Luigi Tornillo

Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis

Hepatocellular nodules in cirrhosis include regenerative (large regenerative, LRN) and dysplastic (low and high grade, LGDN and HGDN) nodules, early and grade 1 HCC (eHCC‐G1), and overt HCC. The differential diagnosis may be particularly difficult when lesions such as HGDN and eHCC‐G1 are involved. We investigated the diagnostic yield of a panel of 3 putative markers of hepatocellular malignancy such as HSP70, glypican 3 (GPC3), and glutamine synthetase (GS). We selected 52 surgically removed nonmalignant nodules (15 LRNs, 15 LGDNs, 22 HGDNs) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2‐3) and immunostained them for HSP70, GPC3, and GS. The sensitivity and specificity of the individual markers for the detection of eHCC‐G1 were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. We identified 2 main phenotypes: (1) all negative, seen in 100% LRN and LGDN, 73% HGDN and 3% eHCC‐G1; (2) all positive, a feature detected in less than half the eHCC‐G1. Using a 3‐marker panel, when at least 2 of them, regardless which, were positive, the sensitivity and specificity for the detection of eHCC‐G1 were respectively 72% and 100%; the most sensitive combination was HSP70+/GPC3+ (59%) when a 2‐marker panel was used. Conclusion: The adopted panel of 3 markers is very helpful in distinguishing eHCC‐G1 from dysplastic nodules arising in cirrhosis. (HEPATOLOGY 2007;45:725–734.)

High frequency of tumor-infiltrating FOXP3+ regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients†

Regulatory T cells (Treg) inhibit the generation of host‐versus‐tumor immunity via suppression of tumor‐specific effector T‐cell responses and development of immune tolerance to neoplastic cells. The transcription factor forkhead box P3 (FOXP3) is an intracellular key molecule for Treg development and function and is considered to represent the most specific Treg cell marker. The aim of this study was to analyze the frequency and prognostic impact of tumor‐infiltrating FOXP3+ Treg in colorectal cancer (CRC) stratified by mismatch‐repair (MMR) status. Using the tissue microarray technique, 1,420 tumor samples were immunohistochemically stained for FOXP3 and stratified into 1,197 MMR‐proficient and 223 MMR‐deficient CRCs. Additionally, the 1,197 MMR‐proficient CRCs were randomized into 2 subgroups (Test Groups 1 and 2; n = 613 and 584, respectively). In both MMR‐proficient CRC subgroups high frequency tumor‐infiltrating FOXP3+ Treg was associated with early T stage (p = 0.001 and <0.001), tumor location (p = 0.01 and 0.045) and increased 5‐year survival rate (p = 0.004 and <0.001), whereas in MMR‐deficient CRCs an association between FOXP3+ Treg and absence of lymph node involvement (p = 0.023), absence of vascular invasion (p = 0.023) and improved 5‐year survival rate (p = 0.029) could be detected. In a multivariable analysis including age, gender, T stage, N stage, tumor grade, vascular invasion, and tumor border configuration, a high FOXP3+ Treg frequency was an independent prognostic factor in both MMR‐proficient CRC subsets (p = 0.019 and p = 0.007), but not in the MMR‐deficient CRCs (p = 0.13). Therefore, high frequency of tumor‐infiltrating FOXP3+ Treg is associated with early T stage and independently predicts improved disease‐specific survival in MMR‐proficient CRC patients.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer.

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P = 0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P = 0.0001) and validation (P = 0.03) sets. A similar trend (P = 0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n = 42) were found to be significantly associated (r = 0.33, P = 0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples.

Several studies have shown that glypican 3 (GPC3) could be a useful diagnostic marker for hepatocellular carcinoma (HCC) and for differentiating HCC from nonneoplastic and preneoplastic liver disease. To systematically investigate the epidemiology of GPC3 expression in the liver and in other organs and tissues, we used tissue microarray technology comprising 4,387 tissue samples from 139 tumor categories and 36 nonneoplastic and preneoplastic tissue types. The immunohistochemical expression of GPC3 was assessed semiquantitatively using a 10% cutoff score and was detected in 9.2% of nonneoplastic liver samples (11/119), 16% of preneoplastic nodular liver lesions (6/38), and 63.6% of HCCs (140/220), underlining the role of GPC3 in hepatocarcinogenesis. Furthermore, several other tumors revealed consistent expression of GPC3, including squamous cell carcinoma of the lung (27/50 [54%]), testicular nonseminomatous germ cell tumors (32/62 [52%]), and liposarcoma (15/29 [52%]).

Is the improved prognosis of p16 positive oropharyngeal squamous cell carcinoma dependent of the treatment modality

The incidence of human papilloma virus (HPV) induced oropharyngeal squamous cell carcinoma (OPSCC) increases in the western countries. These OPSCC show distinct molecular characteristics and are characterized by an overexpression of p16, considered a surrogate marker for HPV infection. When compared to patients with p16 negative OPSCC, patients with HPV induced p16 positive OPSCC show a significantly better prognosis, which is reported to be caused by increased radiosensitivity. The objective of the present study was to analyze the impact of p16 expression status on the prognosis of OPSCC treated by either radiotherapy (RT) or primary surgery. Results are based upon a tissue microarray (TMA) of 365 head neck squamous cell carcinomas (HNSCC) including 85 OPSCC with clinico‐pathological and follow‐up data. p16 positivity correlated significantly with oropharyngeal tumor localization (p < 0.001). Patients with p16 positive OPSCC exhibited a significantly better overall survival than those with p16 negative tumors (p = 0.007). In a multivariate analysis, survival benefit of patients with p16 positive OPSCC was independent of clinico‐pathological parameters such as cT and cN classification and treatment modality. The improved prognosis of p16 positive OPSCC is found after RT as well as after surgery.

The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas

CDX2 is a homeobox domain-containing transcription factor that is important in the development and differentiation of the intestines. Based on recent studies, CDX2 expression is immunohistochemically detectable in normal colonic enterocytes and is retained in most, but not all, colorectal adenocarcinomas. CDX2 expression has also been documented in a subset of adenocarcinomas arising in the stomach, esophagus and ovary. In this study, we examined CDX2 expression in a series of large tissue microarrays representing 4652 samples of normal and neoplastic tissues. Strong nuclear staining for CDX2 was observed in 97.9% of 140 colonic adenomas, 85.7% of 1109 colonic adenocarcinomas overall and 81.8% of 55 mucinous variants. There was no significant difference in the staining of well-differentiated (96%) and moderately differentiated tumors (90.8%, P=0.18), but poorly differentiated tumors showed reduced overall expression (56.0%, P<0.000001). Correspondingly, there was an inverse correlation between CDX2 expression and tumor stage, with a significant decrease in staining between pT2 and pT3 tumors (95.8 vs 89.0%, P<0.012), and between pT3 and pT4 tumors (89.0 vs 79.8%, P<0.016). Analysis of 140 locally advanced, CDX2-positive colorectal adenocarcinomas coarrayed with their matching lymph node metastases revealed that expression of this marker was retained in 82.1% of the metastases. Consistent with previous reports, CDX2 staining was observed in gastric adenocarcinomas (n=71), more commonly in the intestinal-type than the diffuse-type (28.9 vs 11.5%, P<0.05). Occasional ovarian carcinomas were positive for CDX2, including mucinous (10.5%), endometrioid (9.3%) and serous variants (2%), but expression was either very rare or absent in primary carcinomas of the lung, breast, thyroid, pancreas, liver, gallbladder, kidney, endometrium and urinary bladder. A low frequency of CDX2 expression in pancreatic and biliary carcinomas observed on the microarrays was pursued further by comparing these tumors with ampullary adenocarcinomas on conventional sections. Ampullary adenocarcinomas were more commonly positive for CDX2 (19/24, 79%) than cholangiocarcinomas (1/11, 9%) and pancreatic carcinomas (3/20, 15%). In summary, CDX2 is a sensitive and specific marker for colorectal adenocarcinoma, although its expression is decreased among higher grade and stage tumors, and it is not invariably present in metastases from positive primaries. CDX2 may also be helpful in distinguishing adenocarcinomas of the ampulla from those arising in the pancreas and biliary tree.

V600E BRAF mutations are alternative early molecular events in a subset of KIT/PDGFRA wild-type gastrointestinal stromal tumours

Background: A small subset (10–15%) of gastrointestinal stromal tumours (GISTs) lack mutations in KIT and PDGFRA (wild-type GIST). Recently, a novel BRAF exon 15 mutation (V600E) was detected in imatinib-naive wild-type high-risk intestinal GISTs (4%). However, the frequency and distribution of BRAF mutations within the spectrum of GISTs, and whether they might represent secondary events acquired during tumour progression, remain unknown. Methods: 69 GISTs (39 KIT mutants, 2 PDGFRA mutants and 28 wild-type) were analysed for mutations in BRAF exon 15 and KRAS exon 2. To assess the stage at which these mutations might occur in GIST, a considerable number of incidental gastric (n = 23) and intestinal (n = 2) tumours were included. Results: BRAF mutations (V600E) were detected in 2 of 28 wild-type GISTs (7%), but in none of the 41 KIT/PDGFRA mutants. No KRAS mutation was detected. The two BRAF-mutated GISTs measured 4 mm in diameter and originated in the gastric body and the jejunum in two men (mean age, 76 years). Both tumours were mitotically inactive KIT-positive spindle-cell GISTs that were indistinguishable histologically from their more common KIT-mutated counterparts. Conclusion: BRAF mutations represent an alternative molecular pathway in the early tumorigenesis of a subset of KIT/PDGFRA wild-type GISTs and are per se not associated with a high risk of malignancy. Mutations in KIT, PDGFRA and BRAF were mutually exclusive in this study. Results from this and a previous study indicate that BRAF-mutated GISTs show a predilection for the small bowel (four of five tumours), but this needs further evaluation in larger studies.

Hepatocyte Paraffin 1 Expression in Human Normal and Neoplastic Tissues Tissue Microarray Analysis on 3,940 Tissue Samples

Hepatocyte paraffin 1 (Hep Par 1) is a monoclonal antibody developed from hepatic tissue from a failed liver allograft. Several studies have shown that Hep Par 1 is a useful marker to differentiate hepatocellular carcinoma (HCC) from other types of adenocarcinoma metastatic to the liver. The aim of our study was the systematic investigation of the epidemiology of Hep Par 1 expression in 3,940 tissue samples using the tissue microarray technique. Strong Hep Par 1 expression was found most frequently in 35 (73%) of 48 HCCs. In nonhepatic tumors, strong Hep Par 1 expression was detected in adenocarcinoma of the lung (2/50), gallbladder (3/31), pancreas (2/48), stomach (3/74), small intestine (1/11), adenoma of the colon with high-grade dysplasia (1/49), adrenal gland carcinoma (1/6), paraganglioma (1/9), and malignant melanoma (2/48). Our data suggest that Hep Par 1 is a highly specific marker for HCC, although several nonhepatic tumors occasionally can show some Hep Par 1 positivity.

Hepatoid adenocarcinoma with liver metastasis mimicking hepatocellular carcinoma: an immunohistochemical and molecular study of eight cases.

Hepatoid adenocarcinoma (HAC) is a special type of extrahepatic adenocarcinoma, which has a striking morphologic similarity to hepatocellular carcinoma. Seven HACs arising in the stomach and one in the lung, all with liver metastasis, were studied. They shared clinical features, such as old age, high serum alpha-fetoprotein level, aggressive behavior, and hepatic tumor in absence of risk factors for hepatocellular carcinoma (HCC). Morphologically, tumors were characterized by an admixture of tubulo-and/or papillary adenocarcinoma with hepatoid foci. In six cases, liver metastases showed an exclusive hepatoid differentiation, virtually indistinguishable from HCC with solid growth pattern. As HAC and HCC cannot be differentiated on the basis of morphology alone, differences in immunohistochemical reaction patterns would be of considerable diagnostic help. Immunostaining for CK7, CK8, CK18, CK19, CK20, alpha-fetoprotein, p-CEA, and HepPar1 revealed that hepatoid areas of both primary and metastatic HAC have a specific immunoprofile, distinctive of this entity. On the one hand, positivity of virtually all HACs for alpha-fetoprotein, CK8, CK18, and the membranous, canalicular staining for polyclonal carcinoembryonic antigen underline its hepatoid nature. On the other hand, positive staining for CK19 and CK20 and frequent negativity for HepPar1 in both primary tumors and their metastases were distinctive features of HAC. Furthermore, HAC differs from combined hepatocellular cholangiocarcinoma, being negative for CK7. In addition, for comparison of immunohistochemical results, we stained with the same antibody panel a tissue microarray of 121 HCCs. Comparative genomic hybridization study of three HAC supports their hepatoid differentiation as aberrations found in HAC are common in HCC (4q−, 8p−), and hepatoblastoma (Xq+), respectively.

Prognostic significance of the wnt signalling pathway molecules APC, β‐catenin and E‐cadherin in colorectal cancer—a tissue microarray‐based analysis

Aims:  To investigate dysregulation of the wnt signalling pathway by assessing β‐catenin expression/increasing expression and loss of cytoplasmic adenomatous polyposis coli (APC) and membranous E‐cadherin in colorectal cancer (CRC) and determining the prognostic significance of these variables.