Mucin Staining Research Articles

Local injection of dsRNA targeting calcitonin receptor-like receptor (CLR) ameliorates Clostridium difficile toxin A-induced ileitis

Enteritis caused by Clostridium difficile toxin (Tx) is a nosocomial disease of increasing clinical concern, but the local mediators of C. difficile TxA inflammation are unknown. The potent vasodilator calcitonin gene-related peptide mediates neurogenic inflammation via the calcitonin receptor-like receptor (CLR). Here we examined the ileum-specific effects of reducing CLR on TxA ileitis by local preinjection of double-stranded RNAs. Treatment with CLR dsRNA for 7 d decreased CLR immunoreactivity, whereas treatment with non-CLR dsRNA did not. Subsequent injection of TxA in the same location increased CLR in rats treated with non-CLR dsRNA but not in rats treated with CLR dsRNA, documenting that local injection of dsRNA is effective in preventing the increase in CLR immunoreactivity in response to local TxA. After non-CLR dsRNA pretreatment, TxA induced robust intestinal secretion, myeloperoxidase activity, and histopathologic indications of inflammation including epithelial damage, congestion, neutrophil infiltration, loss of mucin from goblet cells, and increase in mast cell numbers. After CLR dsRNA pretreatment, TxA-induced changes in intestinal secretion and histopathologic inflammation were improved, including normal mucin staining and fewer resident mast cells. Loss of CLR prevented TxA-mediated activation of NF-κB and concomitant increases in pERK1/2 and TNF-α mRNA. Locally produced CLR plays a proinflammatory role in TxA ileitis via MAPK signaling and TNF-α. The results reported here strongly suggest that a local injection of dsRNA targeting CLR could be an effective local therapeutic approach at the inflammation site in the treatment of a growing, clinically relevant hospital-acquired disease, C. difficile infection.

Mucin profile of Upper Gastrointestinal Tract Lesions

Background: Mucin secretions reflect the composition changes in the functional state of the mucosa in health and disease of the upper gastrointestinal tract. Objectives: To study the different types of upper gastrointestinal tract lesions and to determine the role of mucin histochemistry in the interpretation of lesions of upper gastrointestinal tract. Methods: A prospective study from September 2006 to August 2007 was carried out. 60 endoscopic biopsy specimens obtained from upper gastrointestinal tract lesions from the department of Gastroenterology at R.L. Jalappa Hospital & Research Centre was sent for histopathological examination. All the sections were stained by routine Haematoxylin and Eosin stain followed by special stains for mucins i.e Alcian blue pH 2.5 / PAS and Hale\'s Colloidal Iron. Results: Age of presentation of various upper gastrointestinal lesions was between 26-90 years. Females dominated the study group i.e. 39 (65%) when compared to males i.e. 21 (35%). Alcian Blue/Periodic Acid Schiff (AB/PAS) staining in oesophageal lesions could detect mixture of acidic mucins & neutral mucins and sulphomucins in Barrett\'s oesophagus. In gastritis, predominantly mixture of acidic and neutral mucins and in one case, sulphomucins were detected. Adenocarcinoma of stomach showed combination of acidic and neutral mucins. Duodenitis showed acidic, neutral and sialomucins. Adenocarcinoma of duodenum showed combination of acidic & neutral mucins. Hale\'s Colloidal Iron (HCI) stain in inflammatory & neoplastic lesions of stomach & duodenum, could detect only acidic mucins. Conclusion: In neoplastic condition of stomach this study suggests that those tumours which secrete neutral and mixed mucins probably arise from gastric epithelium and neck cells of the gastric glands. In those cases where the tumour produces acidic mucins it is possible the tumour arose from areas of intestinal metaplasia. Key words: Mucins, Histochemistry, Alcian Blue / Periodic Acid Schiff, Hale\'s Colloidal Iron

A Procedure for Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Intraductal papillary neoplasm of the bile duct: A biliary equivalent to intraductal papillary mucinous neoplasm of the pancreas?

Intraductal papillary neoplasm of the bile duct (IPNB) is a variant of bile duct carcinoma characterized by intraductal growth and better outcome compared with the more common nodular-sclerosing type. IPNB is a recognized precursor of invasive carcinoma, but its pathogenesis and natural history are ill-defined. This study examines the clinicopathologic features and outcomes of IPNB. A consecutive cohort of patients with bile duct cancer (hilar, intrahepatic, or distal) was reviewed, and those with papillary histologic features identified. Histopathologic findings and immunohistochemical staining for tumor markers and for cytokeratin and mucin proteins were used to classify IPNB into subtypes. Survival data were analyzed and correlated with clinical and pathologic parameters. Thirty-nine IPNBs were identified in hilar (23/144), intrahepatic (4/86), and distal (12/113) bile duct specimens between 1991 and 2010. Histopathologic examination revealed 27 pancreatobiliary, four gastric, two intestinal, and six oncocytic subtypes; results of cytokeratin and mucin staining were similar to those of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Invasive carcinoma was seen in 29/39 (74%) IPNBs. Overall median survival was 62 months and was not different between IPNB locations or subtypes. Factors associated with a worse median survival included presence and depth of tumor invasion, margin-positive resection, and expression of MUC1 and CEA. IPNBs are an uncommon variant of bile duct cancer, representing approximately 10% of all resectable cases. They occur throughout the biliary tract, share some histologic and clinical features with IPMNs of the pancreas, and may represent a carcinogenesis pathway different from that of conventional bile duct carcinomas arising from flat dysplasia. Given their significant risk of harboring invasive carcinoma, they should be treated with complete resection.

Myxoglobulosis of Appendix A Rare Entiy

We describe a case of the extremely rare entity of myxoglobulosis of the appendix in an 18-year-old man who underwent laparoscopic appendectomy with the clinical diagnosis of recurrent appendicitis. Transaction of the appendix after ligation resulted in extrusion of numerous whitish egg-like globules ranging in size from 0.2 to 0.7cm in diameter. It was initially thought to be hydatid of appendix, resulting in emergent usage of povidine iodine as scolicidal after suction of all the cysts/eggs. On microscopic examination, the globules consisted of faintly eosinophilic laminations of mucin surrounding an amorphous granular core were found. The mucin was identified by positivity with histochemical mucin stains. After thorough microscopic examination of the appendix, our case was diagnosed as myxoglobulosis due to mucosal hyperplasia, associated with luminal obstruction with possibly a membrane.

Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells

Airway goblet cell hyperplasia (GCH)—detectable by mucin staining—and abnormal macrophage infiltrate are pathological features present in many chronic respiratory disorders. However, it is unknown if both factors are associated. Using in-vivo and in-vitro models, we investigated whether macrophages are related with GCH and changes in mucin immunophenotypes. Lung sections from Sprague-Dawley rats treated for 48 h with one intra-tracheal dose of PBS or LPS (n=4–6 per group) were immunophenotyped for rat-goblet cells, immune, and proliferation markers. Human monocyte-derived macrophages (MDM) were pre-treated with or without LPS, immunophenotyped, and their supernatant, as well as cytokines at levels equivalent to supernatant were used to challenge primary culture of normal human bronchus epithelial cells (HBEC) in air–liquid interface, followed by MUC5B and MUC5AC mucin immunostaining. An association between increased bronchiolar goblet cells and terminal-bronchiolar proliferative epithelial cells confirmed the presence of GCH in our LPS rat model, which was related with augmented bronchiolar CD68 macrophage infiltration. The in-vitro experiments have shown that MUC5AC phenotype was inhibited when HBEC were challenged with supernatant from MDM pre-treated with or without LPS. In contrast, TNF-α and interleukin-1β at levels equivalent to supernatant from LPS-treated MDM increased MUC5AC. MUC5B was induced by LPS, supernatant from LPS-treated MDM, a mix of cytokines including TNF-α and TNF-α alone at levels present in supernatant from LPS-treated MDM. We demonstrated that macrophages are related with bronchiolar GCH, and that they induced MUC5B and inhibited MUC5AC in HBEC, suggesting a role for them in the pathogenesis of airway MUC5B-related GCH.

Mo1213 Differentiation Between Chronic Pancreatitis and Pancreatic Cancer by Using Metabolomic Analysis

Background Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are associated with substantially increased risk of malignant transformation when compared to other types of pancreatic lesions such as serous cystadenoma (SCA) and mucinous cystadenoma (MCA). A recent study suggests that detection of a mutation in codon 201 of GNAS in pancreatic cyst fluid DNA may be able to distinguish IPMN from other lesions of the pancreas, including both MCA and SCA [1]. This study found the detection of a GNAS mutation to be 100% specific, and the presence of either a GNAS or KRAS mutation to be 96% sensitive for IPMN. We examined the diagnostic significance of GNAS mutations in a cohort of pancreatic cyst fluid specimens and correlated the results with the associated diagnostic information. Methods 200 residual pancreatic cyst fluid DNA specimens from 198 distinct patients were retrieved from specimen archives and tested. These specimens were chosen so that there was an equal mix of KRAS mutated and wildtype results, and a range of CEA results above and below 192 ng/mL. DNA was amplified with primers targeting the codon 201 region, and sequenced using capillary electrophoresis and Sanger sequencing. The clonality of the mutation was identified as the ratio of mutant to wildtype, with a limit of detection of 10%. IPMN was determined to be present if there was duct dilation or connectivity to the duct system together with observation of mucinous characteristics of the fluid (e.g. gross mucin, mucin stain, elevated CEA, etc.). Results GNAS mutations were found in 50/200 (25%) specimens. A significant association between the presence of KRAS mutation and GNAS mutation was found, with more GNAS mutations present among KRAS-mutated than KRASwildtype cysts (odds ratio=4, p=7.0e-5). There was no significant difference in mean CEA values between according to GNAS mutation status. Two small ( CAT) that has not been previously reported in IPMN; this mutation was present in only that one specimen. In 20/50 (40%) of GNAS mutated-specimens neither duct dilation nor cyst communication with the duct system was observed. Conclusions Based on the present evaluation, GNAS is found to be mutated in a significant number of cases lacking the classic features of IPMNs. Further work is needed to better understand the role of GNAS in discriminating IPMNs from other cystic neoplasms of the pancreas and in evaluating the malignant potential of either; additional surgical outcomes would be valuable for this assessment. 1. Sci. Transl. Med. 3, 92ra66 (2011).

Mo1212 Correlation of GNAS Mutation With Diagnostic Information in Pancreatic Cyst Fluids

Background Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are associated with substantially increased risk of malignant transformation when compared to other types of pancreatic lesions such as serous cystadenoma (SCA) and mucinous cystadenoma (MCA). A recent study suggests that detection of a mutation in codon 201 of GNAS in pancreatic cyst fluid DNA may be able to distinguish IPMN from other lesions of the pancreas, including both MCA and SCA [1]. This study found the detection of a GNAS mutation to be 100% specific, and the presence of either a GNAS or KRAS mutation to be 96% sensitive for IPMN. We examined the diagnostic significance of GNAS mutations in a cohort of pancreatic cyst fluid specimens and correlated the results with the associated diagnostic information. Methods 200 residual pancreatic cyst fluid DNA specimens from 198 distinct patients were retrieved from specimen archives and tested. These specimens were chosen so that there was an equal mix of KRAS mutated and wildtype results, and a range of CEA results above and below 192 ng/mL. DNA was amplified with primers targeting the codon 201 region, and sequenced using capillary electrophoresis and Sanger sequencing. The clonality of the mutation was identified as the ratio of mutant to wildtype, with a limit of detection of 10%. IPMN was determined to be present if there was duct dilation or connectivity to the duct system together with observation of mucinous characteristics of the fluid (e.g. gross mucin, mucin stain, elevated CEA, etc.). Results GNAS mutations were found in 50/200 (25%) specimens. A significant association between the presence of KRAS mutation and GNAS mutation was found, with more GNAS mutations present among KRAS-mutated than KRASwildtype cysts (odds ratio=4, p=7.0e-5). There was no significant difference in mean CEA values between according to GNAS mutation status. Two small ( CAT) that has not been previously reported in IPMN; this mutation was present in only that one specimen. In 20/50 (40%) of GNAS mutated-specimens neither duct dilation nor cyst communication with the duct system was observed. Conclusions Based on the present evaluation, GNAS is found to be mutated in a significant number of cases lacking the classic features of IPMNs. Further work is needed to better understand the role of GNAS in discriminating IPMNs from other cystic neoplasms of the pancreas and in evaluating the malignant potential of either; additional surgical outcomes would be valuable for this assessment. 1. Sci. Transl. Med. 3, 92ra66 (2011).

Su1367 Clinical Impact of Pancreatic Duct Lavage Cytology With Cell Block Method for Discriminating Benign and Malignant Intraductal Papillary Mucinous Neoplasm of the Pancreas

Su1367 Clinical Impact of Pancreatic Duct Lavage Cytology With Cell Block Method for Discriminating Benign and Malignant Intraductal Papillary Mucinous Neoplasm of the Pancreas Jitsuko Maruki*, Jin Kan Sai, Yuji Matsumura, Tomoyasu Ito, Ryo Kanazawa, Sumio Watanabe Gastroenterology, juntendo university, Tokyo, Japan Background: The aim of the present study was to examine the usefulness of pancreatic duct lavage cytology with cell block method for discriminating benign and malignant intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Methods: Between January 2009 and June 2011, pancreatic duct lavage cytology with cell block method was investigated in 23 patients with IPMNs preoperatively. The cell block sections were prepared using sodium alginate method and subjected to hematoxylin and eosin (HE) staining and immunohistochemical stainings (MUC1, 2, 6). The sensitivity and specificity of the cytology results were assessed. The agreement of cytological and histological resuls of mucin staining was also examined. Results: The cell block diagnosis was cancer positive (class V or class IV) in 7, and negative (class I, II, III, and non-informative) in 16. The sensitivity, specificity, positive and negative predictive value of the lavage cytology with cell block method was 88%, 100%, 100%, and 94%, respectively. The cytological and histological results of MUC1, 2, 5AC and 6 were in agreement in 89% (8/9), 89% (8/9), 100% (9/9), and 100% (9/9), respectively. Conclusion: Pancreatic duct lavage cytology with cell block method may be useful not only for the differentiation between benign and malignant IPMN, but also for the diagnosis of its mucin type.

Mucin-rich salivary duct carcinoma with signet-ring cell feature ex pleomorphic adenoma of the submandibular gland: a case report of an unusual histology with immunohistochemical analysis and review of the literature

Salivary duct carcinoma (SDC) is a highly aggressive salivary gland carcinoma, and the mucin-rich variant of SDC (mSDC) is extremely rare. We report herein one case of salivary mSDC, showing predominantly signet-ring cell features. The patient was an 84-year-old Japanese woman, who noticed swelling in the left submandibular region. The tumor consisted of two components: one was of mSDC, which contained numerous signet-ring cells in large mucinous lakes, whereas the other was of preexisting pleomorphic adenoma (PA), which showed a hyalinized nodule. mSDC was markedly positive for mucin staining. Signetring cells in the mSDC component were immunopositive for androgen receptor, gross cystic disease fluid protein-15, MUC1, MUC2, MUC5AC, MUC5B, and MUC6, but negative for Her-2 and myoepithelial markers. This case was diagnosed as mSDC with the signet-ring cell feature ex PA. We discuss herein the significance of the signet-ring cell feature in mSDC and the mucin pattern of mSDC.

Modulation of intestinal mucin composition and mucosal morphology by dietary phytogenic inclusion level in broilers

The effect of a dietary phytogenic feed additive (PFA) inclusion level in mucin monosaccharide composition, mucosal morphometry and mucus histochemistry along the broiler intestinal tract was studied. Cobb male broilers (n = 525) were allocated into five experimental treatments that, depending on the type of addition in the basal diet (BD), were labeled as follows: C (BD based on maize–soybean meal with no other additions), E1 (80 mg PFA/kg BD), E2 (125 mg PFA/kg BD), E3 (250 mg PFA/kg of BD) and A (2.5 mg avilamycin/kg BD). Samples from duodenum, ileum and cecum of 14- and 42-day-old broilers were collected and analyzed. In 14-day-old broilers, treatments E2 and E3 had higher (P < 0.01) duodenal mannose than treatments C, E1 and A. Ileal mannose was lower (P < 0.05) in treatment C compared with PFA treatments, and ileal galactose (Gal) was higher (P < 0.01) in treatments E2 and E3 compared with C and A. Polynomial contrast analysis with respect to PFA inclusion level showed that in 14-day-old broilers there was a linear increase (P = 0.001) in duodenal mannose and a quadratic effect (P = 0.038) in duodenal N-acetyl-galactosamine with increasing PFA level. Ileal Gal and mannose increased linearly (P = 0.002 and P = 0.012, respectively) with PFA inclusion level. There were no significant differences between treatments in mucin monosaccharide molar ratios of 42-day-old broilers. However, increasing PFA inclusion level resulted in a linear decrease of ileal fucose (P = 0.021) and cecal N-acetylgalactosamine (P = 0.036). Experimental treatments did not differ (P > 0.05) regarding duodenal villus height (Vh), crypt depth (Cd) and Vh/Cd ratio, irrespective of broiler age and the intestinal segment examined. However, increasing dietary PFA inclusion level showed a pattern of linear increase of duodenal Vh/Cd ratio in 14-day-old broilers and ileal Vh in 42-day-old broilers (P = 0.039 and P = 0.039, respectively). Alcian Blue–Periodic Acid-Schiff (pH 2.5) staining of neutral and acidic mucins showed that the staining intensity of mucus layer in villi was fragment (i.e. tip, midsection and base) dependent, whereas in crypts it was dependent both on intestinal segment (i.e. duodenum, ileum and cecum) and fragment. Finally, mucus layer thickness did not differ (P > 0.05) between treatments, yet a pattern of linear increase (P < 0.05) with PFA inclusion level was observed in the duodenum of 42-day-old broilers. In conclusion, the dietary inclusion level of PFA modulated broiler intestinal mucin composition and morphology. Further studies are required to elucidate the physiological implications of such changes in host–microflora interactions.

Crystalloids in Submandibular Sialadenitis

Fine needle aspiration cytology of the salivary glands plays an important role in the diagnosis of neoplastic and non neoplastic lesions of salivary gland. Crystalloids in salivary gland lesions are a rare occurrence. A case of submandibular sialomegaly in an elderly patient with a clinical suspicion of metastasis was subjected to FNAC which showed crystalloids morphologically resembling amylase occurring in a background of sialadenitis. These crystals are non birefrigent, eosinophilic structures with a variety of geometric shapes including needles, rods, rectangular structures that do not stain with mucin stains. Other important crystalloids which can be observed in neoplastic and non-neoplastic salivary gland lesions are tyrosine-rich crystalloids, collagenous crystalloids, and oxalate crystals. These can be distinguished both morphologically and on the basis of several staining techniques in addition to biochemical studies. FNAC plays a major role in the diagnosis of these lesions and helps in avoiding unnecessary surgery.

Frequency of Subtypes of Biliary Intraductal Papillary Mucinous Neoplasm and Their MUC1, MUC2, and DPC4 Expression Patterns Differ from Pancreatic Intraductal Papillary Mucinous Neoplasm

Biliary intraductal papillary mucinous neoplasm (B-IPMN) has been proposed as a unique clinicopathologic disease with distinct histopathologic features, although wide acceptance remains controversial. A recent consensus conference classified pancreatic IPMN (P-IPMN) into 4 subtypes (ie, gastric, intestinal, pancreatobiliary, oncocytic) based on morphologic appearance and mucin (MUC) staining properties. The aim of this study was to determine whether B-IPMN has similar histopathologic and immunologic subtypes to P-IPMN. Specific immunostaining for MUC1, MUC2, and deleted for pancreas cancer, locus 4 were performed on specimens from 19 patients with a histopathologic diagnosis of B-IPMN. Immunostaining patterns of B-IPMN were correlated with histopathology. Based on histopathology, the following subtypes of B-IPMN were identified: pancreatobiliary n = 9 (47%), intestinal n = 8 (42%), oncocytic n = 2 (11%), and gastric n = 0 (0%). Pancreatobiliary and oncocytic subtypes of B-IPMN were positive for MUC1 and negative for MUC2, and intestinal subtypes were positive for MUC2 and negative for MUC1. Thirteen of the 19 B-IPMN were associated with invasive carcinoma; loss of deleted for pancreas cancer, locus 4 was found in 6 of 13 invasive components and in 3 of 19 noninvasive components of B-IPMN. Five-year survival for patients with resected B-IPMN and invasive carcinoma was 38%, which is similar to that for resected P-IPMN with invasive carcinoma. Histopathologic subtypes and type-specific MUC expression patterns of B-IPMN resemble those of P-IPMN. MUC1 expression and/or absence of MUC2 expression, which correlate with aggressive features of P-IPMN, were found in B-IPMN and correlate with invasive B-IPMN. Loss of deleted for pancreas cancer, locus 4 parallels the findings observed in P-IPMN. These findings provide additional support that B-IPMN is a unique entity with similarities to main duct P-IPMN.

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.

Elevated Alpha-Fetoprotein Level: A Dilemma

To the Editor: This letter is in regard to the article, “Hepatoid esophageal carcinoma: a rare cause of elevated alpha fetoprotein” published in Journal of Gastrointestinal Cancer by Atiq et al. [1]. We share our experience in managing a patient who presented with esophageal cancer, liver metastasis, and elevated alpha-fetoprotein (AFP). A 51-year-old Caucasian male presented in the emergency room with increasing difficulty swallowing, pain in his right upper quadrant, and stating that he had lost 20 lbs over the last 2 months. He was also complaining of nausea, food sticking in his lower chest, and spitting up after eating. The patient’s history was positive for gastroesophageal reflux disease, smoking for 40 pack-years and alcoholism, which he quit 20 years ago, and a family history of esophageal cancer. He denies any constipation, diarrhea, melena, or bleeding from the rectum. His pertinent physical examination was negative for enlarged liver, ascites, or swollen testicles. A computed tomography (CT) was done in the emergency room, which showed at least two dozens of variably sized liver metastases. His complete blood count, comprehensive metabolic panel, and viral hepatitis panel were nonsignificant/negative. His serum AFP level was 24,430 ng/mL, and carcinoembryonic antigen was 7.5 ng/mL. CT scan of the chest was negative. His esophagogastroduodenoscopy was positive for a large fungating lower esophageal mass. Esophageal biopsy showed poorly differentiated adenocarcinoma with areas of signet ring cells (Fig. 1). Periodic acid-Schiff (PAS) and mucin stains were positive. The tumor cells were strongly positive with Ber-EP4 and p53 immunostains and weakly positive for Her2/NEU. CK5/6, AFP, HCG, and TTF-1 immunostains were negative. Rare tumor cells were positive for HepPar-1. There was a high proliferative fraction of greater than 75%, as assessed by Ki-67, indicative of probably biologically aggressive behavior. Due to elevated AFP, liver biopsy was done, which showed tumor to be positive with CK20, CDX2, MOC31, and Mucicarmine (Fig. 2). It was negative with stains including CK7, TTF-1, chromogranin, synaptophysis, and CK5/6.

Intraductal papillary neoplasm of the bile duct (IPNB): A biliary equivalent to IPMN of the pancreas?

201 Background: Intraductal papillary neoplasm of the bile duct (IPNB) is a type of cholangiocarcinoma characterized by intraductal growth, mucin production and a better outcome compared to the more common nodular-sclerosing type. IPNB may be analogous to IPMN of the pancreas and may be a precursor of invasive cholangiocarcinoma, but its pathogenesis and natural history are ill-defined. This study examines the incidence, clinicopathologic features and outcome of IPNB in a single center. Methods: A consecutive cohort of patients with bile duct cancer (hilar, intrahepatic or distal) was reviewed and those with papillary features identified. Histopathologic morphology and immunohistochemical staining for cytokeratin and mucin proteins were utilized to classify IPNB into subtypes. Survival data were analyzed and correlated with clinicopathological parameters. Results: Between 1993 and 2008, 40 IPNBs were identified in hilar (24/144), intrahepatic (4/86) and distal (12/113) bile duct cancer specimens (11.7%). Histopathologic examination revealed 27 pancreatobiliary, 4 gastric, 3 intestinal, and 6 oncocytic subtypes; cytokeratin and mucin staining was similar to that of IPMNs of the pancreas. Invasive carcinoma was seen in 29/40 (72%) IPNBs. Overall median survival was 59 months and was not different between IPNB locations or subtypes. Factors associated with a worse median survival included depth of invasion (39 months for &gt; 5mm, 128 months for &lt; 5mm, and 144 months for none, p &lt;0.05), R1 vs R0 resection (36 months vs 82 months, p &lt;0.05), MUC1 expression (53 months for positive vs 144 months for negative, p &lt;0.006), and CEA expression (42 months for positive vs 128 for negative, p&lt;0.02). Expression of MUC2, MUC5A, MUC6, CDX2, mesothelin, p53, Ki67, HepPar1, and B72.3 were not predictive of outcome. Conclusions: IPNBs are an uncommon variant of bile duct cancer, representing approximately 10% of all cases, occur throughout the biliary tract and share histologic and clinical features with IPMNs of the pancreas. These lesions may represent an alternative carcinogenesis pathway. Given their significant malignant potential, they should be treated aggressively with margin-negative resection. No significant financial relationships to disclose.

What’s new in non-small cell lung cancer for pathologists the importance of accurate subtyping, EGFR mutations and ALK rearrangements

In the past, the only critical point of distinction in the pathological diagnosis of lung cancer was between small cell and non-small cell lung cancer (NSCLC). The emergence of new targeted therapies and clinical trials demonstrating differing efficacy and toxicity of treatments according to specific histological subtypes of NSCLC, has resulted in an increasing need for improvements in pathological diagnosis. Accurate distinction between adenocarcinoma and squamous cell carcinoma is now critical as histological subtyping has the potential to influence clinical decision making and impact on patient outcome. While morphological criteria remain the most important feature to distinguish NSCLC subtypes, use of mucin and immunohistochemical stains (TTF-1, p63 and CK5/6) can be of assistance in difficult small biopsy cases. With the emergence of selective kinase inhibitors targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), there is a corresponding need to identify the subset of NSCLCs harbouring specific genetic mutations associated with sensitivity to these agents, almost all of which are found in adenocarcinomas. In this review, the importance of accurately subtyping NSCLC is discussed, along with a suggested approach for distinguishing histological subtypes in small biopsy specimens. The significance of EGFR and ALK mutations in NSCLC and the impact of these genotypes on pathology and clinical practice are also reviewed.

An Immunohistochemical Profile of the So-called Bile Duct Adenoma

The so-called bile duct adenoma and peribiliary glands are characterized by the expression of two foregut antigens (designated D10 and 1F6) and secretion of acid mucin. On account of this similarity in phenotype and their frequent close association with a large caliber bile duct, it was earlier suggested that bile duct adenoma represent a peribiliary gland hamartoma. Here, we compare the expression of 13 tissue antigens in bile duct adenomas, other benign bile duct lesions, and various foregut-derived tissues, to further investigate the bile duct adenoma phenotype and pathogenesis. Antibodies to 4 intestinal mucins, 3 cytokeratins, and CDX2, were not informative. Five foregut antigens (D10, 1F6, MUC6, MUC5AC, and TFF2) and secretion of acid mucin were of use in distinguishing bile duct adenoma from other hepatic lesions. 1F6 and MUC6 were normally present in bile ductules and canals of Hering, whereas the epithelium lining the larger bile ducts stained focally for D10, MUC5AC, MUC6, or TFF2 in, respectively, 21%, 36%, 43%, and 100% of the livers examined. Thirty-six bile duct adenomas examined were distinguished by expression of MUC6 (94% of bile duct adenoma), MUC5AC (90%), TFF2 (80%), D10 (67%), and 1F6 (61%), and varying degrees of acid mucin secretion (100%). Of 30 bile duct adenoma tested for all 5 antigens, 40% expressed all 5, 27% expressed 4, 17% expressed 3, 13% expressed 2, and 1 expressed only MUC6. Peribiliary glands invariably expressed D10, 1F6, MUC6, and TFF2, and showed acid mucin secretion, with MUC5AC present in the inflamed peribiliary glands of 3/4 livers with recurrent pyogenic cholangitis, but none of the glands of the other 23 normal or diseased livers tested. The acini of pyloric gland metaplasia in gallbladder and terminal ileum also stained for D10, 1F6, MUC6, and TFF2, with MUC5AC focally present in the gastric foveolar metaplasia overlying the pyloric gland metaplasia but not in the metaplastic glands. MUC6 was expressed in 92% of ductular reactions, 1F6 in 42%, and D10 in 25%. Focal expression of MUC6, or TFF2 was observed in 1 or 2 examples of 14 von Meyenburg complexes and 6 polycystic livers, with staining for acid mucin generally obvious only in the glycocalyx of the epithelium of these two types of lesions. The distinguishing feature of so-called bile duct adenoma is their display of the same phenotype as pyloric gland metaplasia. It is concluded that they develop as a localized biliary healing response equivalent to the function of a peribiliary gland or pyloric gland metaplasia in the foregut.

Subtyping of Undifferentiated Non-small Cell Carcinomas in Bronchial Biopsy Specimens

The emergence of treatments for non-small cell lung carcinoma (NSCLC) with differential efficacy and toxicity between subtypes has highlighted the importance of specific pathologic NSCLC subtyping. Most NSCLCs are inoperable, and pathologic diagnosis is made only on small tissue samples that are prone to diagnostic inaccuracy. In a substantial proportion of cases, standard morphology cannot specifically subtype the tumor, necessitating a diagnosis of NSCLC-not otherwise specified (NOS). Histochemical staining for mucin and immunohistochemical (IHC) identification of NSCLC subtype-associated markers could help predict the final subtype of resected NSCLCs diagnosed as NSCLC-NOS on preoperative bronchial biopsy samples. Paraffin sections of 44 bronchial biopsy samples diagnosed as NSCLC-NOS were stained for mucin (Alcian blue/periodic acid Schiff) and thyroid transcription factor 1 by IHC-(markers of adenocarcinoma), and for S100A7, cytokeratin 5/6, high molecular weight cytokeratins, and p63 proteins-markers of squamous cell carcinoma. A predictive staining panel was derived from statistical analysis after comparing staining profiles with the final postsurgical NSCLC subtype. This panel was prospectively applied to 82 small biopsy samples containing NSCLC. True NSCLC subtype of undifferentiated NSCLC samples was best predicted using Alcian blue/periodic acid Schiff plus p63 and thyroid transcription factor 1 IHC, allowing specific subtyping in 73% of NSCLC-NOS cases with 86% accuracy. When applied prospectively, this staining panel showed 100% concordance with specific NSCLC morphologic subtyping in small biopsies. This approach can facilitate treatment selection by accurately predicting the subtype in undifferentiated NSCLC biopsies, reducing to 7% the proportion of cases without a definite or probable histologic subtype.

Refining the Diagnosis and EGFR Status of Non-small Cell Lung Carcinoma in Biopsy and Cytologic Material, Using a Panel of Mucin Staining, TTF-1, Cytokeratin 5/6, and P63, and EGFR Mutation Analysis

The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.