Giovanni Tuccari

Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: Correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis

Although microsatellite instability (MSI) testing is a useful tool for molecular screening of hereditary nonpolyposis colorectal cancer (HNPCC) carcinomas, conflicting results have been obtained in colorectal adenomas. This might result from different techniques of tissue sampling and MSI analysis. Alternatively, some HNPCC-associated adenomas may follow a molecular route that differs from the MSI pathway. In the present study we examined the MSI status of 18 adenomas from 17 HNPCC patients by comparing manual adenoma dissection under gross visual control with laser microdissection of single adenoma crypts. After manual gross dissection, 50% (9 of 18) and 11.1% (2 of 18) of the adenomas displayed high-level (MSI-H) and low-level (MSI-L) MSI, respectively. The same set of adenomas split into 83.3% (15 of 18) MSI-H and 5.6% (1 of 18) MSI-L after laser microdissection. The expression pattern of mismatch repair (MMR) proteins showed a higher concordance rate with the MSI status in laser-dissected (94%) than gross-dissected (47%) adenomas. Whereas two adenomas remained microsatellite stable (MSS) and MMR proficient even after laser-assisted dissection, two MSI-H cases showed either rare instabilities at coding microsatellites or intratumoral heterogeneity of MSI with and without MSH2 expression. This suggests that in some adenomas development of MMR dysfunction occurs stepwise with MSI, arising before complete loss of MMR gene expression, whereas other HNPCC-associated adenomas might develop independently of MMR deficiency.

Colorectal carcinoma grading by quantifying poorly differentiated cell clusters is more reproducible and provides more robust prognostic information than conventional grading

The most widely used system to define the histological grade of colorectal carcinoma (CRC) is based on the degree of gland formation. This system suffers from significant interobserver variability which may limit its prognostic value and consequently better standardized criteria for the assessment of histological grading of CRC are needed. The present study aims to evaluate and to compare, in a cohort of postsurgical pTNM stage I CRC, conventional histological grading, and a novel grading system based on the number of poorly differentiated clusters of neoplastic cells, in terms of interobserver reproducibility, prognostic significance on progression-free survival, and association with other clinicopathological characteristics. Grading with both systems was performed by two pathologists independently and blinded to the clinicopathological data. Interobserver agreement was higher when grade was assessed by counting poorly differentiated clusters than by assessing the relative proportion of the glandular component. Contrary to conventional grading, the novel system provided significant prognostic information in terms of progression-free survival and was significantly associated with budding, invasive growth, lymphatic invasion, and occult nodal metastases of CRC. In conclusion, our findings suggest that a tumor grading system based on the number of poorly differentiated clusters is more reproducible and provides better prognostic stratification of pTNM stage I CRC patients than conventional grading.

Immunohistochemical demonstration of lactoferrin in follicular adenomas and thyroid carcinomas

By immunohistochemistry, the presence of lactoferrin was investigated in follicular adenomas (10 cases) and carcinomas of the thyroid gland (23 cases). Normal thyroid tissue was also tested as control. Follicular adenomas showed a consistant negativity, whereas follicular and papillary carcinomas exhibited various degrees of positivity for lactoferrin. Incorporated organoid structures observed in anaplastic carcinomas were strongly stained; the spindle cell parts of these cancers were always negative for this iron-binding protein. Medullary carcinomas were also unstained. These findings are discussed in relation to the distribution pattern of thyroglobulin. The authors emphasize the possibility that lactoferrin may be useful in clarifying some diagnostic problems in neoplastic thyroid pathology.

Histologic grading based on counting poorly differentiated clusters in preoperative biopsy predicts nodal involvement and pTNM stage in colorectal cancer patients

Histologic grading is commonly assessed in colorectal cancer preoperative biopsies. Nevertheless, its clinical impact is limited by low interobserver reproducibility and poor concordance with grading found in the final resection specimen. In the present study, we aimed to investigate the reproducibility, accuracy, and predictive value on lymph node status or pTNM stage of a novel grading system based on the number of poorly differentiated clusters in colorectal cancer preoperative endoscopic biopsies. Grading based on counting poorly differentiated clusters was assessed in 163 colorectal cancer endoscopic biopsies and corresponding surgical specimens. With this system, 152 biopsies could be graded with good interobserver agreement (κ = 0.735). In comparison with the surgical specimens, 75% of colorectal cancers were correctly graded in the biopsy, and 81% of poorly differentiated colorectal cancers were identified at initial biopsy. High poorly differentiated clusters grade in the biopsy was significantly associated with nodal metastasis, high pTNM stage (P < .0001), or histologic features suggestive of more aggressive behavior (tumor budding, perineural invasion, vascular invasion, and infiltrating tumor border) in the surgical specimen. Furthermore, this system identified colorectal cancer with nodal involvement or high pTNM stage with a 78% positive predictive value and 71% and 69% negative predictive values, respectively. Our findings suggest that a grading system based on the quantification of poorly differentiated clusters is feasible in most colorectal cancer endoscopic biopsies. In view of its good reproducibility, accuracy, and predictive value on the anatomical extent of the disease, it may be taken into account for decision-making in colorectal cancer treatment.

Immunohistochemical expression of metallothionein in normal human colorectal mucosa, in adenomas and in adenocarcinomas and their associated metastases

The immunohistochemial distribution pattern of metallothionein, a low molecular weight protein with strong affinity for divalent heavy metal ions, has been investigated in normal and neoplastic conditions of the large bowel. Utilizing a monoclonal mouse antibody the following formalin‐fixed paraffin‐embedded surgical or biopsy samples were studied: tubulo‐villous adenomas (8 cases); adenocarcinomas with various degree of differentiation (85), nine of which were mucinous‐type; synchronous tubular or tubulo‐villous adenomas separate from carcinomas (30); transitional mucosa (45); metastases in lymph nodes (43); and distant metastases (45). Twenty biopsies from the right and left colon of 10 patients affected by irritable bowel syndrome were also analyzed. Normal colonic mucosa as well as transitional mucosa showed metallothionein immunopositivity in enterocytes at the luminal surface and crypts. Evident nuclear and cytoplasmic staining was encountered in tubulo‐villous adenomas; the same reactivity was noted in the basal glandular component of colorectal carcinomas‐synchronous adenomas, while less intense staining was noted in the apical villous portions. A variable metallothionein immunostaining was observed in adenocarcinomas (62.3%), in lymph node (55.8%) and distant hepatic (17.2%) and omental (43.8%) metastases, although it was not always concordant with that reported in the corresponding primary tumour. Whether the metallothionein positivity observed in normal and neoplastic cells is the result of expression of a stable form of the protein or an accumulation in the nucleus and cytoplasm remains to be clarified.

Increased ratio of vascular endothelial growth factor to semaphorin3A is a negative prognostic factor in human meningiomas

Semaphorin3A (SEMA3A) is an anti‐angiogenic factor which is expressed in human meningiomas in association with low microvessel density (MVD). It competes with vascular endothelial growth factor (VEGF) for receptor neuropilin‐1 (NRP‐1). The ratio between VEGF and SEMA3A has been recently demonstrated to regulate neo‐angiogenesis, proliferation and progression of tumors. To clarify the involvement of these proteins in the above‐mentioned phenomena, we analyzed the immunohistochemical expression of SEMA3A, VEGF and NRP‐1 and their correlation with MVD in a series of 48 cases of meningioma with different histotype and histological grade. SEMA3A and VEGF expression was encountered in about half the cases, although at different levels. NRP‐1 staining was evidenced in the vessels within all but two tumors and in the neoplastic cells of 18/48 meningiomas. A negative significant correlation emerged between SEMA3A amount and MVD; on the other hand, high VEGF levels appeared to be significantly associated with high MVD. A high VEGF/SEMA3A was significantly associated with high histological grade, proliferation index and MVD as well as with a higher recurrence rate of the meningiomas. Present data suggest that the balance between the expression of the pro‐angiogenic factor VEGF and the anti‐angiogenic SEMA3A may be involved in the regulation of neo‐angiogenesis and proliferation in meningiomas, representing also a predictor of recurrences in these tumors. Further validation of our results may open the way for the use of drugs targeting not only VEGF, but also NRP‐1 and SEMA3A to prevent recurrences of meningiomas.

Lactoferrin in benign hypertrophy and carcinomas of the prostatic gland

Using immunoperoxidase procedures, the presence of lactoferrin was investigated in benign hypertrophy (40 cases), differentiated adenocarcinomas (20 cases) and undifferentiated carcinomas (10 cases) of the prostate. In benign hypertrophy, the glandular epithelium and the secretory product were consistently negative. Strong staining for lactoferrin was always observed in neoplastic cells of differentiated adenocarcinomas and in their endoluminal material; in contrast, a very slight positivity was noted in undifferentiated carcinomas. These findings are discussed in relation to the different degree of neoplastic glandular differentiation and functional activity of prostatic carcinomas.

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.

The expression of adiponectin receptors Adipo-R1 and Adipo-R2 is associated with an intestinal histotype and longer survival in gastric carcinoma

Background: Adiponectin (ApN) is a 30 kDa adipocytokine which mediates an antineoplastic effect after binding to its receptors, Adipo-R1 and Adipo-R2. The expression of these receptors has been documented in gastric cancer (GC) cell lines, but only a few data exist on their expression in GC neoplastic tissue. Aim: To investigate the expression of Adipo-R1 and Adipo-R2 in a series of surgically resected GCs and to assess its association with various tumour clinicopathological characteristics as well as with patient survival. Methods: Forty-nine surgically resected GCs were submitted to immunohistochemical assays for Adipo-R1, Adipo-R2 and ApN. Results: Adipo-R1 and Adipo-R2 immunoexpression was found in 22/49 GCs and in intestinal metaplasia areas near the tumours, whereas only slight immunoreactivity for these proteins was found in adjacent normal gastric epithelium. No ApN expression was encountered in any of the cases analysed. Adipo-R1/Adipo-R2 expression was significantly associated with an intestinal histotype of the tumours and with longer overall survival of the patients. Conclusions: Intestinal-type GCs often express Adipo-R1/R2 in association with a better prognosis. The presence of these receptors could be exploited for novel anticancer therapies based on ApN addition in GC.

Brachyury: A Diagnostic Marker for the Differential Diagnosis of Chordoma and Hemangioblastoma versus Neoplastic Histological Mimickers

Brachyury is a transcription factor which is required for posterior mesoderm formation and differentiation as well as for notochord development during embryogenesis. Due to its expression in the neoplastic cells of chordoma, a malignant tumour deriving from notochordal remnants, but not in tumors showing a similar histology, brachyury has been proposed as a diagnostic marker of this neoplasia. Though commonly considered a hallmark of chordoma, the expression of brachyury has been also documented in the stromal cells of hemangioblastoma (HBL), a slow growing tumor which may involve the central nervous system (CNS) and, rarely, the kidney. Herein we review the role of brachyury immunohistochemical detection in the identification and differential diagnosis of chordoma and HBL towards histological mimickers and suggest that brachyury is added to the panel of immunohistochemical markers for the recognition of HBL in routinary practice, principally in unusual sites.