Biomarkers in tumors of the central nervous system – a review

Until recently, diagnostics of brain tumors were almost solely based on morphology and immunohistochemical stainings for relatively unspecific lineage markers. Although certain molecular markers have been known for longer than a decade (combined loss of chromosome 1p and 19q in oligodendrogliomas), molecular biomarkers were not included in the WHO scheme until 2016. Now, the classification of diffuse gliomas rests on an integration of morphology and molecular results. Also, for many other central nervous system tumor entities, specific diagnostic, prognostic and predictive biomarkers have been detected and continue to emerge. Previously, we considered brain tumors with similar histology to represent a single disease entity. We now realize that histologically identical tumors might show alterations in different molecular pathways, and often represent separate diseases with different natural history and response to treatment. Hence, knowledge about specific biomarkers is of great importance for individualized treatment and follow‐up. In this paper we review the biomarkers that we currently use in the diagnostic work‐up of brain tumors.     

Intraductal papillary mucinous neoplasms of the pancreas: reporting clinically relevant features

Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas can exhibit a wide spectrum of macroscopic and microscopic appearances. This not only causes occasional difficulties for the reporting pathologist in distinguishing these tumours from other lesions, but is also relevant clinically. As evidence accumulates, it becomes clear that multiple macroscopic and histological features of these neoplasms are relevant to the risk for malignant transformation and, consequently, of prime importance for clinical patient management. The need for detailed reporting is therefore increasing. This review discusses the panoply of gross and microscopic features of IPMN as well as the recommendations from recent consensus meetings regarding the pathology reporting on this tumour entity.     

The discrete nature and distinguishing molecular features of pancreatic intraductal tubulopapillary neoplasms and intraductal papillary mucinous neoplasms of the gastric type,pyloric gland variant

Intraductal tubulopapillary neoplasms (ITPNs) are composed of tubulopapillary glands with high‐grade dysplasia in the pancreatic duct. Intraductal papillary mucinous neoplasms of the gastric type, pyloric gland variant (IPMN‐PGs) are composed of tubular glands mimicking pyloric glands with low‐grade dysplasia and were formerly called intraductal tubular adenomas. Because of their apparent common tubular morphology, IPMN‐PGs and ITPNs could be associated. While the former might progress to the latter, this has not been fully assessed. In this study, we compared the molecular features of ITPNs and IPMN‐PGs to determine their association using formalin‐fixed, paraffin‐embedded tissues of 14 ITPNs and 15 IPMN‐PGs. Somatic mutations in PIK3CA, GNAS, KRAS, and BRAF were determined by Sanger sequencing. Expression of phosphorylated AKT was examined by immunohistochemistry. Somatic PIK3CA mutations were found in 3 of 14 ITPNs (21.4%) but in none of the IPMN‐PGs (p = 0.0996). In contrast, GNAS mutations were found in none of the ITPNs but in 9 of 15 IPMN‐PGs (60.0%; p < 0.001). KRAS mutations were detected in 1 of 14 ITPNs (7.1%) and 12 of 15 IPMN‐PGs (80.0%; p < 0.001). BRAF mutation was found in one ITPN but in none of the IPMN‐PGs. Phosphorylated AKT expression in ITPNs was significantly more evident than that in IPMN‐PGs (p = 0.0401). These results indicate that ITPNs and IPMN‐PGs are molecularly distinct, suggesting that IPMN‐PG does not progress to ITPN. Furthermore, the molecular features of IPMN‐PGs are confirmed to be identical to those of IPMNs reported elsewhere. These results validate the current World Health Organization system that classifies pancreatic intraductal neoplasms into IPMN and ITPN and confirm that IPMN‐PG is not a benign counterpart of ITPN. The term ‘intraductal tubular adenoma’ should be eliminated and replaced with IPMN‐PG. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MUC1/Y基因在胃肠道肿瘤中的表达及诱导抗肿瘤免疫

目的 研究MUC1／Y基因异常表达在消化道肿瘤中的临床意义；构建MUC1／YcDNA全长载体，修饰树突状细胞(DC)诱导杀伤细胞治疗消化道肿瘤。方法 采用RT-PCR方法检测标本中MUC1／YmRNA表达。选取8例HLA-A2^ 消化道肿瘤患者，体外诱导DC，以构建的MUCl／YcDNA真核表达载体pcDNA3．1-MUC1／Y修饰DC诱导CTL。通过检测对靶细胞SW620和Raji的杀伤作用和诱导靶细胞凋亡的能力来评价抗肿瘤免疫反应。结果 93．88％肿瘤组织表达较高水平的MUC1／Y。分析表明：消化道肿瘤中MUC1／YmRNA表达与组织学分型、肿瘤生长方式、浸润程度及TNM临床分期有关；与性别、肿瘤发生部位及分化程度无关。pcDNA3．1-MUC1／Y修饰DC诱导的CTL对特异性靶细胞的杀伤活性显著高于对照组，并能有效诱导靶细胞凋亡。结论 MUC1／YmRNA表达在消化道肿瘤组织具有重要意义；经pcDNA3．1-MUC1／Y修饰的DC可有效诱导特异性抗肿瘤免疫应答。     

MUC1/Y基因体外冲击树突状细胞诱导抗瘤免疫反应

目的制备含MUC1/Y cDNA质粒转染的树突状细胞(DC),体外诱导杀伤细胞,研究其治疗消化道肿瘤的效果.方法构建MUC1/Y cDNA真核表达载体pIRES2-EGFP-MUC1/Y、pcDNA3.1-MUC1/Y.以pcDNA3.1-MUC/Y电转染8例HLA-A2(+)消化道肿瘤患者单个核细胞衍生的DC后,与自体T细胞混合培养,诱导CTL(T-pcDAN3.1-MUC1/Y).以SW620细胞[HLA-A2(+)、MUC1/Y(+)]为特异性靶细胞,Raji细胞[HLA-A2(-)、MUC1/Y(-)]和Lovo细胞[HLA-A2(-)、MUC1/Y(+)]为非特异性靶细胞,通过乳酸脱氢酶(LDH)释放实验测定杀伤活性,ELISA法检测基因修饰后DC刺激自体T细胞产生IFN-γ的能力,并以ANNEXIN V-FITC试剂盒检测特异性CTL诱导靶细胞凋亡情况.结果pIRES2-EGFP-MUC1/Y转染效率为8%左右.T-pcDAN3.1-MUC1/Y诱导的杀伤作用显著高于T-pcDNA3.1[pcDNA3.1(+)修饰DC诱导的CTL]和T-IL-2(IL-2刺激外周血单个核细胞产生的CTL),P＜0.05.而且T-pcDNA3.1-MUC1/Y对靶细胞的杀伤和诱导凋亡的能力显著高于对照组.基因修饰后的DC能刺激自体T细胞分泌高水平IFN-γ,与未转染的DC相比具有显著差异(P＜0.05).结论成功构建MUC1/Y全长cDNA真核表达载体.pIRES2-EGFP-MUC1/Y可用于真核细胞转染,通过观察转染效率,易于筛选阳性克隆;经pcDNA3.1-MUC1/Y修饰的DC可有效诱导特异性抗肿瘤免疫应答.     

卵巢浆液性和黏液性肿瘤MUC1、MUC2的表达及其意义

目的探讨卵巢浆液性和黏液性肿瘤中黏蛋白MUC1、MUC2的表达与临床病理特征的相关性。方法免疫组化S—P法检测90例卵巢浆液性和黏液性肿瘤的黏蛋白MUC1、MUC2的表达,并对其中50例恶性病例作生存分析。结果（1）交界性与恶性卵巢肿瘤中黏蛋白MUC1的表达阳性率明显高于良性肿瘤,差异有显著性（P〈0．001）;黏蛋白MUC1与WHO病理分级、FIGO临床分期、大网膜转移显著相关（P〈0．05）。（2）黏蛋白MUC2与组织学类型、WHO病理分级相关（P〈0．05）。（3）黏蛋白MUC1与MUC2呈负相关（P〈0．05）。（4）对50例恶性浆液性和黏液性肿瘤进行的生存分析中,单因素分析显示：WHO病理分级、FIGO临床分期、大网膜转移、MUC1表达程度与预后相关（P〈0．05）,而多因素分析中只有FIGO临床分期、MUC1表达程度具有独立的预后意义（P〈0．05）,Kaplan—Meier生存曲线分析显示,Ⅲ、Ⅳ期较Ⅰ、Ⅱ期生存率差异有显著（P〈0．01）,MUC1阳性组和阴性组生存率差异有显著性（P〈0．01）。结论黏蛋白MUC1、MUC2与恶性卵巢浆液性和黏液性肿瘤的浸润、转移相关,Ⅲ、Ⅳ期肿瘤、MUC1强表达可作为恶性卵巢浆液性和黏液性肿瘤预后不良的可行性指标。     

K-Ras and microsatellite marker analysis of fine-needle aspirates from intraductal papillary mucinous neoplasms of the pancreas

Preoperative diagnosis of pancreatic cystic lesions is difficult despite the combination of cytomorphology, radiographic imaging characteristics, and fluid tumor markers such as carcinoembryonic antigen. Intraductal papillary mucinous neoplasms (IPMNs) represent a subset of preinvasive pancreatic cystic neoplasms and are associated with accumulated genetic mutations, especially K-ras and tumor suppressor genes such as p53. Application of molecular techniques to cyst fluid obtained by endoscopic ultrasound guided fine-needle aspiration (EUSFNA) may contribute to preoperative assessment.Sixteen patients with pancreatic cystic lesions had cyst fluid obtained by preoperative pancreatic EUSFNA or intraoperative aspiration. All patients subsequently underwent surgical resection of the pancreas and IPMN was documented in all (6 adenomas, 6 borderline tumors, and 4 carcinomas). DNA was extracted from the cyst fluids and mutational analysis for K-ras point mutations and loss of heterozygosity (LOH) analysis using a preselected panel of genomic loci were performed. LOH was observed in 3 of 4 carcinomas as compared to 4 of 11 adenomas and borderline lesions (1 was QNS). LOH and K-ras mutations were both acquired in 2 of 4 carcinomas and in 1 of 12 adenoma/borderline lesions.Although the study is small, molecular analysis for LOH and K-ras mutations is useful in the preoperative evaluation of cystic pancreatic lesions. Increasing degree of neoplasia appears to correlate with increased genetic abnormality using a panel of selected genomic markers.     

MUC-1 mucin expression in invasive areas of intraductal papillary mucinous tumors of the pancreas

The expression of MUC-1 mucin (membrane-associated mucin) and MUG2 much (secretory mucin) were immunohistochemically examined in 46 invasive ductal carcinomas (IDC) and 16 intraductal papillary mucinous tumors (IPMT) of the pancreas. lntraductal papillary mucinous tumors usually reveal expansive growth. However, of the 16 IPMT examined in the present study, three showed an invasive growth pattern, which was similar to 'mucinous carcinoma', around the non-invasive growth areas. Of 46 IDC, MUCl much detected by monoclonal antibodies, DF3 and MY.1E12, was expressed in 44 cases (96%) and in 45 cases (98%), respectively, whereas MUC-2 mucin detected by polyclonal antibody, anti-MRP, was not expressed in any of the cases (0%). In contrast, in the non-invasive growth areas of the 16 IPMT, MUG1 much detected by DF3 and MY.1 E12 was expressed in four cases (25%) and in six cases (38%), respectively, whereas MUG2 mucin detected by anti-MRP was expressed in 13 cases (81%). The invasive growth areas of the three IPMT showed positive expression of MUG-1 mucins detected by DF3 and MY.1E12, although the non-invasive growth areas showed negative expression of MUG1 muclns, except for their focal positive expression in one of the three cases. These findings indicate that the invasive growth areas of IPMT acquire a characteristic of MUC-1 much expression that is usually seen In IDC.     

Subtyping of male breast cancer by PAM50 and immunohistochemistry: a pilot study of a consecutive Danish cohort

Male breast cancer (MBC) is a rare disease that is still to be fully understood. In female breast cancer, molecular subtyping by gene expression has proven its significance. In this study, we characterize a consecutive cohort of MBC patients surgically treated from 1997 to 2017, identified at our institution (N = 37), and report the association between molecular subtypes found by a surrogate panel of immunohistochemical (IHC) markers, and the PAM50 signature, as well as risk of recurrence score and overall survival for the different subtypes. PAM50 subtypes were determined using the nCounter FLEX system instrument and software. The distribution of molecular subtypes according to the PAM50 signature was as follows: 56% luminal B, 39% luminal A, and 5% basal-like. None of the tumors were HER2-enriched. Using IHC surrogate markers, we found 80% luminal B-like, 15% luminal A-like, and 5% basal-like. None were HER2-positive (non-luminal). We found a strong statistical association between subtypes found by PAM50 signature and the IHC surrogate markers (p < 0.001). Furthermore, we found luminal A tumors to be smaller in size compared to luminal B tumors (p = 0.04). Patients with luminal A subtype tumors had the lowest ROR scores with a mean of 39, whereas patients with luminal B subtype tumors had a mean ROR score of 69. Significant worse overall survival for luminal B tumors compared to luminal A tumors was seen (p = 0.02). Male breast cancer seems to be a mainly luminal disease, with luminal B being the most frequent subtype. Further studies are needed to ensure correct therapeutic strategies for this select group of patients.     

Molecular characteristics and biological behaviours of the oncocytic and pancreatobiliary subtypes of intraductal papillary mucinous neoplasms

Intraductal papillary mucinous neoplasm (IPMN) consists of four epithelial subtypes. Of those, pancreatobiliary and oncocytic types are recently recognized and relatively uncommon, and usually exhibit high-grade dysplasia. The biological properties and molecular characteristics of these two types have not been well documented. The few molecular studies of the oncocytic type showed absence of KRAS mutations commonly seen in the other subtypes, raising the possibility that the oncocytic type is distinct from the other subtypes. Thus, we examined clinicopathological features and molecular alterations of the two subtypes. The study cohort consisted of 12 pancreatobiliary and 18 oncocytic IPMN cases. KRAS, BRAF, and PIK3CA mutations and TP53, SMAD4, and β-catenin expression were analysed, and the results of molecular and clinicopathological profiles were compared between the two subtypes. KRAS mutations were identified in the oncocytic type, but less frequently than the pancreatobiliary type (17% versus 58%, p = 0.048). BRAF mutation was found in a single oncocytic tumour, and no PIK3CA mutations were seen in any of the study cohort. TP53 overexpression was less frequent in the oncocytic type than in the pancreatobiliary type (11% versus 58%, p = 0.013). Invasive components were present in 50% of the oncocytic and 92% of the pancreatobiliary types, with lymph node metastasis more frequently seen in the latter, corresponding to better outcomes in the former (5-year survival rates: 93% versus 32%, p = 0.014). Our demonstration of KRAS and BRAF mutations in the oncocytic-type IPMN supports a role for the activation of the RAS-MAPK pathway in this tumour type. However, the less frequent TP53 overexpression associated with the significantly lower rates of invasion and nodal disease in the oncocytic type correlates with better outcomes compared to the pancreatobiliary type.     

Immunohistochemical analysis of steroidogenic enzymes in ovarian‐type stroma of pancreatic mucinous cystic neoplasms: Comparative study of subepithelial stromal cells in intraductal papillary mucinous neoplasms of the pancreas

Mucinous cystic neoplasms (MCNs) are generally defined as cyst‐forming epithelial neoplasms that arise in the pancreas and harbor characteristic ovarian‐type stroma beneath the epithelium. In this study, we compared the immunoreactivity of steroid‐related factors in these subepithelial stromal cells in MCNs to those in intraductal papillary mucinous neoplasms (IPMNs) to further characterize this unique MCN ovarian‐type stroma through evaluation of sex steroid biosynthesis. Twenty MCNs and twenty IPMNs were examined. Immunoreactivity of steroid hormone receptors, including estrogen receptor (ERα and ERβ), progesterone receptor (PR, PR‐A, and PR‐B), and androgen receptor (AR), was more frequently detected in MCN ovarian‐type stromal cells than in IPMN stromal cells (P < 0.01). The H‐scores (mean ± SD) of steroidogenic factor (SF)‐1 were also significantly higher in MCNs (112.3 ± 33.1) than in IPMNs (0.9 ± 1.2) (P < 0.01). The steroidogenic enzymes cytochrome P450 cholesterol side‐chain cleavage enzyme (P450scc), cytochrome P450 17 alpha‐hydroxylase (P450c17) and 3β‐hydroxysteroid dehydrogenase (3β‐HSD) showed immunoreactivity in 9/20 (45.0 %), 15/20 (75.0 %) and 13/20 (65.0 %), respectively, of ovarian‐type stroma from MCN cases. These results demonstrate that the ovarian‐type stroma of MCNs can express steroidogenic enzymes. Thus, the ovarian‐type stroma of MCNs can produce sex steroids that may also act on these cells.     

Reflections on MUC1 glycoprotein: the hidden potential of isoforms in carcinogenesis

Mucin 1 (MUC1) has been described as the renaissance molecule due to the large set of functions it displays in both normal and neoplastic cells. This membrane‐tethered glycoprotein is overexpressed and aberrantly glycosylated in most epithelial cancers, being involved in several processes related with malignant phenotype acquisition. With a highly polymorphic structure, both in the polypeptide and glycan counterparts, MUC1 variability has been associated with susceptibility to several diseases, including cancer. Biochemical features and biological functions have been characterized upon the full‐length MUC1 protein, remaining to clarify the real impact on cell dynamics of the plethora of MUC1 isoforms. This review aims to encompass a detailed characterization of MUC1 role in carcinogenesis, highlighting recent findings in cell differentiation and uncovering new evidences of MUC1 isoforms involvement in malignant phenotype.     

Determination of malignant and invasive predictors in branch duct type intraductal papillary mucinous neoplasms of the pancreas: a suggested scoring formula

Prediction of malignancy or invasiveness of branch duct type intraductal papillary mucinous neoplasm (Br-IPMN) is difficult, and proper treatment strategy has not been well established. The authors investigated the characteristics of Br-IPMN and explored its malignancy or invasiveness predicting factors to suggest a scoring formula for predicting pathologic results. From 1994 to 2008, 237 patients who were diagnosed as Br-IPMN at 11 tertiary referral centers in Korea were retrospectively reviewed. The patients' mean age was 63.1 ± 9.2 yr. One hundred ninty-eight (83.5%) patients had nonmalignant IPMN (81 adenoma, 117 borderline atypia), and 39 (16.5%) had malignant IPMN (13 carcinoma in situ, 26 invasive carcinoma). Cyst size and mural nodule were malignancy determining factors by multivariate analysis. Elevated CEA, cyst size and mural nodule were factors determining invasiveness by multivariate analysis. Using the regression coefficient for significant predictors on multivariate analysis, we constructed a malignancy-predicting scoring formula: 22.4 (mural nodule [0 or 1]) + 0.5 (cyst size [mm]). In invasive IPMN, the formula was expressed as invasiveness-predicting score = 36.6 (mural nodule [0 or 1]) + 32.2 (elevated serum CEA [0 or 1]) + 0.6 (cyst size [mm]). Here we present a scoring formula for prediction of malignancy or invasiveness of Br-IPMN which can be used to determine a proper treatment strategy.     

Targeted next‐generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas

Intraductal neoplasms are important precursors to invasive pancreatic cancer and provide an opportunity to detect and treat pancreatic neoplasia before an invasive carcinoma develops. The diagnostic evaluation of these lesions is challenging, as diagnostic imaging and cytological sampling do not provide accurate information on lesion classification, the grade of dysplasia or the presence of invasion. Moreover, the molecular driver gene mutations of these precursor lesions have yet to be fully characterized. Fifty‐two intraductal papillary neoplasms, including 48 intraductal papillary mucinous neoplasms (IPMNs) and four intraductal tubulopapillary neoplasms (ITPNs), were subjected to the mutation assessment in 51 cancer‐associated genes, using ion torrent semiconductor‐based next‐generation sequencing. P16 and Smad4 immunohistochemistry was performed on 34 IPMNs and 17 IPMN‐associated carcinomas. At least one somatic mutation was observed in 46/48 (96%) IPMNs; 29 (60%) had multiple gene alterations. GNAS and/or KRAS mutations were found in 44/48 (92%) of IPMNs. GNAS was mutated in 38/48 (79%) IPMNs, KRAS in 24/48 (50%) and these mutations coexisted in 18/48 (37.5%) of IPMNs. RNF43 was the third most commonly mutated gene and was always associated with GNAS and/or KRAS mutations, as were virtually all the low‐frequency mutations found in other genes. Mutations in TP53 and BRAF genes (10% and 6%) were only observed in high‐grade IPMNs. P16 was lost in 7/34 IPMNs and 9/17 IPMN‐associated carcinomas; Smad4 was lost in 1/34 IPMNs and 5/17 IPMN‐associated carcinomas. In contrast to IPMNs, only one of four ITPNs had detectable driver gene (GNAS and NRAS) mutations. Deep sequencing DNA from seven cyst fluid aspirates identified 10 of the 13 mutations detected in their associated IPMN. Using next‐generation sequencing to detect cyst fluid mutations has the potential to improve the diagnostic and prognostic stratification of pancreatic cystic neoplasms. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

MUC1模拟表位肽治疗表达人MUC1 T739荷瘤小鼠的实验研究

目的观察MUC1的模拟表位肽对表达人MUC1 T739荷瘤小鼠的治疗作用。方法建立稳定表达人MUC1 T739荷瘤小鼠,培养T739小鼠成熟树突状细胞,荷瘤小鼠随机分为4组,用负载模拟表位肽成熟DC免疫表达人MUC1 T739荷瘤小鼠,第1组皮下注射等渗盐水,第2组注射空DC,第3组注射负载1号肽的DC,第4组注射负载2号肽的DC,测量肿瘤大小,计算肿瘤体积,称瘤重,并观察小鼠存活期。结果两组DC+肽免疫的小鼠瘤体积减小明显,与1、2组相比差异非常显著(P<0.01)。平均瘤重1、2组与3、4组相比差异非常显著(P<0.01)。第1～4组荷瘤鼠的生存期分别为(34.6±3.507)d,(35.2±3.564)d,(58.4±5.595)d,(59±6.819)d,3、4两组荷瘤小鼠生存期较1、2组明显延长,差异有显著性意义(P<0.01)。结论 MUC1模拟表位肽能明显抑制表达人MUC1 T739荷瘤小鼠肿瘤生长,显著延长T739荷瘤小鼠生存时间。