Carme Mallofré

KIT expression in chromophobe renal cell carcinoma: Comparative immunohistochemical analysis of KIT expression in different renal cell neoplasms

The overexpression of c-Kit in chromophobe renal cell carcinoma (ChRCC) has been described by comparative gene expression analyses and has been proposed as a possible specific hallmark of this neoplasm. The aim of our study was to establish its immunohistochemical expression in a large series of ChRCC and to compare it with other renal neoplasms. In our study, immunohistochemical characterization of KIT was performed in 87 renal neoplasms including 25 cases of ChRCC, 13 cases of renal oncocytoma, and 39 renal cell carcinomas (21 cases of conventional RCC [CRCC], 8 cases of CRCC with granular cell differentiation, and 10 cases of papillary RCC [PRCC]). Eighty-eight percent ChRCC and 71% oncocytomas showed immunohistochemical expression of KIT, while the other types of RCC studied were all negative. The meaning of immunohistochemical expression of KIT in ChRCC and oncocytomas is still unknown, but its immunohistochemical staining appears to be useful in distinguishing ChRCC from PRCC, CRCC, and its granular cell variant. Moreover, our findings support current models that consider that there is a histopathogenic relationship between oncocytoma and ChRCC. Finally, it should be determined whether KIT plays a role in the tumorigenesis of ChRCC and oncocytoma and whether targeted therapy with STI-571, an inhibitor of KIT, could be effective in exceptional cases of ChRCC with metastatic extension or recurrence.

Immunohistochemical expression of CK20, p53, and Ki-67 as objective markers of urothelial dysplasia

Urothelial dysplasia and carcinoma in situ (CIS) are related to recurrence and progression of urothelial carcinoma. Distinguishing CIS and dysplasia from reactive atypia is often difficult on the basis of histological features alone. Cytokeratin 20 (CK20), p53, and Ki-67 are related either to neoplastic change or prognosis in urothelial proliferations. The objective of the present study was to establish the immunohistochemical pattern of these three antibodies in urothelial dysplasia and CIS. Three groups of patients were evaluated: 40 nonneoplastic urothelial samples, 50 cases with histologically incontrovertible CIS, and 30 samples with nonconclusive atypical changes (atypia of unknown significance). Monoclonal antibodies (MoAb) against CK20, p53, and Ki-67 (MIB-1) were used on paraffin-embedded samples. Nonneoplastic urothelium showed no reactivity to CK20 except for umbrella cells; p53 and Ki-67 were negative or weakly positive in <10% of basal cells. In the CIS group, 42% showed positivity for all three MoAb; 44%, for two; and 14%, only for one. CK20 was positive through the full thickness of the urothelium in 72% of cases, p53 was positive in 80% of cases, and Ki-67, in 94% of cases. In the third group, the suspected dysplastic cells showed strong positivity in scattered cells through the epithelium in 75% of cases. Aberrant CK20 expression in urothelial cells plus overexpression of p53 and Ki-67 are indicators of dysplastic change in urothelial mucosa. Thus, immunohistochemistry is a useful tool to confirm the diagnosis of CIS and could be helpful to distinguish dysplastic changes from reactive atypia.

Differential expression of cdc25 cell-cycle-activating phosphatases in human colorectal carcinoma.

cdc25 is a family of cell-cycle phosphatases that activate the cyclin-dependent kinases. cdc25A and B, but not C, have oncogenic potential in vitro. In this study, we analyzed the possible implication of cdc25 genes in the progression of colorectal tumors. RNA and DNA were extracted from 34 paired tumor and normal colorectal tissues and examined by Northern blot, RT-PCR, and Southern blot, respectively. Protein expression was analyzed by Western blot in a subset of normal and tumor samples. The expression levels were correlated with the clinicopathologic characteristics and survival of the patients. cdc25B mRNA was overexpressed in 19 carcinomas (56%). A significant correlation was observed between high cdc25B mRNA levels and the relapse-free, overall, and cancer-related survival of the patients. The cdc25B2 splicing variant was detected in 27 carcinomas (79%) but only in 9 normal samples (26%) and was associated with the grade of the differentiation of the tumors. cdc25A mRNA was overexpressed in four tumors (12%) and cdc25C1 mRNA was overexpressed in nine tumors (26%). A new cdc25C2 splicing variant lacking exon 4 and 5 was identified in all of the tumors and in 56% of the normal samples. No amplifications or gene rearrangements of these genes were detected. In conclusion, these findings indicate that cdc25 isoforms and splicing variants are differentially regulated in colorectal carcinomas and may participate in the development of these tumors. Additionally, the correlation between cdc25B mRNA levels and the survival of the patients also suggest that the cdc25B isoform may be involved in the progression of the disease.

Usefulness of histological analysis for predicting the presence of microorganisms at the time of reimplantation after hip resection arthroplasty for the treatment of infection.

BACKGROUND Appropriate interpretation of a frozen section has a relatively high specificity and sensitivity for the diagnosis of infection when septic loosening of a prosthesis is suspected. However, its usefulness for predicting the presence of microorganisms at the time of reimplantation after hip resection arthroplasty for the treatment of infection is not well defined. The aim of the present study was to evaluate the usefulness of histological analysis in this situation. METHODS From January 2002 to February 2006, a total of twenty-one patients underwent reimplantation after hip resection arthroplasty for the treatment of infection. Histological studies and cultures of specimens of periprosthetic tissue that had been obtained at the time of reimplantation were retrospectively reviewed. The results of culture were considered positive when the same microorganism was isolated in at least two samples. Two histological criteria were used to diagnose infection: (1) Criterion A (the Feldman criterion), defined as the presence of at least five neutrophils per high-power field (x400) in at least five separate microscopic fields and (2) Criterion B (the Athanasou criterion), defined as the presence of at least one neutrophil per high-power field (x400), on average, after examination of ten microscopic fields. The sensitivity, specificity, positive predictive value, and negative predictive value of each of these criteria were calculated with use of microbiological results as the gold standard for defining infection. RESULTS Seven of the twenty-one patients had a positive result on culture, and the most common microorganism was coagulase-negative staphylococcus. The sensitivity, specificity, positive predictive value, and negative predictive value of frozen-section analysis were 28.5%, 100%, 100%, and 73.6%, respectively, according to the Feldman criterion and 71.4%, 64.2%, 50%, and 81.8%, respectively, according to the Athanasou criterion. The numbers of lymphocytes and plasma cells did not help in the diagnosis of infection. Fibrosis was more common in patients without an infection. CONCLUSIONS The probability of infection is high when at least five neutrophils per high-power field are found in the periprosthetic tissue, but it is not possible to rule out infection when the number of neutrophils is less than five. LEVEL OF EVIDENCE Diagnostic Level I.

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx.

p21WAF1/Cip1 is a recently identified gene involved in cell cycle regulation through cyclin‐CDK‐complex inhibition. The expression of this gene in several cell lines seems to be induced by wild‐type, but not mutant, p53. p21WAF1/Cip1 expression has been studied at both mRNA and protein levels in a series of 49 normal mucosae and squamous cell carcinomas of the larynx. A significant association was found between mRNA and protein expression in tumours (P<0·0001). p21WAF1/Cip1 expression was strongly associated with squamous cell differentiation of carcinomas, because six of seven (86 per cent) undifferentiated carcinomas (grade 4) showed very low levels of p21WAF1/Cip1 expression, whereas 41 out of 42 (98 per cent) carcinomas with squamous cell differentiation (grades 1–3) had normal or high levels of p21WAF1/Cip1 expression (P<0·0001). In addition, p21WAF1/Cip1 expression was topologically related to the squamous differentiation of tumour cells with a distribution similar to that seen in normal squamous epithelium. No correlation was found between p21WAF1/Cip1 expression and the global S‐phase of the carcinomas. p53 mutations (exons 5–9) were found in ten carcinomas with p21WAF1/Cip1 expression, but no p53 mutations were detected in three p21WAF1/Cip1‐negative tumours. In conclusion, p21WAF1/Cip1 expression is frequently upregulated in squamous cell carcinomas of the larynx and is associated with tumour cell differentiation. p21WAF1/Cip1 expression in these tumours is independent of p53 gene mutations.

Low sensitivity of histology to predict the presence of microorganisms in suspected aseptic loosening of a joint prosthesis.

Intraoperative histology has a high specificity and sensitivity when a septic prosthesis loosening is suspected. However, its usefulness to predict the presence of microorganisms when aseptic loosening is suspected is not well defined. Intraoperative histology and cultures from periprosthetic tissue of 61 revision arthroplasties performed owing to suspected aseptic loosening were retrospectively reviewed. Frozen sections were evaluated following Mirras criteria (adapted by Feldman). Culture was considered positive when the same microorganism was isolated in at least two samples. The cultures were positive in 12 cases and coagulase-negative staphylococci were the most common microorganisms (11 cases). In six out of 12 cases (50%), the histology revealed more than five polymorphonuclear leukocytes per high-power field. The sensitivity, specificity, positive and negative predictive value of histology to detect the presence of microorganisms was 50, 81, 40 and 86%, respectively. In conclusion, intraoperative histology using Mirras criteria had a low sensitivity to predict the presence of microorganisms in samples from suspected aseptic prosthetic loosening.

Neutrophils in frozen section and type of microorganism isolated at the time of resection arthroplasty for the treatment of infection

IntroductionThe histology of periprosthetic tissue is a gold standard for the diagnosis of periprosthetic joint infection. However, the specificity and sensitivity of histology has never been 100%. In the present study we hypothesized that the type of microorganism could be related to the effectiveness of histology in the detection of infection.Material and methodsFrozen sections and cultures from periprosthetic tissue of 38 revision arthroplasties taken at the time of resection arthroplasty for the treatment of infection were retrospectively reviewed. Frozen sections were evaluated following Feldman’s criteria. A culture was considered positive when the same microorganism was isolated in at least two samples or when pus was present around the prosthesis. The literature providing information on histology and microbiology of arthroplasty-associated infection was reviewed.ResultsCoagulase-negative staphylococcus (CNS) was the etiology in 13 cases, Gram-negative bacilli in 8, Staphylococcus aureus in 7, Candida sp in 2, Peptococcus sp in 2, Enterococcussp in 1 and S. pneumoniae in 1. No microorganism was isolated in four cases. Almost all the frozen sections in the 38 cases were positive except in 2 of the 13 that were caused by CNS (15.3%). The articles reviewed supported our findings.ConclusionFrozen section is a useful test to intraoperatively confirm an infection when preoperative septic loosening is suspected. However, histology has false-negative results when the infection is due to low-virulence microorganisms.

Interface membrane is the best sample for histological study to diagnose prosthetic joint infection

The objective of our study was to study which is the most accurate specimen for histological diagnosis of prosthetic joint infections (pseudocapsule or interface membrane). This is a prospective study including hip revision arthroplasties performed from January 2007 to June 2009. Specimens from pseudocapsule and from interface membrane were obtained from each patient. The histology was considered positive for infection when ≥5 neutrophils per high-power field ( × 40) were found. Definitive diagnosis of infection was considered when ≥2 cultures were positive for the same microorganism. According to the definition of infection, patients were classified in two groups: (A) patients with aseptic loosening in whom cultures obtained during surgery were negative and (B) patients with prosthetic joint infection. A total of 69 revisions were included in the study; 57 were classified in group A and 12 in group B. In group B, the percentage of positive interface membrane histology was significantly higher than the percentage of positive pseudocapsule histology (83 vs 42%, P=0.04, Fishers exact test). The results suggest that periprosthetic interface membrane is the best specimen for the histological diagnosis of prosthetic joint infection.

Adhesion molecules α, β and γ‐catenin as prognostic factors of tumour progression in upper urinary tract urothelial tumours: the role of AKT‐P/GSK‐3β/β‐catenin pathway

To evaluate α, β and γ‐catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway.

Expression and mutational analyses of KIT and PDGFR-α in sarcomatoid renal cell carcinoma

Sir: Sarcomatoid renal cell carcinoma (SRCC), first described by Farrow et al. in 1967, is currently not regarded as a distinct histological category but as a high-grade transformation occurring in different renal cell carcinoma (RCC) subtypes. Prognosis of patients with SRCC is very poor because of high stage at presentation and ineffective treatment in advanced stages. The report of KIT expression in sarcomatoid differentiation of RCC raises the potential use of Imatinib for the treatment of this neoplasm. This hypothesis is studied more deeply in this work. Imatinib is a well-known targeted therapy against tyrosine kinases BCR-ABL, KIT and platelet-derived growth factor receptor (PDGFR) that is effective in chronic myelogenous leukaemia (CML) and gastrointestinal stromal tumour (GIST). In GIST, KIT or PDGFR-a (PDGFRA) gain of function mutations play a crucial role in its pathogenesis and are responsible for the success of Imatinib. This example among others provides a model of how expression and oncogenic activation of tyrosine kinases sustains the rationale and clinical implementation of biological treatments. Taking this model as a reference, we assessed the immunohistochemical expression of KIT and PDGFRA, both targeted by Imatinib (KIT: A 4502; Dako, Carpinteria, CA, USA, dilution 1:100; and PDGFRA: sc-338; Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:100) in a series of 16 SRCCs assessing the epithelial and the sarcomatoid part independently. The epithelial components of the SRCC cases were classified according to the World Health Organization classification 2002 into Clear cell RCC (nine cases), Papillary RCC (one case), Chromophobe RCC (five cases) and Unclassified RCC (one case). For KIT, the extent of immunoreactivity was semiquantitatively categorized as focal (<25% of tumour cells positive), moderate (25–50% of tumour cells positive) and diffuse (>50% of tumour cells positive). For PDGFRA, a score of immunoreactivity in three categories (negative, low and high expression) was made up taking into account extension and intensity (Table 1). The extent of positivity was also regarded semiquantitatively in the same manner as for KIT. The immunoreactivity of renal tubules served as a reference of intensity and lower intensities were regarded as weak and intensities higher or the same as renal tubules were considered strong. Higher expression of both tyrosine kinase receptors was observed in the sarcomatoid part (81.2% KIT and 81.2% PDGFRA) in comparison with the epithelial part (31.2% KIT and 43.7% PDGFRA) (Table 1, Figure 1). Of note, cases positive for KIT in the epithelial part were of Chromophobe RCC subtype in accordance with already published data (Figure 1 (A1)). Since the approval of Imatinib as an effective treatment against GIST and CML, many studies have been published characterizing expression of Imatinib targets on different neoplasms with the purpose of increasing the range of tumours in which Imatinib could be a therapeutic option. Recent data support the concept that mutation-mediated activation of these two tyrosine kinase receptors is a prerequisite for successful treatment with Imatinib and that sensitivity to Imatinib can not be inferred based solely on positive immunohistochemical expression of its targets. Based on these guidelines, we searched for mutational status of both KIT and PDGFRA in a subset SRCC cases where sarcomatoid differentiation showed overexpression of these proteins. In our analysis, we concentrated on exons 9 and 11 of KIT and 12 and 18 of PDGFRA, where activating mutations in KIT and PDGFRA have been most commonly found in GIST. Therefore, sequencing of KIT exons 9, 11 was done in the sarcomatoid part of six cases, two of which expressed KIT focally, one moderately and three diffusely. Mutational analysis of PDGFRA exons 12 and 18 was done in the sarcomatoid part of seven cases, all of which showed high expression of PDGFRA. None of the cases analysed harboured mutations in KIT or in PDGFRA in the sequenced exons. These results are in concordance with those of Sengupta et al., who reported absence of exon 9, 11, 13 and 17 KIT mutations in seven KIT positive high-grade RCCs, five of which exhibited sarcomatoid differentiation. In conclusion, our work shows that KIT and PDGFRA expression is a common finding in sarcomatoid differentiation of RCC. The molecular mechanism underlying this expression and whether this could have an active role in the clinical aggressiveness of this highgrade transformation is still unknown. We sought for specific mutations in KIT and PDGFR exons that have been involved in GIST tumorigenesis and response to