Comprehensive genomic profiling of salivary gland carcinoma: Analysis of the Center for Cancer Genomics and Advanced Therapeutics database in Japan.

Comprehensive information on genetic alterations in salivary gland cancer (SGC) is limited. This study aimed to elucidate the genetic and clinical characteristics of patients with SGC using the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database, a Japanese national genomic database. We analyzed data of 776 patients with SGC registered in the C-CAT database between June 1, 2019, and June 30, 2023. Adenoid cystic carcinoma was the most common histologic type, followed by salivary duct carcinoma (SDC) and adenocarcinoma not otherwise specified. Genetic data of 681 patients receiving FoundationOne® CDx were analyzed. We identified specific features of the combination of TP53 and CDKN2A alterations among the histological types. Specific LYN amplification was mainly detected in carcinoma ex pleomorphic adenoma and myoepithelial carcinoma. For SDC, the frequency of ERBB2 and BRAF alterations were higher in cases with metastatic lesions than in those with primary lesions. Although 28.6% patients were offered recommended treatment options, only 6.8% received the recommended treatments. This study highlights the differences in genetic alterations among the histological types of SGC, with comprehensive genomic profiling tests revealing lower drug accessibility. These findings could contribute to the development of personalized treatment for patients with SGC.

The Landscape of MYB/MYBL1- and Peri-MYB/MYBL1-Associated Rearrangements in Adenoid Cystic Carcinoma.

Approximately 60% of adenoid cystic carcinoma (AdCC) cases are positive for MYB::NFIB or MYBL1::NFIB, whereas MYB/MYBL1 oncoprotein, a key driver of AdCC, is overexpressed in most cases. Juxtaposition of superenhancer regions in NFIB and other genes into the MYB/MYBL1 locus is an attractive oncogenic hypothesis for AdCC cases, either negative or positive for MYB/MYBL1::NFIB. However, evidence supporting this hypothesis is insufficient. We examined 160 salivary AdCC cases for rearrangements in MYB/MYBL1 loci and peri-MYB/MYBL1 areas (centromeric and telomeric areas of 10 Mb each) using formalin-fixed, paraffin-embedded tumor sections. For the detection of the rearrangements, we employed conventional fluorescence in situ hybridization split and fusion assays and a 5 Mb fluorescence in situ hybridization split assay. The latter is a novel assay that enabled us to detect any possible splits within a 5 Mb distance of a chromosome. We found MYB/MYBL1- and peri-MYB/MYBL1-associated rearrangements in 149/160 patients (93%). AdCC cases positive for rearrangements in MYB, MYBL1, the peri-MYB area, and the peri-MYBL1 area numbered 105 (66%), 20 (13%), 19 (12%), and 5 (3%), respectively. In 24 peri-MYB/MYBL1 rearrangement-positive cases, 14 (58%) were found to have a juxtaposition of the NFIB or RAD51B locus into the MYB/MYBL1 loci. On comparing with a tumor group positive for MYB::NFIB, a hallmark of AdCC, other genetically classified tumor groups had similar features of overexpression of the MYB transcript and MYB oncoprotein as detected by semiquantitative RT-qPCR and immunohistochemistry, respectively. In addition, clinicopathological and prognostic features were similar among these groups. Our study suggests that peri-MYB/MYBL1 rearrangements may be a frequent event in AdCC and may result in biological and clinicopathological consequences comparable to MYB/MYBL1 rearrangements. The landscape of MYB/MYBL1 and peri-MYB/MYBL1 rearrangements shown here strongly suggests that juxtaposition of superenhancers into MYB/MYBL1 or peri-MYB/MYBL1 loci is an alteration that acts as a key driver for AdCC oncogenesis and may unify MYB/MYBL1 rearrangement-positive and negative cases.

Salivary mucoepidermoid carcinoma: histological variants, grading systems, CRTC1/3-MAML2 fusions, and clinicopathological features.

AIMS: To investigate the histological diversity of salivary mucoepidermoid carcinoma (MEC), its clinicopathological features, and its associations with CRTC1/3-MAML2 fusions. METHODS AND RESULTS: Salivary MEC cases (n = 177) were examined for CRTC1/3-MAML2 fusions, histological variants were classified, and tumours were graded according to four different grading systems. Adverse histological features considered to be unusual in MEC were also investigated. Of the 177 MEC cases, 110 were positive for CRTC1/3-MAML2 fusions. The classical variant was the most frequent in the fusion-positive case group, the fusion-negative case group, and the total case group. The clear/oncocytic variant was the second most frequent in the fusion-positive and total case groups. Oncocytic, Warthin-like and spindle variants were seen in the fusion-positive case group only. Clear cell, sclerosing, mucinous and central variants were seen in both the fusion-positive case group and the fusion-negative case group. No case was classified as a ciliated variant, as a mucoacinar variant, or as a high-grade transformation. As compared with the classical variant, non-classical variants were characterised by frequent CRTC1/3-MAML2 fusions and a low clinical stage in all cases. Of the four histological features considered to be unusual in MEC, marked nuclear atypia, frequent mitoses (>10/10 high-power fields) and extensive necrosis were found independently of the fusion status, and were present in 3-5% of all cases. However, none of the cases showed overt keratinisation. On comparison, the Armed Forces Institute of Pathology and modified Healey grading systems downgraded tumours, the Brandwein system upgraded tumours, and the Memorial Sloan Kettering system provided a moderate means of assessment. CONCLUSION: Recognition of the histological diversity of MEC, its clinicopathological features and its associations with CRTC1/3-MAML2 fusions is helpful for an accurate diagnosis of this carcinoma.

CCR7 alterations associated with inferior outcome of adult T-cell leukemia/lymphoma under mogamulizumab treatment.

Adult T-cell leukemia/lymphoma (ATL) patients have a very poor prognosis. The humanized anti-CCR4 therapeutic monoclonal antibody, mogamulizumab, is a key agent for ATL treatment. Our previous integrated molecular analysis demonstrated that among all the driver genes in ATL, CCR7 gene alterations were significantly associated with clinical response to mogamulizumab. Accordingly, here we investigated the detailed clinical impact of CCR7 alterations in a larger cohort of ATL patients. These CCR7 alterations, most of which lead to C-terminus truncations, were observed in 27 of 223 patients (12%). For patients receiving mogamulizumab but not allogeneic hematopoietic stem cell transplantation (HSCT), CCR7 alterations were significantly associated with worse survival (median survival from the first dose of mogamulizumab of 0.7 years for 12 patients with CCR7 alterations vs. 1.6 years for 72 patients without, p = 0.020). On the other hand, the presence or absence of CCR7 alterations had no significant impact on survival in the entire cohort (median overall survival of 1.4 and 1.8 years, respectively, p = 0.901), or on the survival of patients receiving allogeneic HSCT (median survival from the day of transplantation of 0.9 years for 6 patients with CCR7 alterations and 1.4 years for 48 without, p = 0.543). Multivariate analysis indicated that patients with CCR4 alterations but lacking CCR7 alterations (n = 20) had significantly better survival after receiving mogamulizumab-containing treatments (hazard ratio for survival, 0.437, 95% confidence interval, 0.192-0.994). This study contributes to the establishment of precision medicine for ATL.

Serum creatinine/cystatin C ratio is a surrogate marker for sarcopenia in patients with idiopathic pulmonary fibrosis.

BACKGROUND: The serum creatinine/cystatin C (Cr/CysC) ratio has attracted attention as a marker for sarcopenia, but has not been studied in patients with idiopathic pulmonary fibrosis (IPF). This study aimed to confirm the utility of the serum Cr/CysC ratio in predicting sarcopenia and investigate its clinical relevance. METHODS: This cross-sectional pilot study prospectively enrolled patients with stable IPF. IPF was diagnosed through multidisciplinary discussions according to the 2018 international guidelines, and sarcopenia was diagnosed according to the 2019 consensus report of the Asian Working Group for Sarcopenia. Patient-reported outcomes (PROs) were evaluated using the modified Medical Research Council (mMRC) dyspnea scale, chronic obstructive pulmonary disease assessment test (CAT), and King's Brief Interstitial Lung Disease (K-BILD) questionnaire. The associations between serum Cr/CysC ratio and the presence of sarcopenia and other clinical parameters, including PROs scores, were examined. RESULTS: The study enrolled 49 Japanese patients with IPF with a mean age of 73.0 ± 7.7 years and a mean percentage of predicted forced vital capacity of 80.4 ± 15.5%. Sarcopenia was diagnosed in 18 patients (36.7%), and the serum Cr/CysC ratio was 0.86 [0.76-0.94] (median [interquartile range]). The receiver operating characteristic curve analyses for the detection of sarcopenia according to the serum Cr/CysC showed that the area under the curve, optimal cutoff value, specificity, and sensitivity were 0.85, 0.88, 0.65, and 0.94, respectively. Sarcopenia was identified in 13% of patients with a high serum Cr/CysC ratio (≥ 0.88) and 60% of patients with a low serum Cr/CysC ratio (< 0.88) (P < 0.001). Multiple linear regression analysis showed that the serum Cr/CysC ratio was an independent predictive marker of worse PROs evaluated using mMRC (P < 0.05), CAT (P < 0.05), and K-BILD (P < 0.05). CONCLUSIONS: This study showed that the serum Cr/CysC ratio may be a surrogate marker of sarcopenia in patients with IPF. Furthermore, it is important to pay attention to the serum Cr/CysC ratio because a lower serum Cr/CysC ratio is associated with worse PROs. Further studies are required to validate these observations to determine whether the Cr/CysC ratio can be used to detect sarcopenia in patients with IPF.

Decreased peak expiratory flow rate associated with mortality in idiopathic pulmonary fibrosis: A preliminary report.

OBJECTIVES: The peak expiratory flow rate (PEFR) is known to decrease in patients with sarcopenia. However, little is known about the clinical impact of the PEFR in idiopathic pulmonary fibrosis (IPF). This study aimed to confirm whether a decrease in PEFR over 6 months was associated with survival in IPF patients. METHODS: Consecutive IPF patients who had been assessed at a single center were retrospectively analyzed. The relative decline in PEFR over 6 months was assessed. Survival analyses were performed by univariate and multivariate Cox proportional hazard models. RESULTS: A total of 61 eligible cases (average age 70 years) were examined, and 21 patients (34.4%) died. The univariate Cox regression analysis showed that the body mass index, baseline % predicted forced vital capacity (FVC), baseline % predicted PEFR, % predicted diffusion capacity for carbon monoxide (DLCO), relative decline in FVC, and relative decline in PEFR were prognostic factors. On multivariate analyses, relative decline in PEFR (hazard ratio [HR] 1.037, p < .05) and baseline % predicted FVC (HR 0.932, p < .001) were independent prognostic factors, whereas relative decline in FVC was not. CONCLUSION: A decrease in PEFR after 6 months may predict worse survival in patients with IPF.

Frequency and impact on clinical outcomes of sarcopenia in patients with idiopathic pulmonary fibrosis.

OBJECTIVES: Sarcopenia is a syndrome characterized by reduced muscle mass and function. It is well-recognized as a complication in chronic diseases such as chronic obstructive pulmonary disease. However, little is known about sarcopenia in patients with idiopathic pulmonary fibrosis (IPF). This study aimed to investigate the clinical characteristics of sarcopenia and the association between quality of life and sarcopenia in patients with IPF. METHODS: In this pilot cross-sectional study, 56 Japanese outpatients with IPF (49 men) were enrolled prospectively. Sarcopenia was diagnosed according to the criteria of the Asian Working Group for Sarcopenia 2019. Its associations with clinical parameters including age, pulmonary functions, physical performance, and patient-reported outcomes (PROs) were examined. RESULTS: The frequency of sarcopenia was 39.3% (n = 22) in this cohort. There were significant differences in St George's Respiratory Questionnaire (p = .005), modified Medical Research Council score (p = .004), and Hospital and Anxiety Depression Scale depression score (p = .030) between the sarcopenic and non-sarcopenic groups. On multivariate regression analysis, 6-min walk distance (6MWD) was an independent factor associated with sarcopenia (odds ratio 1.241, 95% confidence interval 1.016-1.515, p = .034). CONCLUSION: Sarcopenia was associated with PROs and physical performance in patients with IPF.

Pathological evaluation of tumor grade for salivary adenoid cystic carcinoma: A proposal of an objective grading system.

Three pathological grading systems advocated by Perzin/Szanto, Spiro, and van Weert are currently used for adenoid cystic carcinoma (AdCC). In these systems, the amount or presence of the solid tumor component in AdCC specimens is an important index. However, the "solid tumor component" has not been well defined. Salivary AdCC cases (N = 195) were collected after a central pathology review. We introduced a novel criterion for solid tumor component, minAmax (minor axis maximum). The largest solid tumor nest in each AdCC case was histologically screened, the maximum oval fitting the solid nest was estimated, and the length of the minor axis of the oval (minAmax) was measured. The prognostic cutoff for the minAmax was determined using training and validation cohorts. All cases were evaluated for the four grading systems, and their prognostic impact and interobserver variability were examined. The cutoff value for the minAmax was set at 0.20 mm. Multivariate prognostic analyses showed the minAmax and van Weert systems to be independent prognostic tools for overall, disease-free, and distant metastasis-free survival while the Perzin/Szanto and Spiro systems were selected for overall survival but not for disease-free or distant metastasis-free survival. The highest hazard ratio for overall survival (11.9) was obtained with the minAmax system. The reproducibility of the minAmax system (kappa coefficient of 0.81) was scored as very good while those of the other three systems were scored as moderate. In conclusion, the minAmax is a simple, objective, and highly reproducible grading system useful for prognostic stratification for salivary AdCC.

Clinical significance of TP53 mutations in adult T-cell leukemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) patients have a poor prognosis. Here, we investigated the impact of TP53 gene mutations on prognosis of ATL treated in different ways. Among 177 patients, we identified 47 single nucleotide variants or insertion-deletions (SNVs/indels) of the TP53 gene in 37 individuals. TP53 copy number variations (CNVs) were observed in 38 patients. Altogether, 67 of 177 patients harboured TP53 SNVs/indels or TP53 CNVs, and were categorized as having TP53 mutations. In the entire cohort, median survival of patients with and without TP53 mutations was 1·0 and 6·7 years respectively (P < 0·001). After allogeneic haematopoietic stem cell transplantation (HSCT), median survival of patients with (n = 16) and without (n = 29) TP53 mutations was 0·4 years and not reached respectively (P = 0·001). For patients receiving mogamulizumab without allogeneic HSCT, the median survival from the first dose of antibody in patients with TP53 mutations (n = 27) was only 0·9 years, but 5·1 years in those without (n = 42; P < 0·001). Thus, TP53 mutations are associated with unfavourable prognosis of ATL, regardless of treatment strategy. The establishment of alternative modalities to overcome the adverse impact of TP53 mutations in patients with ATL is required.

Expression of cancer testis antigens in thymic epithelial tumors.

Cancer testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. However, to our knowledge, expression of the antigens in thymic epithelial tumors has not been examined yet. We examined the immunohistochemical expression of five CTAs (MAGE-A, NY-ESO-1, MAGE-C1, SAGE and GAGE7) in 192 cases of thymic epithelial tumor. The CTAs were variably expressed in the thymic epithelial tumors. Type B component of type AB thymomas, type B1/B2/B3 thymomas, and thymic carcinomas showed a generally positive correlation between the malignancy grades and positive expression rates in four CTAs other than MAGE-C1. In thymic squamous cell carcinomas (SqCCs), four antigens except for MAGE-C1 showed high expression rates ranging from 23.1% to 43.6%. In the prognostic analysis, a positive expression of SAGE (P = 0.0485) and GAGE7 (P = 0.0289) were associated with a shorter overall survival in type B2/B3 thymomas, respectively. In thymic SqCC, a positive MAGE-A expression was significantly associated with an increased level of programmed death ligand in tumor-infiltrating lymphocytes (P = 0.0181). We showed (i) a frequent CTA expression, (ii) a general correlation of CTA expression with tumor malignancy grades and (iii) a prognostic impact in some of the CTAs.

Prognostic impact of CRTC1/3-MAML2 fusions in salivary gland mucoepidermoid carcinoma: A multiinstitutional retrospective study.

Mucoepidermoid carcinoma (MEC) is rare, but the most common primary malignancy of the salivary gland and not infrequent in young individuals. CRTC1/3-MAML2 fusions are frequently detected in MEC and are useful as a diagnostic biomarker. However, there has been debate as to whether the fusions have prognostic significance. In this study, we retrospectively collected 153 salivary gland MEC cases from 11 tertiary hospitals in Japan. As inclusion criteria, the MEC patients in this study had curative surgery as the initial treatment, received no preoperative treatment, and had no distant metastasis at the time of the initial surgery. The MEC diagnosis was validated by a central pathology review by five expert salivary gland pathologists. The CRTC1/3-MAML2 fusions were detected using FISH and RT-PCR. In 153 MEC cases, 90 (58.8%) were positive for CRTC1/3-MAML2 fusions. During the follow-up period, 28 (18.3%) patients showed tumor recurrence and 12 (7.8%) patients died. The presence of the fusions was associated with favorable tumor features. Of note, none of the fusion-positive patients died during the follow-up period. Statistical analysis showed that the presence of the fusions was a prognostic indicator of a better overall survival in the total and advanced-stage MEC cohorts, but not in the early-stage MEC cohort. In conclusion, CRTC1/3-MAML2 fusions are an excellent biomarker for favorable overall survival of patients with salivary gland MEC.

Clinicopathological significance of EGFR pathway gene mutations and CRTC1/3-MAML2 fusions in salivary gland mucoepidermoid carcinoma.

AIMS: Mucoepidermoid carcinoma (MEC) is one of the most common salivary gland carcinomas. Epidermal growth factor receptor (EGFR) signalling pathway gene mutations are important in predicting a patient's prognosis, selecting molecularly targeted drugs and estimating the efficacy of a molecular therapy. However, their significance in MEC have been poorly clarified. CRTC1/3-MAML2 fusions are specific to MEC and may be associated with favourable characteristics in these patients. METHODS AND RESULTS: We looked for CRTC1/3-MAML2 fusions and gene alterations in the EGFR, RAS family (KRAS, HRAS and NRAS), PIK3CA, BRAF and AKT1 in 101 MEC cases. We also examined mutations in TP53. CRTC1/3-MAML2 fusions were found in 62.4% of the cases. KRAS, HRAS and PIK3CA mutations were detected in 6.9%, 2.0% and 6.9%, respectively, but other EGFR pathway genes were not mutated. In total, gene mutations (RAS/PIK3CA) in the EGFR pathway were detected in 14.9% of the cases. TP53 mutations were found in 20.8%. CRTC1/3-MAML2 fusions were associated with a better prognosis and RAS/PIK3CA mutations a worse prognosis of the patients, respectively, and both were selected as independent prognostic factors for the overall survival of the patients. TP53 mutations had no prognostic impact. CRTC1/3-MAML2 fusion-positive rates were inversely associated with the patients' age and the fusions were found in 82% of patients aged < 30 years. CONCLUSIONS: RAS/PIK3CA mutations were frequently detected, and may be a biomarker for a poorer prognosis in MEC patients. CTRC1/3-MAML2 fusions were positive in most of the young MEC patients.

Next-generation sequencing assay in salivary gland cytology: A pilot study.

BACKGROUND: Preoperative diagnosis of salivary gland tumors (SGTs) by fine-needle aspiration (FNA) cytology is challenging. Next-generation sequencing (NGS)-based assays for somatic mutations have a great advantage in that a large number of genes can be analyzed simultaneously. Although NGS may have an enormous diagnostic potential in cytology, to our knowledge, the significance of NGS in SGT cytology remains to be clarified. METHODS: In this pilot study, we retrospectively examined 32 frozen SGT samples obtained at surgery (14 malignant and 18 benign). After the stored frozen tumor tissues were thawed, aspirate samples were obtained using 22-gauge needles and subjected to smear tumor samples and to DNA extraction for an NGS assay employing the Illumina AmpliSeq Cancer Hotspot Panel v2. The results were correlated to preoperative cytological diagnosis. RESULTS: The preoperative diagnoses obtained by FNA cytology included 23 negative lesions (no malignancy in 6 and benign tumor in 17) and nine positive lesions (suspicious for malignancy in 4 and malignancy in five), providing a sensitivity and a specificity of 9/14 (64%) and 18/18 (100%), respectively. The NGS assay detected somatic mutations in 10/14 malignant and 1/18 benign SGT cases, providing a sensitivity and a specificity of 71% and 94%, respectively. CONCLUSION: The NGS assay may be helpful for detecting the malignant potential in SGT cases and can be used as an ancillary test for SGT cytology.

The impact of clinicopathological factors on clinical outcomes in patients with salivary gland adenoid cystic carcinoma: a multi-institutional analysis in Japan.

BACKGROUND: Owing to the low incidence of adenoid cystic carcinoma (AdCC), reliable survival estimates and prognostic factors remained unclarified. METHODS: In this multi-institutional retrospective analysis, we collected 192 AdCC cases, and investigated the impact of clinicopathological factors on clinical outcomes of the patients. All AdCC cases were of salivary gland origin and were surgically treated with curative intent. Diagnoses of AdCC were validated by a central pathology review by expert pathologists. RESULTS: The 5-year overall survival (OS) and disease-free survival (DFS) rates were 92.5 and 50.0%, respectively. Treatment failure occurred in 89 patients (46%) with the distant failures in 65 (34%). Multivariate analysis indicated that pN2 and a pathologically positive surgical margin were independent prognostic factors for both OS and DFS. Histological grade III was an independent prognostic factor for OS. A primary site in the submandibular gland, pT3/4, pN1, and histological grade II were independent prognostic factors for DFS. Postoperative radiation therapy (PORT) improved the locoregional control (LRC) rate. Prophylactic neck dissection was not associated with a better OS or better LRC among patients with cN0. Facial nerve dissection did not improve clinical outcomes in parotid AdCC cases without facial nerve palsy. CONCLUSIONS: A higher TN classification, a pathologically positive surgical margin, and a higher histological grade were associated with a lower OS. PORT improved LRC rates but neck dissection failed to improve clinical outcomes in patients with cN0. As the distant metastasis was frequent, effective systemic therapy is imperative to improve the survival of AdCC patients.

Immunohistochemistry for identification of CCND1, NSD2, and MAF gene rearrangements in plasma cell myeloma.

The t(11;14)/CCND1-IGH, t(4;14)/NSD2(MMSET)-IGH, and t(14;16)/IGH-MAF gene rearrangements detected by fluorescence in situ hybridization (FISH) are used for risk stratification in patients with multiple myeloma (MM). Compared with conventional FISH techniques using fresh cells, immunohistochemistry (IHC) is much more cost- and time-efficient, and can be readily applied to routinely prepared formalin-fixed, paraffin-embedded (FFPE) materials. In this study, we performed tissue FISH and IHC employing FFPE specimens, and examined the usefulness of IHC as a tool for detecting CCND1, NSD2, and MAF gene rearrangements. CD138 signals were used to identify plasma cells in tissue FISH and IHC analyses. With cohort 1 (n = 70), we performed tissue FISH and subsequently IHC, and determined IHC cut-off points. In this cohort, the sensitivity and specificity for the 3 molecules were ≥.90 and ≥.96, respectively. With cohort 2, using MM cases with an unknown gene status (n = 120), we performed IHC, and the gene status was estimated using the cut-off points determined with cohort 1. The subsequent FISH analysis showed that the sensitivity and specificity for the 3 molecules were ≥.92 and ≥.98, respectively. CCND1, NSD2, and MAF gene rearrangements were estimated accurately by IHC, suggesting that conventional FISH assays can be replaced by IHC.

A mutation analysis of the EGFR pathway genes, RAS, EGFR, PIK3CA, AKT1 and BRAF, and TP53 gene in thymic carcinoma and thymoma type A/B3.

AIMS: Thymic carcinoma is rare and usually has a fatal outcome. Gene mutations in the epidermal growth factor receptor (EGFR) signalling pathway and TP53 have not been well analysed in thymic carcinoma. METHODS AND RESULTS: We examined a large cohort of thymic carcinoma and thymoma type A/B3 and looked for gene mutations in the RAS family, EGFR, PIK3CA, AKT1, BRAF and TP53. Among 54 thymic carcinoma cases, RAS family mutations were detected in 10 cases, EGFR in two, PIK3CA in one, AKT1 in one, BRAF in none and TP53 in five. Among 33 thymoma type A/B3 cases, HRAS gene mutation were found in one, PIK3CA in two and AKT1 in one. All these mutations were those of missense type activating mutations. RAS family mutations were significantly more frequent in thymic carcinoma than in thymoma type A/B3 (P = 0.0461). A prognostic analysis focusing on thymic squamous cell carcinoma cases (n = 44) showed that the overall survival was significantly shorter in patients with EGFR pathway mutations (n = 9) than in those without in a univariate analysis (P = 0.0173). Subsequently, EGFR pathway mutations were selected as an independent factor for a poor overall survival in a multivariate analysis (P = 0.0389). CONCLUSIONS: Mutations in the EGFR pathway and TP53 in thymic carcinoma may be frequent, and the EGFR pathway mutations may be associated with a poor prognosis in thymic squamous cell carcinoma patients. The therapeutic significance of gene mutations in thymic carcinoma should be further clarified.

Improved clonality detection in Hodgkin lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH and IGK rearrangements: A paraffin-embedded tissue study.

The BIOMED-2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin-embedded tumor sections. We previously described the usefulness of the semi-nested BIOMED-2 IGH assay in B-cell malignancies. In this study, we devised a novel semi-nested BIOMED-2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED-2, BIOMED-2 followed by BIOMED-2 re-amplification, and BIOMED-2 followed by semi-nested BIOMED-2, all targeting IGH and IGK, using paraffin-embedded tissues. In both IGH and IGK assays, semi-nested assays yielded significantly higher clonality detection rates than the standard assays and re-amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi-nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30-positive cell density, CD20-positive cell density, or Epstein-Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two-thirds of classical HL cases using semi-nested BIOMED-2 IGH/IGK assays using paraffin tumor sections. These semi-nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs.

MYB, MYBL1, MYBL2 and NFIB gene alterations and MYC overexpression in salivary gland adenoid cystic carcinoma.

AIMS: Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and the long-term prognosis is poor. In this study, we examined alterations of AdCC-associated genes, MYB, MYBL1, MYBL2 and NFIB, and their target molecules, including MYC. The results were correlated to clinicopathological profile of the patients. METHODS AND RESULTS: Using paraffin tumour sections from 33 cases of salivary gland AdCC, we performed a detailed fluorescence in-situ hybridization (FISH) analysis for gene splits and fusions of MYB, MYBL1, MYBL2 and NFIB. We found that 29 of 33 (88%) AdCC cases showed gene splits in either MYB, MYBL1 or NFIB. None of the cases showed an MYBL2 gene alteration. AdCCs were divided genetically into six gene groups, MYB-NFIB (n = 16), MYB-X (n = 4), MYBL1-NFIB (n = 2), MYBL1-X (n = 1), NFIB-X (n = 6) and gene-split-negative (n = 4). AdCC patients showing the MYB or MYBL1 gene splits were associated with microscopically positive surgical margins (P = 0.0148) and overexpression of MYC (P = 0.0164). MYC expression was detected in both ductal and myoepithelial tumour cells, and MYC overexpression was associated with shorter disease-free survival of the patients (P = 0.0268). CONCLUSIONS: The present study suggests that (1) nearly 90% of AdCCs may have gene alterations of either MYB, MYBL1 or NFIB, suggesting the diagnostic utility of the FISH assay, (2) MYB or MYBL1 gene splits may be associated with local aggressiveness of the tumours and overexpression of MYC, which is one of the oncogenic MYB/MYBL1 targets and (3) MYC overexpression may be a risk factor for disease-free survival in AdCC.

Expression of cancer/testis antigens in salivary gland carcinomas with reference to MAGE-A and NY-ESO-1 expression in adenoid cystic carcinoma.

AIMS: Cancer/testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. CTAs are highly immunogenic proteins, and thus represent ideal targets for cytotoxic T-lymphocyte-mediated specific immune therapy. The aim of this study was to screen CTA expression in various types of salivary gland carcinoma and to clarify clinicopathological significance of MAGE-A and NY-ESO-1 expression in adenoid cystic carcinomas (AdCCs) of the salivary gland, which is one of the most common salivary gland carcinomas, and usually has a fatal outcome. METHODS AND RESULTS: We used immunohistochemistry to examine the expression of four CTAs (MAGE-A, NY-ESO-1, CT7, and GAGE7) in various types of salivary gland carcinoma (n = 95). When carcinoma cases were divided into low-grade and intermediate/high-grade types, NY-ESO-1 and CT7 were expressed more frequently in intermediate/high-grade carcinomas. We then focused on MAGE-A and NY-ESO-1 expression in a large cohort of adenoid cystic carcinomas (AdCCs) (n = 46). MAGE-A and NY-ESO-1 were frequently expressed in AdCC; specifically, MAGE-A was expressed in >60% of the AdCC cases. MAGE-A expression and tumour site (minor salivary gland) were identified as independent risk factors for locoregional tumour recurrence. CONCLUSIONS: These findings suggest that CTAs may be expressed in a variety of salivary gland carcinomas, especially in those with higher histological grades. In addition, MAGE-A, which is frequently expressed in AdCC cases, may be a useful prognostic factor for poorer locoregional recurrence-free survival.

Improved clonality detection in B-cell lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH rearrangement: A paraffin-embedded tissue study.

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas.