatic cancer at the early stage. METHODS: Using a novel combination of hollow fiber membrane-based low-molecular-weight protein enrichment and LC-MS-based quantitative shotgun proteomics, we compared the plasma proteome between 24 patients with pancreatic cancer and 21 healthy controls (training cohort). An identified biomarker candidate was then subjected to a large blinded independent validation (n = 237, validation cohort) using a high-density reverse-phase protein microarray. RESULTS: Among a total of 53,009 MS peaks, we identified a peptide derived from CXC chemokine ligand 7 (CXCL7) that was significantly reduced in pancreatic cancer patients, showing an area under curve (AUC) value of 0.84 and a P value of 0.00005 (Mann-Whitney U test). Reduction of the CXCL7 protein was consistently observed in pancreatic cancer patients including those with stage I and II disease in the validation cohort (P < 0.0001). The plasma level of CXCL7 was independent from that of CA19-9 (Pearson's r = 0.289), and combination with CXCL7 significantly improved the AUC value of CA19-9 to 0.961 (P = 0.002). CONCLUSIONS: We identified a significant decrease of the plasma CXCL7 level in patients with pancreatic cancer, and combination of CA19-9 with CXCL7 improved the discriminatory power of the former for pancreatic cancer. IMPACT: The present findings may provide a new diagnostic option for pancreatic cancer and facilitate early detection of the disease.

Glossodynia from Candida-associated lesions, burning mouth syndrome, or mixed causes.

OBJECTIVE: Candida-associated lesions (CALs) and burning mouth syndrome (BMS) may induce glossodynia without objective manifestations. We investigated patients with glossodynia to examine the relationship between CAL and BMS. PATIENTS AND METHODS: A visual analog scale was used to divide 95 patients with glossodynia into three groups according to intensity of pain at rest and when eating. Group A was the functional pain group; group B was the nonfunctional pain group; and group C was a mixed pain group. Antifungal treatment was scheduled for patients with suspected Candida infection by clinical, mycological, or cytological criteria. RESULTS: Culture tests and direct examination results indicated that group A had high Candida positivity (73.0% by culture and 59.5% by direct examination), and showed a good response to antifungal treatment (75.7%). Antifungal treatment was not useful in group B. This was supported by a low Candida infection rate, as determined by direct examination (3.1%). For group C, Candida positivity and antifungal treatment effectiveness were between groups A and B. Furthermore, six patients in group C showed complete remission of functional pain by antifungal treatment only. Favorable outcomes were obtained for 23 patients (10 in group B and 13 in group C), who received antidepressant treatment. CONCLUSION: These results suggested that glossodynia was Candida-associated in group A, and BMS-induced in group B, while group C contained patients with both CAL and BMS.

Prolyl 4-hydroxylation of alpha-fibrinogen: a novel protein modification revealed by plasma proteomics.

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed "Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)," is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of alpha-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated alpha-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated alpha-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated alpha-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.

Usefulness of culture test and direct examination for the diagnosis of oral atrophic candidiasis.

BACKGROUND: Culture test and direct microscopy, which are currently used in the diagnosis of oral candidiasis, can yield false-negative results. METHODS: Forty patients with atrophic candidiasis of the tongue were evaluated. The diagnosis was confirmed by a favorable outcome consisting of tongue pain improvement and regeneration of filiform papilla after antifungal treatment in all patients. Specimens were examined by fungal culture and direct microscopy following rapid staining; the usefulness of these procedures for diagnosis was reevaluated retrospectively after treatment. RESULTS: In the culture test, 30 patients (75.0%) were positive for candidal species, most of which were confirmed to be Candida albicans. Twenty-three (57.5%) were positive for pseudohyphae of fungi on direct examination. Twenty-two (55.0%) were positive and nine (22.5%) were negative for both. With regard to the diagnosis of oral atrophic candidiasis, these examinations revealed false-negative results of 25% in the culture examination and 42.5% in the direct examination. CONCLUSION: Careful clinical observation of the patient for signs, such as prolonged disease duration, pain on eating, and no benefit from topical steroid treatment, and cytologic examination are important in the diagnosis of this disease.

Epidemiological study of malignant tumors in the oral and maxillofacial region: survey of member institutions of the Japanese Society of Oral and Maxillofacial Surgeons, 2002.

We studied 1809 patients with oral cancer who visited and were treated, in 2002, at the 148 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons. Of these institutions, 39 are dental university hospitals, 44 are medical university hospitals, 64 are general hospitals, and for 1 institution, the classification was not known. The patients consisted of 1071 (59.2%) males and 738 (40.8%) females (male: female ratio, 1.45:1), who had a average age of 65.2 years. The tongue (40.2%) was the most common site affected, followed by the gingiva (32.7%), buccal mucosa (10.1%), and oral floor (9.0%). There were 6 cases of multiple intraoral cancers. On histopathological examinations, squamous cell carcinoma (88.7%) was the most common type found, followed by adenoid cystic carcinoma (2.1%), and mucoepidermoid carcinoma (1.7%). Cases classified as T2N0 were the most common (32.1%), followed by T1N0 (21.4%), T4N0 (8.0%), and T2N1 (7.6%). Distant metastasis occurred in 17 patients (1.0%). Nonepithelial tumors, among which malignant melanoma was the most common type, accounted for 1.8% of the tumors. The sizes of the nonepithelial malignant tumors ranged from 1.0 to 7.0 cm, with an average size of 3.7 cm.

Boron neuron capture therapy using epithermal neutrons for recurrent cancer in the oral cavity and cervical lymph node metastasis.

The purpose of this clinical trial was to evaluate the utility of boron neutron capture therapy (BNCT) using epithermal neutrons for cases of recurrent cancer in the oral cavity, which are not indicated for a conventional treatment modality. We enrolled four patients with local recurrence or metastasis to the regional lymph nodes after completion of initial treatments, including surgery, chemotherapy and radiotherapy. Before receiving BNCT, patients underwent 18F-p-bononophenylalanine (BPA) positron emission tomography (PET) examinations to assess the BPA accumulation ratios in tumors and normal tissues. All patients showed at least a tentative partial response, while a marked improvement in quality of life was seen in one patient. Before BNCT, that patient could not be discharged from the hospital because of eating difficulties and malaise; after treatment, he was comfortably discharged. Mild malaise, oral mucositis and alopecia were seen as mild adverse effects; however, no life-threatening systemic symptoms were observed in any of the cases. Our results suggested that BNCT is a useful treatment modality for recurrent or regionally metastasized oral cancer.

Relationships between dynamic contrast-enhanced MRI findings and pattern of invasion for tongue carcinoma.

The purpose of this study was to evaluate the effectiveness of dynamic contrast-enhanced MRI (DC-MRI) for assessing the pattern of invasion of tongue carcinomas. We studied 20 cases of squamous cell carcinoma of the tongue that showed peripheral enhancement patterns on DC-MRI. The diameter of each tumor was >2.0 cm and no apparent artifacts were seen. The signal enhancement to noise ratio (SE/N) of the regions of interest, which were located in the central and peripheral regions of the tumor, were measured using DC-MRI, while maximum SE/N, %-wash out, and the ascending rate of SE/N were also calculated. The histopathological pattern of invasion was assessed in each case and used to classify them into clear (13 cases) and diffuse (7 cases) groups, after which the 4 parameters were compared between the 2 groups. The ascending rate of SE/N in the peripheral region was significantly lower in the diffuse group (p=0.047). There were no other significant differences between the 2 groups for any parameter in either tumor region. These results suggest that DC-MRI is able to show the histopathological pattern of invasion into surrounding structures.

Atrophic tongue associated with Candida.

BACKGROUND: Traditionally, total atrophic tongue has been due to nutritional deficiencies, such as vitamin B12, folic acid, or iron deficiencies, and partial atrophic tongue has been well known as median rhomboid glossitis or geographic tongue. The other cause of atrophic tongue is oral candidiasis. METHODS: Forty patients with atrophic change of the tongue were examined on a relation to candidiasis. All of them complained of tongue pain on spicy or hot diet. Laboratory examinations included blood examination for diabetes and anemia, culture test and direct cytologic examination. The intensity of tongue pain was evaluated pre- and post-treatment using visual analogue scale (VAS). RESULTS: Twenty-four of 40 (60%) had pre-disposing factors of candidiasis including diabetes mellitus, malignancy, systemic steroid therapy, long-term antibiotic therapy and others in their medical history. Blood examinations revealed mild anemia and/or Fe deficiency in 5 (12.5%), mild diabetes in 4 (10.0%), both in two, while residual 29 patients (72.5%) were within reference levels. In the culture examination, candidal species were isolated in 72.5%, and almost all of them were candida albicans. The direct cytologic examination performed in 17 of 40 patients, witch revealed pseudohyphae of fungi in 14 patients (82.4%). After the antifungal treatment, the tongue pain disappeared or improved markedly in 80%. Simultaneously, the regenerative tendency of filifolm papilla of the tongue dorsum was observed in these patients. CONCLUSION: Atrophic tongue associated with pain at eating, even though it is mild atrophic change, has a high probability of being a candida-induced lesion. Long disease duration and no benefit by topical steroids are suggestive and diagnostic factors of this disease.

Evaluation of speech intelligibility after a secondary dehiscence operation using an artificial graft in patients with speech disorders after partial glossectomy.

The purpose of this study was to evaluate speech intelligibility after a dehiscence operation using artificial grafts for patients with speech disorders after partial glossectomy that were caused by scars resulting from the primary operation. The subjects were six men and three women, who had had a partial glossectomy for tongue cancer followed by direct closure without reconstruction. They were operated on a second time operation to mobilise the residual tongue by dividing the cicatrix. An artificial graft was applied to the wound to maintain the dehiscence. Speech intelligibility was evaluated by a standardised Japanese speech intelligibility test before, and 6 and 12 months after the second operation. The intelligibility scores significantly improved during the first 6 months after the second operation, and continued to improve slightly during the following 6 months. This study suggests that the dehiscence operation using an artificial graft could improve speech in patients after partial glossectomy.