HELLS regulates transcription in T-cell lymphomas by reducing unscheduled R-loops and by facilitating RNAPII progression.

Chromatin modifiers are emerging as major determinants of many types of cancers, including Anaplastic Large Cell Lymphomas (ALCL), a family of highly heterogeneous T-cell lymphomas for which therapeutic options are still limited. HELLS is a multifunctional chromatin remodeling protein that affects genomic instability by participating in the DNA damage response. Although the transcriptional function of HELLS has been suggested, no clues on how HELLS controls transcription are currently available. In this study, by integrating different multi-omics and functional approaches, we characterized the transcriptional landscape of HELLS in ALCL. We explored the clinical impact of its transcriptional program in a large cohort of 44 patients with ALCL. We demonstrated that HELLS, loaded at the level of intronic regions of target promoters, facilitates RNA Polymerase II (RNAPII) progression along the gene bodies by reducing the persistence of co-transcriptional R-loops and promoting DNA damage resolution. Importantly, selective knockdown of HELLS sensitizes ALCL cells to different chemotherapeutic agents, showing a synergistic effect. Collectively, our work unveils the role of HELLS in acting as a gatekeeper of ALCL genome stability providing a rationale for drug design.

Discordant Eosinophilic/T-Cell Chorionic Vasculitis in a Dichorionic Diamniotic Placenta.

Eosinophilic/T-cell chorionic vasculitis (ETCV) is an idiopathic lesion composed of eosinophils, CD3+ T lymphocytes, and histiocytes. In twins, ETCV may affect only one chorionic plate, a feature defined as "discordant". We present a case of ETCV discordance in a diamniotic dichorionic placenta at 38 weeks of gestation, in which the female twin was small for gestational age, weighing 2670 g (25th percentile). The corresponding placental territory presented ETCV in two close chorionic vessels with concordance of the fetal inflammatory response. Immunohistochemistry showed an abundance of CD3+/CD4+/CD25+T lymphocytes, CD68 PG M1+ macrophages, and scattered CD8+ T cells with focal TIA-1 positivity. Granzyme B, CD20 B lymphocytes, and CD56 natural killer cells were negative. High-grade villitis of unknown etiology (VUE) was additionally found and displayed comparable ETCV findings, except for an equivalent ratio of CD4+/CD8+ T cells, but TIA-1 was focally expressed. VUE was associated with chronic histiocytic intervillositis (CHI). The combination of ETCV, VUE, and CHI may have been responsible for reduced fetal growth. Concordance was observed in the ETCV and TIA-1 expression, both in ETCV and in VUE, which is a maternal response. These findings may suggest a common antigen or chemokine pathway to which both mother and fetus accordingly responded.

Pembrolizumab-Induced Fatal Myasthenia, Myocarditis, and Myositis in a Patient with Metastatic Melanoma: Autopsy, Histological, and Immunohistochemical Findings-A Case Report and Literature Review.

Immune checkpoint inhibitors (ICIs) represent a major advance in cancer treatment. The lowered immune tolerance induced by ICIs brought to light a series of immune-related adverse events (irAEs). Pembrolizumab belongs to the ICI class and is a humanized IgG4 anti-PD-1 antibody that blocks the interaction between PD-1 and PD-L1. The ICI-related irAEs involving various organ systems and myocarditis are uncommon (incidence of 0.04% to 1.14%), but they are associated with a high reported mortality. Unlike idiopathic inflammatory myositis, ICI-related myositis has been reported to frequently co-occur with myocarditis. The triad of myasthenia, myositis, and myocarditis must not be underestimated as they can rapidly deteriorate, leading to death. Herein we report a case of a patient with metastatic melanoma who fatally developed myasthenia gravis, myocarditis, and myositis, after a single cycle of pembrolizumab. Considering evidence from the literature review, autopsy, histological, and immunohistochemical investigations on heart and skeletal muscle are presented and discussed, also from a medical-legal perspective.

Interleukin-6 expression in inflamed and non-inflamed temporal arteries from patients with giant cell arteritis.

OBJECTIVES: To evaluate whether interleukin-6 expression in the temporal arteries could be a more sensitive marker of active inflammation compared to the presence of an inflammatory infiltrate. METHODS: Sixty-three formalin-fixed, paraffin-embedded temporal artery biopsies performed between 2009 and 2012 from 32 patients with biopsy-proven giant cell arteritis, 8 patients with a negative biopsy but with a final diagnosis of giant cell arteritis, and 23 controls (patients with an initial clinical suspicion of giant cell arteritis in whom an alternative diagnosis subsequently was made) were examined. Biopsy specimens showing a transmural inflammatory infiltrate were considered positive for giant cell arteritis. Immunochemistry was performed to detect interleukin-6 in the temporal artery specimens. Slides of temporal artery biopsies were independently assessed by five readers. Interleukin-6 expression was graded as 0 (absent), 1 (mild), 2 (moderate) and 3 (marked). We considered anti-IL-6 staining positive if staining was of grade 2 or 3. RESULTS: Temporal artery biopsies specimens from patients with biopsy-proven giant cell arteritis, biopsy-negative giant cell arteritis and controls were positive for anti-interleukin-6 staining in 59%, 13% and 48% of cases, respectively. CONCLUSIONS: Interleukin-6 expression does not increase the sensitivity of temporal artery biopsy in patients with giant cell arteritis who have morphologically uninflamed arteries.

Strong Notch activation hinders bevacizumab efficacy in advanced colorectal cancer.

AIM: To assess the role of Notch activation in predicting bevacizumab efficacy in colorectal cancer (CRC). MATERIALS & METHODS: Notch activation was evaluated by immunohistochemistry (IHC) on 65 CRC enrolled within randomized clinical trials assessing first-line bevacizumab-based chemotherapy and on 21 CRC treated with chemotherapy alone. RESULTS: Strong Notch (IHC 3+) activation was negatively associated with response (18 vs 62% in low Notch cases [IHC 0, 1, 2+]; p = 0.016), progression-free survival (4.9 vs 12.1 months; p = 0.002) and overall survival (19.3 vs 30.4 months; p = 0.039). No correlation was found between Notch activation and clinical outcome in CRC treated with chemotherapy alone. CONCLUSION: A potential role of Notch activation in the antitumor activity of bevacizumab could be hypothesized.

Resveratrol-mediated apoptosis of hodgkin lymphoma cells involves SIRT1 inhibition and FOXO3a hyperacetylation.

Resveratrol (RSV), a plant-derived stilbene, induces cell death in Hodgkin lymphoma (HL)-derived L-428 cells in a dose-dependent manner (IC50 = 27 µM, trypan blue exclusion assay). At a lower range (25 µM), RSV treatment for 48 hr causes arrest in the S-phase of the cell cycle, while at a higher concentration range (50 µM), apoptosis can be detected, with activation of caspase-3. The histone/protein deacetylase SIRT1 has been described as a putative target of RSV action in other model systems, even though its role in cancer cells is still controversial. Here we show that RSV, at both concentration ranges, leads to a marked increase in p53, while a decrease of SIRT1 expression level, as well as enzyme activity, only occurred at the higher concentration range. Concomitantly, however, treatments at both concentration ranges resulted in a marked increase in K373-acetylated p53 and lysine-acetylated FOXO3a. Immunohistochemical stainings of human lymph nodes show a preferential distribution of SIRT1 in the germinal center of the follicles while the mantle zone shows nearly no staining to few positive cells. The classical HL-affected lymph nodes show a strong positivity of the diagnostic Hodgkin Reed-Sternberg cells. Notably, both the HL-derived cell lines and the Hodgkin Reed-Sternberg cells of the affected lymph nodes derive from germinal center-derived B cells. The study of SIRT1 distribution and expression on a larger number of biopsies might disclose a novel role for this histone/protein deacetylase as therapeutic target.

Survival and Recurrence in Vitreoretinal Lymphoma Simulating Uveitis at Presentation: The Possible Role of Combined Chemotherapy.

PURPOSE: To investigate the role of combined systemic and local chemotherapy in improving the survival of patients with vitreoretinal lymphoma (VRL). METHODS: Patients with VRL consecutively seen from 2006 to 2020 were retrospectively reviewed; data on the presence and time of central nervous system (CNS) involvement and treatment regimen (systemic, local or combined chemotherapy) were collected. Overall survival (OS) and progression-free survival (PFS) were calculated for each group. RESULTS: Forty-three eyes of 22 subjects with histology-proven VRL were included. Mean time of survival was 64.8 months (SE±10.8). Twelve patients (57%) presented CNS involvement, which was significantly associated with progression (r = 0.48, P = .03) and death (r = 0.56, P = .009). The isolated primary VRL group had a 5-year OS of 80%. Combined systemic and local chemotherapy reduced the risk of death by 82% (hazard ratio 0.18[0.04- 0.85]) in the entire cohort. CONCLUSION: Combined systemic and local chemotherapy significantly improved OS but not PFS of patients affected by VRL.

EBV-Driven Lymphoproliferative Disorders and Lymphomas of the Gastrointestinal Tract: A Spectrum of Entities with a Common Denominator (Part 3).

EBV is the first known oncogenic virus involved in the development of several tumors. The majority of the global population are infected with the virus early in life and the virus persists throughout life, in a latent stage, and usually within B lymphocytes. Despite the worldwide diffusion of EBV infection, EBV-associated diseases develop in only in a small subset of individuals often when conditions of immunosuppression disrupt the balance between the infection and host immune system. EBV-driven lymphoid proliferations are either of B-cell or T/NK-cell origin, and range from disorders with an indolent behavior to aggressive lymphomas. In this review, which is divided in three parts, we provide an update of EBV-associated lymphoid disorders developing in the gastrointestinal tract, often representing a challenging diagnostic and therapeutic issue. Our aim is to provide a practical diagnostic approach to clinicians and pathologists who face this complex spectrum of disorders in their daily practice. In this part of the review, the chronic active EBV infection of T-cell and NK-cell type, its systemic form; extranodal NK/T-cell lymphoma, nasal type and post-transplant lymphoproliferative disorders are discussed.

Pathophysiology of Hyperechogenic Bowel in Congenitally Human Cytomegalovirus Infected Fetuses.

Hyperechogenic bowel (HB) is a nonspecific ultrasound finding that can be associated with human cytomegalovirus (CMV) congenital infection. In this study, we investigated HB pathophysiology in CMV-infected fetuses. We examined small and large intestine as well as pancreas in 8 fetuses at 22 weeks of gestation with congenital CMV infection. Ultrasound findings showed 4 fetuses with HB and 4 without. As negative group, 4 fetuses without CMV infection and without HB were studied. Immunohistochemistry for CMV, lymphocytic infiltrate, B-cell leukemia/lymphoma-2 (bcl-2), CD-117, cystic fibrosis transmembrane regulator (CFTR) were performed. HB fetuses showed multiple and sequential CMV-positive ganglion cells of Auerbach's myenteric plexus. In the ganglia, bcl-2 was weakly expressed representing a reduced neuronal functionality. CD-117 revealed a regular distribution of Cajal cells, the pacemakers of intestinal contractility. Pancreas showed normal CFTR staining, indicating a preserved exocrine secretion, thus unlikely a contributory factor in HB. In CMV-infected fetuses without HB, CMV-positive cells were scatteredly found in ganglion cells and bcl-2 was strongly expressed. Intestinal CD-117 and pancreatic CFTR expression were similar to fetuses with HB. In conclusion, fetal CMV infection of the bowel may lead to peristalsis impairment (paralytic ileus) due to intestinal plexus involvement, which at ultrasound appeared as HB.

High levels of Notch intracellular cleaved domain are associated with stemness and reduced bevacizumab efficacy in patients with advanced colon cancer.

δ­like ligand 4 (DLL4)­Notch signaling is associated with tumor resistance to anti­vascular endothelial growth factor (VEGF) therapy. Furthermore, Notch signaling is critical for the maintenance of colon cancer stem cells (CSCs), which are relevant in drug resistance and tumor angiogenesis. CD44 is a transmembrane glycoprotein and is considered a putative marker of CSCs. To assess the association of Notch intracellular cleaved domain (NICD), DLL4 and CD44 expression with the efficacy of anti­angiogenic drugs, a series of samples derived from patients with advanced colon cancer enrolled in prospective clinical trials were analyzed. Histological samples from 51 primary tumors that originated from patients treated with bevacizumab­based first­line chemotherapy were analyzed by immunohistochemistry for NICD, DLL4 and CD44 expression, and CD31 for microvessel count. The expression levels of genes relevant for angiogenesis [angiopoietin (ANGPT)1, ANGPT2, fibroblast growth factor (FGF)1, FGF2, epidermal growth factor, placental growth factor, VEGFA and DLL4] were detected by reverse transcription­quantitative PCR using RNA extracted from the frozen tissues of four tumors with low and four tumors with high NICD expression. Strong NICD levels were observed in 12/51 (24%) of the patients, whereas 16/51 (31%) of the colon cancer subjects exhibited high CD44 expression. Strong CD44 staining was associated with high NICD levels compared with the CD44 expression levels noted in samples with low NICD levels (67 vs. 20%, P=0.005). No association was observed with regards to the expression levels of NICD, CD44 and the other aforementioned biomarkers. High expression levels of NICD and CD44 predicted reduced progression­free survival (P<0.001) and overall survival (P=0.002). No significant differences in the expression of angiogenesis­related genes were detected between low and high NICD­expressing tumors. In conclusion, NICD and CD44 tissue levels exhibited an association and may be related to a reduced survival rate in patients with advanced colon cancer treated with bevacizumab.

Methylation changes of SIRT1, KLF4, DAPK1 and SPG20 in B-lymphocytes derived from follicular and diffuse large B-cell lymphoma.

Diffuse large-B cell lymphomas (DLBCL) and follicular lymphomas (FL) are the most represented subtypes among mature B-cell neoplasms and originate from malignant B lymphocytes. Methylation represents one of the major epigenetic mechanisms of gene regulation. Silent information regulator 1 (SIRT1) is a class III lysine-deacetylase playing several functions and considered to be a context-dependent tumor promoter. We present the quantitative methylation, gene expression and tissue distribution of SIRT1 and some key mediators related to lymphoma pathogenesis in B lymphocytes purified from biopsies of follicular hyperplasias, FL and DLBCL. SIRT1 mRNA levels are higher in FL than follicular hyperplasias and DLBCL. B cell lymphoma 6 (BCL6) positively correlates with SIRT1. SIRT1 promoter shows a methylation decrease in the order: follicular hyperplasia - FL - DLBCL. Kruppel-like factor 4 (KLF4), Death-associated protein kinase 1 (DAPK1) and Spastic Paraplegia 20 (SPG20) methylation increase significantly in FL and DLBCL compared to follicular hyperplasias. Gene expression of DAPK1 and SPG20 inversely correlates with their degree of methylation. Our findings evidence a positive correlation between SIRT1 and BCL6 expression increase in FL. SIRT1 methylation decreases in FL and DLBCL accordingly and this parallels the increase of KLF4, DAPK1 and SPG20 methylation.

No detection of varicella-zoster virus in temporal arteries of patients with giant cell arteritis.

OBJECTIVE: Data on the presence of varicella-zoster virus (VZV) in temporal arteries of patients with giant cell arteritis (GCA) are controversial. We analyzed VZV infection in temporal arteries from Italian patients with temporal artery biopsy (TAB)-positive GCA, TAB-negative GCA, and controls. METHODS: A total of 79 formalin-fixed, paraffin-embedded (FFPE) TABs performed between 2009 and 2012 at a single institution from 34 TAB-positive GCA patients, 15 TAB-negative GCA patients, and 30 controls were retrieved. Six 5-µm sections of all FFPE TABs were cut. The first section was analyzed by immunohistochemistry using mouse monoclonal anti-VZVgE IgG1 antibody. DNA was extracted from the remaining five sections and analyzed by real-time polymerase chain reaction (PCR) for the presence of VZV DNA. For 10 of the 34 TAB-positive GCA patients, an additional 2-mm piece of frozen TAB was available. DNA was extracted from the entire 2-mm length frozen specimen and analyzed by PCR for the presence of VZV DNA. Thirty additional 5-µm sections were cut from the FFPE TABs of these 10 patients and analyzed by immunohistochemistry for the presence of VZV antigen. RESULTS: Immunohistochemical analysis detected VZV antigen in 1/34 (3%) TAB-positive GCA, 0/15 TAB-negative GCA, and 0/30 controls, and in none of the 300 sections cut from the 10 FFPE TABs positive for GCA for which the frozen specimens were available. DNA obtained from all TABs was amplifiable. VZV DNA was neither found in any of the FFPE TABs nor found in frozen TABs. CONCLUSION: Our data do not support in Italian patients a possible role for VZV infection in the etiopathogenesis of GCA.

Inter-relationship between PD-L1 expression and clinic-pathological features and driver gene mutations in pulmonary sarcomatoid carcinomas.

INTRODUCTION: Pulmonary Sarcomatoid Carcinoma (PSC) is a rare subset of NSCLC, associated with worse prognosis and resistant to platinum-based regimens. Recent investigations have shown high levels of PD-L1 expression in PSC, providing a rationale for the potential use of immunotherapy. In this study, we investigated whether the PD-L1 expression was related to clinico-pathologic and molecular characteristics. MATERIALS AND METHODS: Fortythree surgically-resected PSCs were selected from 2006 to 2014 and clinical information retrieved. PD-L1 expression was analyzed by immunohistochemistry and correlated with the clinic-pathologic features and driver gene mutations analyzed by Next-Generation-Sequencing. Correlation of clinical, pathological and genetic variants with PD-L1 expression positivity were tested by Fisher's exact test analysis. RESULTS: About 25% of PSCs showed a significant expression of PD-L1 (positive staining defined as staining in ≥10% of tumor cells). PD-L1 expression was associated with aggressive pathological features of PSCs including N2-involvement (PD-L1 positive in 83.3% of N2-PSCs vs in 16.2% of N0/N1-PSCs, p=0.003) and presence of either local (p=0.038) and distant metastases (p=0.022). Furthermore, PD-L1 expression was significantly associated with the overall mutational load of the tumors (PD-L1 positivity only in PSCs with at least one mutational event) and in particular with the presence of KRAS mutation (PD-L1 positive in 44.4% of KRAS-Mut PSCs vs 12.0% in KRAS-Wild PSCs). The correlation between PD-L1 expression and KRAS-mutation were found at univariate analysis (p=0.031), even considering PD-L1 as a continuous variable (p=0.018), and confirmed at multivariate analysis (p=0.035). The mutational status of the other genes explored in the NGS-panel (EGFR, APC, PTEN, PIK3CA, TP53 and STK11) did not correlate with PD-L1 expression. CONCLUSIONS: PD-L1 expression significantly correlates with overall mutational load and KRAS mutational status in pulmonary sarcomatoid carcinomas.

Does Immunohistochemistry Represent a Robust Alternative Technique in Determining Drugable Predictive Gene Alterations in Non-Small Cell Lung Cancer?

Immunohistochemistry (IHC) is a widely-tested, low-cost and rapid ancillary technique available in all laboratories of pathology. This method is generally used for diagnostic purposes, but several studies have investigated the sensitivity and specificity of different immunohistochemical antibodies as a surrogate test in the determination of predictive biomarkers in non-small cell lung cancer (NSCLC), particularly for Epidermal Growth Factor Receptor (EGFR) gene mutations, Anaplastic Lymphoma Kinase (ALK) gene and ROS1 rearrangements. In this review, a critical examination of the works comparing the consistency of IHC expression and conventional molecular techniques to identify genetic alterations with predictive value in NSCLC is discussed. Summarizing, data on sensitivity and specificity of antibodies against ALK and ROS1 are very consistent and time has come to trust in IHC at least as a cost-effective screening tool to identify patients with rearranged tumors in clinical practice. On the other hand, mutant-specific antibodies against EGFR demonstrate a good specificity but a lowto- fair sensitivity, raising some cautions on their employment as robust predictive biomarkers. A brief comment on preliminary experiences with antibodies against BRAF, RET, HER2 and c-MET is also included.

Diamond: immunohistochemistry versus sequencing in EGFR analysis of lung adenocarcinomas.

AIMS: Identification of epidermal growth factor receptor (EGFR) mutations in lung adenocarcinomas is the single most important predictor of clinical response and outcome using EGFR tyrosine kinase inhibitors (TKIs). EGFR E746-A750del and L858R mutations are the most common gene alterations, also predicting the best clinical response to TKIs. We evaluated the accuracy of EGFR mutation-specific antibodies in a large cohort of lung adenocarcinomas, with different molecular settings and types of tissue samples. METHODS: 300 lung adenocarcinomas diagnosed on cytology (48 cell blocks), biopsy (157 cases) and surgical resections (95 cases) were selected. All cases were investigated for EGFR by sequencing and two mutation-specific antibodies (clone 6B6 for E746-A750del; clone 43B2 for L858R) were tested using an automated immunostainer. Discordant results were investigated by next-generation sequencing (NGS). RESULTS: Overall sensitivity and specificity of mutant-specific antibodies were 58.6% and 98.0%, respectively, and they increased up to 84% and 100% if only tumours harbouring E746-A750del were considered. In 13 discordant cases, NGS confirmed immunohistochemistry results in eight samples. CONCLUSIONS: The EGFR mutation-specific antibodies have a fair/good sensitivity and good/high specificity in identifying classic mutations, but they cannot replace molecular tests. The antibodies work equally well on biopsies and cell blocks, possibly permitting a rapid screening in cases with poor material.

Cadherin 6 is a new RUNX2 target in TGF-ß signalling pathway.

Modifications in adhesion molecules profile may change the way tumor cells interact with the surrounding microenvironment. The Cadherin family is a large group of transmembrane proteins that dictate the specificity of the cellular interactions. The Cadherin switch that takes place during epithelial-mesenchymal transition (EMT) contributes to loosening the rigid organization of epithelial tissues and to enhancing motility and invasiveness of tumor cells. Recently, we found Cadherin-6 (CDH6, also known as K-CAD) highly expressed in thyroid tumor cells that display mesenchymal features and aggressive phenotype, following the overexpression of the transcriptional regulator Id1. In this work, we explored the possibility that CDH6 is part of the EMT program in thyroid tumors. We demonstrate that CDH6 is a new transforming growth factor-ß (TGF-ß) target and that its expression is modulated similarly to other EMT mesenchymal markers, both in vitro and in thyroid tumor patients. We show for the first time that CDH6 is expressed in human thyroid carcinomas and that its expression is enhanced at the invasive front of the tumor. Finally, we show that CDH6 is under the control of the transcription factor RUNX2, which we previously described as a crucial mediator of the Id1 pro-invasive function in thyroid tumor cells. Overall, these observations provide novel information on the mechanism of the EMT program in tumor progression and indicate CDH6 as a potential regulator of invasiveness in thyroid tumors.

Runx2 isoform I controls a panel of proinvasive genes driving aggressiveness of papillary thyroid carcinomas.

CONTEXT: The ability of tumor cells to invade adjacent tissues is governed by a complicated network of molecular signals, most of which have not yet been identified. In a recent work, we reported that the transcriptional regulator Id1 contributes to thyroid cancer progression by powering the invasion capacity of tumor cells. OBJECTIVE: The intent of this work was to further investigate the biology of invasive thyroid tumors, through the analysis of the molecular interactions existing between Id1 and some of its target genes and through the characterization of the function of these factors in the progression of thyroid tumors. RESULTS: We showed that Id1 controls the expression of the Runx2 isoform I and that this transcription factor plays a central role in mediating the Id1 proinvasive function in thyroid tumor cells. We demonstrated that Runx2 regulates proliferation, migration, and invasiveness by activating a panel of genes involved in matrix degradation and cellular invasion, which we previously identified as Id1 target genes in thyroid tumor cells. Finally, we show that Runx2 is strongly expressed in metastatic human thyroid tumors both at the primary site and in metastases. CONCLUSION: Overall, our experiments demonstrate the existence of a previously unknown molecular axis that controls thyroid tumor invasiveness by altering the ability of tumor cells to interact with the surrounding microenvironment. These factors could prove to be valuable markers that permit early diagnosis of aggressive thyroid tumors.

Epidermal growth factor receptor (EGFR) gene copy number in colorectal adenoma-carcinoma progression.

Adenomas are the easily identifiable precursors of the vast majority of colorectal cancers. Some of their morphological features, such as dysplasia, are predictive of their biological evolution toward adenocarcinomas. A large body of evidence has demonstrated that the epidermal growth factor receptor (EGFR) signaling pathway is commonly activated in colorectal cancer and EGFR-target therapies have improved the outcome for colorectal cancer patients. Nevertheless, the mechanisms underlying the role of EGFR in the adenoma-carcinoma sequence are not entirely clear. We retrospectively analyzed EGFR gene copy number by fluorescence in situ hybridization (FISH) in paraffin-embedded tissue from 215 patients recruited through a prospective colorectal cancer screening procedure and undergoing surgical colectomy. We observed that in human colorectal carcinogenesis, EGFR copy number increases progressively, from adenomas with high-grade dysplasia to locally advanced adenocarcinomas, through early invasive adenocarcinomas, suggesting that deregulation of EGFR may correlate with the malignant progression.