Autophagy in advanced low- and high-grade tubular adenocarcinomas of the stomach: An ultrastructural investigation.

Autophagy represents a catabolic process in which cellular protein and organelles are engulfed into autophagosomes, digested in lysosomes and reutilized for the cellular metabolism. In neoplastic conditions, autophagy may act either as a tumour suppressor avoiding the accumulation of damaged proteins and organelles or as a mechanism of cell survival promoting the tumour growth. Although enhanced autophagy has been reported in hypoxic areas of solid tumors, there are only few ultrastructural reports concerning the relationships between autophagy and tumor grade. In the present study, we have performed an ultrastructural investigation aimed to document autophagy in a cohort of advanced gastric carcinomas of tubular type, correlating the observed findings with low and high tumor grade. Among 71 surgically resected cases of advanced gastric carcinomas, we have selected twelve low-grade and thirteen high-grade tubular adenocarcinomas. Autophagic vacuoles (AV) were only occasionally found in low-grade tubular carcinomas, while they constituted a frequent finding in high-grade ones (p < 0.01). Moreover, in high-grade tubular adenocarcinomas, our data revealed a morphologic association between autophagy and nuclear changes, such as multinucleation, micronucleation and nuclear buds, largely considered as ultrastructural aspects of mitotic instability. However, an increased autophagy was associated with organelle-poor cytoplasm or a senescent phenotype, characterized by lipofuscin granules and cytoplasmic vacuoles. In the light of our observations, it may be suggested that autophagy should be considered a phenomenon mainly related to the cellular differentiation and tumor progression.

Cytohistological and immunohistochemical characteristics of spindle-shaped mesenchymal neoplasms occurring in the gastrointestinal tract.

The purpose of the present review is to analyze the cytohistological and immunohistochemical characteristics of spindle-shaped mesenchymal gastrointestinal neoplams (MGNs), a group of unusual neoplastic conditions with different biological behavior. These tumors exhibit clinical pictures strictly related to the site of origin and dimensions, even if they appear generally with an intramural localization. This latter point may suggest an useful application of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), mainly followed by the cell-block procedure (CBP) in the differential diagnostic approach. First of all, we discuss the most common entity of MGNs represented by gastrointestinal stromal tumors (GISTs), analyzing the morphologic characteristics and stressing the strength of immunohistochemical algorithm for diagnostic purposes. Successively, we have reported the less common group of spindle-shaped MGNs comprehensive of those arising elsewhere the soft tissues, such as leiomyomas, leiomyosarcomas, schwannomas, inflammatory myofibroblastic tumor and intra-abdominal desmoid fibromatosis. Finally, very uncommon spindle-shaped MGNs, like clear cell, follicular dendritic cell, undifferentiated pleomorphic and radiation-induced sarcomas as well as spindle cell dedifferentiated liposarcomas, have been briefly mentioned.

Morphological and Cellular Features of Innate Immune Reaction in Helicobacter pylori Gastritis: A Brief Review.

Innate and adaptive immunity are both involved in acute and chronic inflammatory processes. The main cellular players in the innate immune system are macrophages, mast cells, dendritic cells, neutrophils, eosinophils, and natural killer (NK), which offer antigen-independent defense against infection. Helicobacter pylori (H. pylori) infection presents peculiar characteristics in gastric mucosa infrequently occurring in other organs; its gastric colonization determines a causal role in both gastric carcinomas and mucosa-associated lymphoid tissue lymphoma. In contrast, an active role for Epstein-Barr virus (EBV) has been identified only in 9% of gastric carcinomas. The aim of the present review is to discuss the role of cellular morphological effectors in innate immunity during H. pylori infection and gastric carcinogenesis.

Eosinophil-Specific Granules in Tumor Cell Cytoplasm: Unusual Ultrastructural Findings in a Case of Diffuse-Type Gastric Carcinoma.

A case of desmoplastic variant of diffuse-type gastric carcinoma in a 72-year-old woman is reported. Microscopic findings included poorly cohesive tumor cells, resembling mononuclear inflammatory cells, prominent diffuse desmoplasia, and tumor-associated tissue eosinophilia. Electron microscopy confirmed the undifferentiated phenotype of tumor cells and disclosed activated eosinophils in the tumor stroma. Eosinophil-specific granules were found either free in the tumor stroma or within the cytoplasm of some tumor cells. Electron microscopy provided also circumstantial evidence of phagocytosis of apoptotic eosinophils by tumor cells. Extracellular, membrane-bound, eosinophil-specific granules have been long recognized in tissues associated with eosinophilia, including allergic diseases, inflammatory responses to helminths, and in stroma of some neoplasms. Our ultrastructural study now extends these findings and provides additional morphological evidence of eosinophil-specific granules within the cytoplasm of gastric carcinoma cells.

Discordance rate of HER2 status in primary gastric carcinomas and synchronous lymph node metastases: a multicenter retrospective analysis.

BACKGROUND: The assessment of human epidermal growth factor receptor 2 (HER2) gene amplification is essential in order to identify those patients affected by advanced gastric cancer who may benefit from Trastuzumab targeted therapy. MATERIALS AND METHODS: With the aim to investigate the concordance rate in HER2 status between primary gastric carcinoma (GC) and synchronous lymphnode metastases, we investigated HER2 status in a cohort of 108 surgical formalin-fixed paraffin-embedded specimens of GC and matched synchronous metastatic lymph nodes collected from three different units of Anatomic Pathology in southern of Italy. Fleiss-Cohen weighted k statistics were used to assess the concordance rate of HER2 status. RESULTS: HER2 amplification was observed in 17% of primary GCs and the overall concordance rate with corresponding nodal metastases was 90.74%. Changes in HER2 status between primary GC and matched synchronous metastases were evidenced in 10 (9.26%) cases. Of these, 6 cases were HER2 amplified in the primary GC and not amplified in the metastases, while 4 were HER2 not amplified in the primary tumour and amplified in the lymph node metastases. CONCLUSIONS: Although at present the simultaneous determination of HER2 in advanced gastric cancer and corresponding metastatic lymph nodes is not mandatory, the possibility that the synchronous metastases of GC have a different HER2 status from that of the primary tumour is of remarkable significance; Indeed this may have influence on the therapeutic management and prognosis of the patients.

Chronic allergic-like inflammation in the tumor stroma of human gastric carcinomas: an ultrastructural study.

Inflammatory cell infiltration around the sites of carcinoma invasion is believed to play important roles in tumor biological behavior. The status of inflammatory cell infiltration at the sites of frank invasion in 92 cases of gastric carcinomas was examined, with special emphasis on tumor-associated tissue eosinophilia (TATE). TATE was found in 7 out of 92 (7.6%) gastric carcinomas (6 of intestinal-type and 1 of diffuse-type). Electron microscopy, selectively performed in the 7 cases of gastric carcinomas with TATE, showed that eosinophils participated in the stromal reaction by interacting with tumor cells, mast cells, and each other. Most of the tumor-infiltrating mast cells exhibited anaphylactic or piecemeal degranulation, indicating that the mast cells had been activated in situ. Some mast cells were noted in close contact to viable tumor cells, suggesting the existence of direct cell-to-cell interactions. There was also extracellular deposition of free eosinophil granules and Charcot-Leyden crystals. These morphologic findings are similar to that described in late/chronic-phase allergic reaction in both human and experimental animals, where angiogenesis and fibrosis/tissue repair are also present. In conclusion, TATE may indicate a chronic allergic-like Th2 host-tumor reaction, and understanding these pathways should create tools to enhance defence and contrast neoplastic disease.

Autophagy-related Prognostic Signature in HER2 Positive Gastric Carcinomas.

BACKGROUND: The immunohistochemical analysis of autophagy-related proteins (ATGs) has been recently applied in human pathology to study differentiation and cancer progression. The aim of the present study is to analyze a cohort of gastric carcinomas (GC) by five ATG antisera (Beclin-1, LC3A/B, p62, ULK-1 and AMBRA-1), also evaluating their possible relationship with clinicopathological parameters, HER2 status and final outcome of patients. METHODS: A cohort of 123 GCs has been studied by ATG antisera utilizing Masuda's criteria that define positive cases in which at least two out of five protein expressions were documented. RESULTS: The immunohistochemical signature for autophagy (A-IHC) was 49.59% as a whole. The percentage of A-IHC ranged from 31% for poorly cohesive carcinomas to 56% for adenocarcinomas. The performance of each ATG immunomarker documented high values for sensitivity, specificity and efficiency for LC3A/B, Beclin-1 and p62. In univariate analysis of GC, grade, stage, Ki67 expression, HER2 status as well as A-IHC appeared as emerged as relevant parameters with a high p-value (p < 0.001). Finally, in multivariate analysis, HER2 status, stage and A-IHC emerged as independent prognostic variables. In the comparison of survival curves, GC cases immunoreactive for A-IHC exhibited a shorter survival with a worse outcome. CONCLUSIONS: We have hypothesized that A-IHC could represent an additional morphological tool to provide prognostic elements in order to identify patients affected by aggressive with shorter survival and worse outcome.

Abnormal nuclear structures (micronuclei, nuclear blebs, strings, and pockets) in a case of anaplastic giant cell carcinoma of the thyroid: an immunohistochemical and ultrastructural study.

The authors report a case of a 70-year-old woman with an anaplastic giant cell thyroid carcinoma, along with immunohistochemical and electron microscopic findings. Histologically, the tumor is characterized by mononucleated and multinucleated giant cells, lack of architectural cohesion, atypical mitoses, and extensive areas of coagulative necrosis. Tumor cells showed AE1/AE3 positivity as well as nuclear overexpression of p53 and ki-67. Semithin sections revealed multiple nuclei with heterogeneous size ranging from micronuclei to large-size (giant) nuclei. Micronuclei were confirmed by electron microscopy that disclosed also the presence of nuclear blebs, strings, and pockets. Morphological findings of these abnormal nuclear structures in conjunction with p53 and Ki-67 nuclear overexpression suggested a faulty mitotic checkpoint/mitotic catastrophe in the progression of anaplastic giant cell thyroid carcinoma.

Ultrastructural descriptions of heterotypic aggregation between eosinophils and tumor cells in human gastric carcinomas.

A histological variant of gastric adenocarcinoma, characterized by an intense tumor-associated tissue eosinophilia (TATE), has been occasionally reported in the literature. The purpose of this ultrastructural study was to determine the interactions between frequently occurring eosinophils and tumor cells in gastric carcinoma characterized by TATE. Fresh tumor tissue of 92 gastric carcinomas was processed for both light and electron microscopic examination. Intense TATE was found in 7 out of 92 (7.6%) gastric carcinomas (6 of intestinal-type and 1 of diffuse-type). Electron microscopy, selectively performed in 7 cases with intense TATE, revealed eosinophils, singly or in groups, in contact with damaged or necrotic tumor cells. Activated eosinophils showing piecemeal degranulation were also found in intimate contact with viable tumor cells, characterized by plasma membrane caveolar invaginations. The authors regard this close morphological relationship as in vivo evidence for possible cross-talk between eosinophil and viable tumor cell, a conclusion that has already been drawn from experimental studies, but until now inadequately supported by ultrastructural observations in a human tumor.

Intraepithelial infiltration by mast cells in human Helicobacter pylori active gastritis.

Recent observations suggest an involvement of mast cells in Helicobacter pylori gastritis, but the mechanism of intraepithelial mast cell activation in H. pylori-infected patients remains to be clarified. Intraepithelial mast cells, identified by immunohistochemistry for CD117, were quantified in antral biopsies from 6 patients with H. pylori "active" chronic gastritis, 7 patients with H. pylori "nonactive" gastritis, and 9 controls. Antral biopsies from patients with H. pylori "active" gastritis showed higher intraepithelial mast cell counts than those from patients with H. pylori "nonactive" gastritis and from controls. Electron microscopy, selectively performed in 6 cases of H. pylori "active" gastritis, confirmed the presence of intraepithelial mast cells and allowed their subdivision into mature cells with intact electron-dense granules or degranulated cells. Other mast cells appeared to migrate through defects in the basement membrane into the epithelial layer. Mast cells in these areas often showed piecemeal degranulation or were characterized by large canaliculi, expanded Golgi areas, and a few granules, a process similar to the phase of recovery from anaphylactic degranulation of isolated human mast cells. The possible significance of these unusual ultrastructural findings is discussed.

Evolutionarily-Related Helicobacter pylori Genotypes and Gastric Intraepithelial Neoplasia in a High-Risk Area of Northern Italy.

Helicobacter pylori (Hp) is the major recognized risk factor for non-cardia gastric cancer (GC), but only a fraction of infected subjects develop GC, thus GC risk might reflect other genetic/environmental cofactors and/or differences in virulence among infectious Hp strains. Focusing on a high GC risk area of Northern Italy (Cremona, Lombardy) and using archived paraffin-embedded biopsies, we investigated the associations between the Hp vacA and cagA genotype variants and gastric intraepithelial neoplasia (GIN, 33 cases) versus non-neoplastic gastroduodenal lesions (NNGDLs, 37 cases). The glmM gene and the cagA and vacA (s and m) genotypes were determined by polymerase chain reaction (PCR) and sequencing. Hp was confirmed in 37/37 (100%) NNGDLs and detected in 9/33 GINs (27%), consistently with the well-known Hp loss in GC. CagA was detected in 4/9 Hp-positive GINs and in 29/37 NNGDLs. The vacA s1a and m1 subtypes were more common in GINs than in NNGDLs (6/7 vs. 12/34, p=0.014, for s1a; 7/7 vs. 18/34, p=0.020 for m1), with significant vacA s genotype-specific variance. The GIN-associated vacA s1a sequences clustered together, suggesting that aggressive Hp strains from a unique founder contribute to GC in the high-risk area studied.

Ultrastructural observations on inflammatory angiogenesis in gastric carcinomas with massive neutrophil infiltration.

Neutrophils are traditionally thought of as terminal effectors of inflammatory reaction, but experimental studies suggest that they play a direct role in the inflammatory angiogenesis of tumors. Thus, further evidence in humans is required regarding the mechanisms by which neutrophils induce tumor angiogenesis. In this study, 4 cases of human gastric carcinomas with massive neutrophil infiltration were studied by light and electron microscopy, focusing on the inflammatory angiogenesis in the tumor stroma. At light microscopy, the tumors were advanced gastric carcinomas in which various degrees of tubular differentiation were present. Under an electron microscope, pericytes exhibited two major differentiated states with distinct ultrastructural features: a contractile phenotype and a synthetic phenotype. The contractile phenotype was characterized by abundant microfilaments. Synthetic pericytes contained abundant rough endoplasmic reticulum, lipid bodies, and numerous membrane-bound vesicles. These ultrastructural findings extend concept of contractile/synthetic phenotype modulation, originally described in smooth muscle cells, to tumor microvascular pericytes. Tumor microvasculature was also characterized by abortive or slit-like lumina, endothelial cell mitoses, and replicating basement membranes. These qualitative and observational transmission electron microscopy findings provide additional morphological evidence of active inflammatory angiogenesis in gastric carcinomas with massive neutrophil infiltration.

Immunohistochemical Expression of Autophagy-Related Proteins in Advanced Tubular Gastric Adenocarcinomas and Its Implications.

In neoplastic conditions, autophagy may act as a tumor suppressor avoiding the accumulation of damaged proteins and organelles or as a mechanism of cell survival promoting the tumor growth. Although ultrastructural analysis has been considered the traditional method to identify autophagy, some proteins such as microtubule-associated protein 1 light chain 3 (LC3A/B), Beclin-1 and activating molecule in Beclin-1-regulated autophagy protein-1 (AMBRA-1) may be considered as markers of autophagy-assisted cancerogenesis. Herein, we analyzed a cohort of advanced tubular gastric adenocarcinomas by the abovementioned immunohistochemical antisera; through immunohistochemistry, autophagy (A-IHC) is diagnosed when at least two out of the three proteins are positive in the samples. Immunostaining for LC3A/B, Beclin-1, and AMBRA-1 was exclusively found in neoplastic elements, but not in surrounding stromal cells. In detail, LC3A/B and Beclin 1 were expressed both in the cytoplasm and in the nucleus of the cancer cells, while AMBRA-1 was preferentially localized in the nucleus, mainly in high grade cases. LC3A/B, Beclin 1, and AMBRA-1 expression were positive in 18 (56.2%), 17 (53.1%), and 12 (37.5%) cases, respectively. The sensibility and specificity of LC3A/B and Beclin-1 ranged from 81.25% to 93.75%, with high efficiency (90.63%) for Beclin-1. Moreover, the ultrastructural autophagic index (AI) was also available in all cases. All high-grade cases documented a Ki-67 labelling index (LI) ≥ 30%, even if three low-grade cases revealed a high Ki-67 value; p53 positivity was encountered in 21/32 (65.62%) of cases, independently of the tumor grade. A statistically significant correlation among A-IHC and clinicopathological parameters such as grade, stage, clinical course, Ki-67 LI and AI was revealed. Univariate analysis documented a significant p-value for the same autophagic variables. Additionally, multivariate survival analysis identified the grade, AI and A-IHC as independent significant variables. Finally, the overall survival curves of all cases of gastric tubular adenocarcinoma were greatly dependent on A-IHC. Therefore, we suggest that autophagic-related proteins might be considered promising predictive prognostic factors of advanced gastric cancer. Further investigations may be required to determine whether new targeted therapies should be addressed to autophagy-related proteins.

Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease.

AIM: To evaluate the role of genetic factors in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), we investigated the single nucleotide polymorphisms (SNPs) of NOD2/CARD15 (R702W, G908R and L1007finsC), and Toll-like receptor 4 (TLR4) genes (D299G and T399I) in a selected inflammatory bowel disease (IBD) population coming from Southern Italy. METHODS: Allele and genotype frequencies of NOD2/CARD15 (R702W, G908R and L1007finsC) and TLR4 (D299G and T399I) SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS: NOD2/CARD15 R702W mutation was significantly more frequent in CD (9.8%) than in controls (2.4%, P = 0.001) and in UC (2.3%, P = 0.03). No significant difference was found between UC patients and control group (P > 0.05). In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD (9.8%) compared with controls (2.9%, P = 0.002) and UC patients (2.3%, P = 0.01). Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort (allele frequencies: D299G-controls 3.9%, CD 3.7%, UC 3.4%, P > 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P > 0.05). CONCLUSION: These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/CARD15, but not TLR4 SNPs, are associated with increased risk of CD.

Abnormal nuclear structures (micronuclei, nucleoplasmic bridges, and nuclear buds) in a pleomorphic giant cell carcinoma of the stomach.

A rare case of pleomorphic giant cell carcinoma of the stomach in a 70-year-old man is reported. Characteristic microscopic findings included a general lack of architectural cohesiveness, aggregates of mononucleated or multinucleated giant cells, extensive areas of coagulative necrosis, and numerous mitoses. Immunohistochemically, tumor cells displayed cytoplasmic immunoreactivity for cytokeratin AE1/AE3 as well as overexpression of p53 and Ki-67. Electron microscopy revealed paranuclear tonofilaments bundles in giant cells confirming their epithelial nature. Furthermore, giant cells contained two or more nuclei with heterogeneous size, nucleoplasmic bridges, nuclear buds, and micronuclei. Similar abnormal nuclear structures have been closely related to breakage-fusion-bridge type of mitotic disturbances in tumor cell lines, and have not been previously reported in a human tumor.

Microvascular changes in human gastric carcinomas with coagulative necrosis: an ultrastructural study.

Ultrastructural findings in three cases of gastric carcinoma with coagulative necrosis are reviewed with special emphasis on microvascular changes. Intratumoral microvasculature revealed more or less stabilized vessels. Some were characterized by a close association between pericytes and endothelial cells, whereas others showed laminated basement membrane, with a loose association between pericytes and endothelial cells. Some mural cells exhibited ultrastructural signs of regressive changes, including lipofuscin granules, swollen mitochondria, and cytoplasmic lucency. These findings are discussed in relationship to a number of recent studies of the microvascular injury caused by hypoxia and reoxygenation, in humans and animals.

Micropapillary carcinoma of the breast with necrosis-like cell death: a case report.

A primary invasive micropapillary carcinoma of the breast in a 46-year-old woman is reported. Histologically, it was composed predominantly of papillary tumor cell clusters without fibrovascular cores, surrounded by a clear space. Tumor cells were positive for cytokeratin (CK) 7, estrogen receptor (ER), and progesterone receptor (PR), but negative for p53, CK 20, CD34, c-Erb-B2, CK5, epidermal growth factor receptor (EGFR), vimentin, and c-kit. MUC1 expression was found at the reversed apical membrane of neoplastic cell clusters. Accordingly, electron microscopy showed the lack of basement membrane and presence of microvilli at the basal surface of the tumor cells. Moreover, ultrastructural examination revealed single tumor cell death characterized by patchy condensations of chromatin throughout the nucleus. These nuclear alterations were associated with the occurrence of empty cytoplasmic vacuoles, conferring a necrosis-like phenotype to this cell death. Alternative programmed cell deaths are reviewed and their morphologic distinction is discussed.

Human Epidermal Growth Factor Receptor 2 Status in Gastric Carcinomas with Distinctive Prevalent Cribriform Component.

OBJECTIVES: A cribriform architectural pattern has been reported in 9% of one unselected consecutively collected series of gastric carcinomas (GC) with unfavourable prognostic outcome. Taking into consideration the biological relevance of the human epidermal growth factor receptor 2 (HER2) status, we have analyzed a cohort of GC with a cribriform component more than 40% (CGC) to evaluate the HER2 amplification rate as a potential target for therapy with trastuzumab. RESULTS: HER2 overexpression was encountered in 21 of 100 (21%) GC; a progressive increase in HER2 amplification was appreciated moving from non-CGC (20.6%) towards CGC cases (21.6%), although this difference does not reach a statistical significance. Nevertheless, either in univariate or in multivariate analyses, stage and HER2 status showed a significant p value (<0.001) in CGC patients. CONCLUSIONS: Our data confirmed a worse prognosis in all CGC patients with HER2 amplification, resulting in a shorter survival time. We invite all pathologists in their daily practice to specify the occurrence of cribriform neoplastic component in GC, either in surgical or in bioptical samples, taking into practical assessment the high HER2 overexpression rate in order to correctly treat these patients with worse behavior.

Poorly Differentiated Clusters: Clinical Impact in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and it still represents a major cause of cancer-related death. Postsurgical Tumor Node Metastasis (pTNM) stage is the main prognostic factor in CRC and it currently drives therapeutic protocols after surgery. However, CRC outcome is not always predictable on the basis of pTNM stage. Hence, there is the need to find additional prognostic factors that may allow to predict the clinical course of CRC regardless of pTNM stage, so that patients may receive the most appropriate treatment for their tumor. In recent years, the prognostic value of a novel grading system based on the counting of poorly differentiated clusters (PDCs) of neoplastic cells in tumor tissue was documented in CRC. In this article, we review the clinical relevance of grading based on PDC counting in CRC. In view of its high prognostic value and reproducibility, PDC grading could be introduced in routine histopathological report of CRC and used for therapeutic decision making. Although there is initial evidence that PDC may be correlated with biomolecular profile of CRC, whether their presence is associated with response to targeted therapies is still unknown and deserves further investigation.

An Updated Review of Cribriform Carcinomas with Emphasis on Histopathological Diagnosis and Prognostic Significance.

Cribriform is a histopathological term used to describe a neoplastic epithelial proliferation in the form of large nests perforated by many quite rounded different-sized spaces. This growth pattern may be seen in carcinomas arising in different organs, and shows important prognostic implications. Therefore, recent data in literature suggest that cribriform carcinoma is a histologically and clinically distinctive type of tumour that should be separated from other similar tumour types. In this article, the pathology of cribriform adenocarcinoma of the prostate, lung, breast, stomach, colon, thyroid, and skin is discussed with particular reference to morphologic and immunohistochemical features, differential diagnosis, and clinical behaviour.