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.

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.

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.

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.

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.

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.

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.

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.

Pleomorphic Carcinoma of the Colon: Morphological and Immunohistochemical Findings.

Pleomorphic carcinoma is an aggressive neoplasm defined by the World Health Organization (WHO) as a poorly differentiated (squamous cell carcinoma or adenocarcinoma) or undifferentiated carcinoma in which at least 10% spindle and/or giant cells are identified, or as a carcinoma constituted purely of spindle and giant cells. Although this entity has initially been shown in the lung, it has been described also in extrapulmonary locations, with only one report for a colonic site. A 65-year-old woman developed a caecal tumour. Gross examination revealed an endophytic/ulcerative mass 7 cm in length. Microscopically, the tumour was a poorly differentiated adenocarcinoma with a pleomorphic component that occupied more than 10% of the specimen. The tumour shared these histopathological findings with pulmonary giant cell carcinoma but differed in other clinicopathological features such as a pushing growth pattern, stage pT3N1, and an uneventful outcome 24 months after operation. The pleomorphic component showed morphological and immunohistochemical features compatible with mitotic catastrophe, a non-apoptotic cell death occurring in cycling cells after aberrant mitosis. These features included multinucleation, micronucleation, atypical mitoses, foci of geographic necrosis, as well as immunohistochemical overexpression of p53 and Ki-67. The interpretation of the pleomorphic component as morphological expression of mitotic catastrophe may be useful in comprehending the pathogenesis of this rare neoplasm, and it may have practical implications as a potential cancer therapeutic target.

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.

Cell-block procedure in endoscopic ultrasound-guided-fine-needle-aspiration of gastrointestinal solid neoplastic lesions.

In the present review we have analyzed the clinical applications of endoscopic ultrasound-guided-fine-needle-aspiration (EUS-FNA) and the methodological aspects obtained by cell-block procedure (CBP) in the diagnostic approach to the gastrointestinal neoplastic pathology. CBP showed numerous advantages in comparison to the cytologic routine smears; in particular, better preservation of cell architecture, achievement of routine haematoxylin-eosin staining equivalent to histological slides and possibility to perform immunohistochemistry or molecular analyses represented the most evident reasons to choose this method. Moreover, by this approach, the differential diagnosis of solid gastrointestinal neoplasias may be more easily achieved and the background of contaminant non-neoplastic gastrointestinal avoided. Finally, biological samples collected by EUS-FNA CBP-assisted should be investigated in order to identify and quantify further potential molecular markers.

Phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

AIM: To identify those with a micropapillary pattern, ascertain relative frequency and document clinicopathological characteristics by reviewing gastric carcinomas. METHODS: One hundred and fifty-one patients diagnosed with gastric cancer who underwent gastrectomy were retrospectively studied and the presence of a regional invasive micropapillary component was evaluated by light microscopy. All available hematoxylin-eosin (HE)-stained slides were histologically reviewed and 5 tumors were selected as putative micropapillary carcinoma when cancer cell clusters without a vascular core within empty lymphatic-like space comprised at least 5% of the tumor. Tumor tissues from these 5 invasive gastric carcinomas were immunostained using an anti-mucin 1 (MUC1) antibody (clone MA695) to detect the characteristic inside-out pattern and with D2-40 antibody to determine the presence of intratumoral lymph vessels. Detection of intraepithelial neutrophil apoptosis was evaluated in consecutive histological tissue sections by three independent methods, namely light microscopy with HE staining, the conventional terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemistry for activated caspase-3 (clone C92-605). RESULTS: Among 151 gastric cancers resected for cure, 5 (3.3%) were adenocarcinomas with a micropapillary component. Four of the patients died of disease from 6 to 23 mo and one patient was alive with metastases at 9 mo. All patients had advanced-stage cancer (≥ pT2) and lymph node metastasis. Positive MUC1 immunostaining on the stroma-facing surface (inside-out pattern) of the carcinomatous cluster cells, together with negative immunostaining for D2-40 in the cells limiting lymphatic-like spaces, confirmed the true micropapillary pattern in these gastric neoplasms. In all five cases, several micropapillae were infiltrated by neutrophils. HE staining, TUNEL assay and immunostaining for caspase-3 demonstrated apoptotic neutrophils within cytoplasmic vacuoles of tumor cells. These data suggest phagocytosis (cannibalism) of apoptotic neutrophils by micropapillary tumor cells. Tumor cell cannibalism is usually found in aggressive tumors with anaplastic morphology. Our data extend these observations to gastric micropapillary carcinoma: a tumor histotype analogously characterized by aggressive behavior and poor prognosis. The results are of interest because they raise the intriguing possibility that neutrophil cannibalism by tumor cells may be one of the mechanisms favoring tumor growth in gastric micropapillary carcinomas. CONCLUSION: This is the first study showing phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

Histological grading in colorectal cancer: new insights and perspectives.

Poor histological differentiation is currently considered among the adverse histopathological factors associated with unfavourable clinical course of colorectal carcinoma (CRC). At present, the histological grade of CRC is assessed based on the percentage of glandular differentiation in the tumor according to the World Health Organization (WHO) criteria. However the prognostic value of the WHO grading system is limited by significant inter-observer variability in its assessment. In addition, the prognostic significance of WHO grading seems to depend on the microsatellite instability (MSI) status of the tumor. Finally, this grading does not apply to rarer histotypes of colorectal adenocarcinomas, such as the micropapillary, medullary, mucinous and signet ring cell variants. Recently a novel grading system based on the counting of clusters of five or more cells lacking a glandular structure (poorly differentiated clusters) and set in the tumor stroma or at invasive edge has been proposed in CRC. There is evidence that grading based on poorly differentiated clusters (PDC) is more reproducible and has more robust prognostic significance compared to WHO grading in CRC. In the present review we discuss the morphological features, criteria for the assessment, prognostic significance and correlation with biomolecular profiles of grading based on PDC counting in CRC.

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.

Mitochondrial DNA alterations in the progression of gastric carcinomas: unexplored issues and future research needs.

Gastric cancer is the second most frequent cause of cancer death worldwide. Patients infected with Helicobacter pylori (H. pylori) are at increased risk of gastric cancer. H. pylori induces genomic instability in both nuclear and mitochondrial (mt) DNA of gastric epithelial cells. Changes in mtDNA represent an early event during gastric tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis in gastric carcinoma.This review article summarizes the mtDNA mutations that have been reported in gastric carcinomas and their precancerous conditions. Unexplored research topics, such as the role of mtDNA alterations in an alternative pathway of gastric carcinogenesis, are identified and directions for future research are suggested.

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.

Inflammatory bowel disease in pediatric and adolescent patients: a biomolecular and histopathological review.

Crohn's disease (CD) and ulcerative colitis (UC) are the two main forms of inflammatory bowel disease (IBD) with both overlapping and distinct clinical, pathological and biomolecular features. It has been suggested that pediatric IBD is a distinct disease entity, with probably different disease subtypes.The aim of this study is to review and summarize the evolution of the current concept of pediatric IBD. The results of this review reinforce the idea that pediatric CD and UC may be further classified in various clinicopathologic entities. For clinicians and pathologists convenience, practical algorithms for the distinction of the various subphenotypes of pediatric IBD are also provided.

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.

HER2 status in unusual histological variants of gastric adenocarcinomas.

AIM: To investigate HER2 status in a cohort of 109 gastric adenocarcinomas also including unusual variants, such as 14 hepatoid (HAS) and 9 mitochondrion-rich (MRC), characterised by an opposing clinical behaviour. METHODS AND RESULTS: Using HercepTest (DAKO) and FISH test (pharmDx DAKO), HER2 overexpression/amplification was encountered in 23 of 109 (21.10%) of all gastric adenocarcinomas. A progressive increase in HER2 overexpression was observed moving from the poorly cohesive histotype to MRC, tubular adenocarcinomas and HAS. A statistically significant difference was found between poorly cohesive carcinomas and the others; a similar significant difference was encountered between HAS and all other variants of adenocarcinoma. HER2 overexpression was significantly associated with high grade, advanced stage, high Ki-67 labelling index value and death from gastric cancer. Multivariate analysis identified HER2 overexpression as an independent unfavourable prognostic variable for adenocarcinomas as a whole and also for the HAS variant. CONCLUSIONS: Trastuzumab has been confirmed as an additional useful therapeutic standard option for patients with HER2-positive advanced gastric cancers, and also in aggressive variants of adenocarcinomas such as HAS.