[Update on precursors of vulvar carcinoma]. / Lésions vulvaires précancéreuses : mise au point.

Vulvar carcinomas represent 4% of all gynaecological cancers with 838 new cases in France in 2018. The precursor lesions of vulvar carcinomas are differentiated vulvar intraepithelial lesion (dVIN) in a context of lichen sclerosus and vulvar high-grade squamous intraepithelial lesion (HSIL) link to human papillomavirus (HPV) infection. Three typical clinical forms of HSIL are described: the Bowenoid papulosis, the Bowen's disease and the confluent VIN. Histopathology cannot differentiate effectively these two types of lesions. P16 and P53 immunostaining are valuable tools to respectively assess HPV infection and divide different types of dVIN. However, P53 immunostaining is still lacking sensibility to detect dVIN. First line therapies are medical treatment excluding the cases with a doubt of invasion. The gold standard treatment for dVIN and vulvar HSIL are respectively topical corticosteroids and imiquimod. Primary prevention for vulvar HSIL and dVIN are respectively HPV vaccination and early treatment of lichen sclerosus. Destructive therapy can be used in case of medical treatment failure such as CO2 laser, cryotherapy, dynamic phototherapy. Surgical indications should be carefully assessed between the risk of recurrence, the spread of the lesions, the aesthetic and functional aspect. Surgical procedures consist in either superficial vulvectomy or radical vulvectomy with or without flap reconstruction. Recurrence rate after surgery is around 20%.

Masses in mammography: what are the underlying anatomopathological lesions?

The semiological description of masses in mammography is based on the BI-RADS system provided by the American College of Radiology. The contour is the most discriminating morphological criterion between benign and malignant masses. Most circumscribed masses are benign. Nevertheless, due to specific histological characteristics, certain malignant lesions or lesions with a risk of malignancy may appear in the mammography in this falsely reassuring form. An indistinct contour in the mammography is suspicious and requires a tissue sample. The positive predictive value of malignancy varies according to the morphology of the contour. It is lower for microlobulated contours, increases for masked, then indistinct contours and reaches 96% for spiculated contours. However, in rare cases, certain benign lesions may appear in the form of spiculated masses. In these specific cases, a correlation between the histological results with the imaging data is essential in order to avoid failing to recognise an underlying malignant lesion that the biopsy may have underestimated.

[The lesions of flat epithelial atypia diagnosed on breast biopsy]. / Les lésions de métaplasies cylindriques atypiques diagnostiquées sur biopsie mammaire.

Among pre-invasive breast diseases, the lesion of flat epithelial atypia has a level of risk that remains unclear. The clinical significance of these lesions and how to behave during their diagnostic biopsy (monitoring vs. surgery) are still uncertain, because few studies (including monitoring) are available and because of the polymorphic spectrum of lesions and their many denominations across the studies in the literature. This article aims to update our knowledge and provide elements for the management of these lesions diagnosed on breast biopsy.

Impact of immunohistochemical markers, CK5/6 and E-cadherin on diagnostic agreement in non-invasive proliferative breast lesions.

AIMS: To assess the impact of cytokeratin (CK) 5/6 and E-cadherin immunohistochemistry on diagnostic agreement of non-invasive proliferative breast lesions. METHODS AND RESULTS: Twenty pathologists classified 105 cases of non-invasive proliferative breast lesions into 10 diagnostic categories. One haematoxylin and eosin (H&E) slide of each case was analysed on a first round and one H&E slide with corresponding CK5/6 and E-cadherin immunohistochemistry was analysed on a second round. Interobserver reproducibility for category-specific and management-specific lesions was measured on each round. CK5/6 and E-cadherin had little impact on diagnostic agreement, which remained moderate between the first and second rounds (overall kappa coefficients of 0.47 and 0.53, respectively, P = NS). Levels of agreement slightly improved for lesions with specific CK5/6 and E-cadherin immunoprofiles (usual ductal hyperplasia, atypical ductal hyperplasia, atypical lobular hyperplasia, lobular carcinoma in situ, non-high-grade ductal carcinoma in situ), but the differences observed were not statistically significant. However, diagnostic agreement improved when lesions were grouped according to their management category (overall kappa coefficients of 0.58 and 0.66 in the first and second rounds, respectively). CONCLUSIONS: CK5/6 and E-cadherin immunohistochemistry has little impact on interobserver reproducibility for non-invasive breast lesions. Diagnostic agreement can, however, be improved by grouping lesions in management categories.

Use of radiology for the pathologist in the management of breast lesions.

Today radiology is an essential step in the pathological analysis of breast biopsies. It is determinant at each stage of the management of non palpable lesions, clusters of microcalcifications and opacities, whether this concerns the needle biopsy or the surgical excision. Firstly, an X-ray is necessary to ensure that the core needle biopsy specimen has been adequately sampled and when samples with microcalcifications are selected by the radiologist, management can be more specific and accurate. In the case of surgical specimens, the X-ray confirms the presence of the radiographic abnormality or the clip indicating the site of the surgical excision which guides sampling. Some radiographic features also provide information on underlying pathologies allowing management to be adapted accordingly. Radiographs are also important to ensure that microscopically detected microcalcifications or lesions exactly correspond to the radiographic abnormality in size and location. The paraffin block can also be X-rayed to select those containing microcalcifications for additional slicing. It is also important to identify the presence of modifications caused by the core needle biopsy (fibrosis, haemorrhage and inflammation) and to carefully recognize displacement of epithelial cells and pseudo-emboli resulting from the needle procedure. Such correlation between radiology and pathology is essential so that appropriate management of the specimen can be adapted and to avoid pitfalls arising from pre-operative procedures.

Stellate images: anatomic and radiologic correlations.

The stellate images are the most well-known and most typical finding in the diagnosis of breast cancer. Its positive predictive value is very high. However, in some cases, the stellate images can correspond to benign lesions. Stellate images require high quality mammograms, precise analysis, and as always in senology, confirmation by clinical examination, ultrasound if necessary, and in the case of palpable lesions, cytology. In this work, we will study malignant and benign stellate images mammographic-pathologic correlation as well as the importance of stellate images in the detection of non-palpable lesions. The typical stellate finding is correlated with the phenomena of fibrosis and elastosis and it is impossible to distinguish benign spicules from malignant spicules on mammography. We will present guidelines in the face of stellate image. Stellate images are for the most part suggestive of malignant lesions, and their discovery should lead to suspicion of cancer until the contrary is proven.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Gene transcript assay by real-time RT-PCR in epithelial breast cancer cells selected by laser microdissection.

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERalpha, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor homogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma.

In a retrospective study of 488 women with primary breast cancer, after a median follow-up of 10 years, we sought interactions between disease-free survival (DFS) and overall survival (OS) and tumor antigen levels of two components of the plasminogen system, urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and the transmembrane growth factor receptor c-erbB-2. We used ELISAs (American Diagnostica, Greenwich, CT, USA) to quantify uPA and PAI-1 antigen levels in cytosols, and a double monoclonal antibody-based assay (EIA) (Ciba Corning Diagnostics, Alameda, CA, USA) to quantify c-erbB-2 in membrane extracts of the same tissues. Weak positive correlations were found between uPA and c-erbB-2 (r(s) = 0.146; p = 0.001) and between PAI-1 and c-erbB-2 (r(s) = 0.154; p < 0.001). In the overall population, using univariate analyses, c-erbB-2 overexpression and high uPA and PAI-1 antigen levels (> 300 IU/mg, > 1.40 ng/mg and > 5.53 ng/mg, respectively) were significantly associated with shorter DFS (p = 0.003, p < 0.001 and p < 0.001, respectively) and OS (p < 0.001 in all cases). Using multivariate analyses, PAI-1, node status and tumor size were independent predictors of DFS and c-erbB-2 was retained in the model only for OS. In the node-negative subgroup, PAI-1 was the strongest significant survival predictor both for OS (p = 0.003; HR 2.52) and DFS (p < 0.001; HR 2.39). This study shows that in primary breast cancer c-erbB-2 offers no additional prognostic information when uPA and/or PAI-1 are candidates in the multivariate analyses.

[HER2 gene amplification assay: is CISH an alternative to FISH?]. / Recherche de l'amplification du gène HER2: la CISH est-elle une alternative à la technique FISH?

The HER2 proto-oncogene encodes a transmembrane protein, which is considered to function as a growth factor receptor. Overexpression of this protein found by immunohistochemistry in about 20% of infiltrating breast carcinomas, has a predictive value of response to treatment by trastuzumab, an anti-HER2 humanized monoclonal antibody. Search for HER2 gene amplification is necessary to adapt the immunohistochemical technique quality and also in the cases of delicate analysis or weak overexpression. It is usually carried out by Fluorescence In Situ Hybridization (FISH). A more recent hybridization technique, named CISH because of its chromogenic revelation is an alternative method, which gives highly correlated results with FISH. We present details of this technique, which may be more familiar for the pathologists than FISH, because reading analysis is similar to that of immunohistochemical staining.

Prognostic value of MIB-1 antibody labeling index to predict response to Bacillus Calmette-Guérin therapy in a high-risk selected population of patients with stage T1 grade G3 bladder cancer.

PURPOSE: Proliferation rate is an important determinant of bladder tumor progression. However, this factor has not yet been correlated to bacillus Calmette-Guérin (BCG) therapy response in a selected high-risk population of patients with stage T1 grade G3 bladder cancer. To assess the predictive value of the proliferation rate, an immunoreactivity test with monoclonal antibodies MIB-1 was carried out. The aim of this study was to evaluate the prognostic value of an MIB-1 labeling index by selecting a group of responsive patients prior to intravesical therapy. MATERIALS AND METHODS: After complete transurethral resection, 35 patients with T1G3 bladder carcinoma received 6 weekly installations of BCG (intravesical Pasteur strain: 75 mg in 50 ml course of BCG therapy). After treatment a cystoscopy and randomized biopsies of the bladder mucosa were carried out and all recurrences were systematically resected. All tissue samples were fixed in Bouin's solution, embedded in paraffin and stained with hematoxylin-eosin-safran. Pathologists had sufficient material to perform immunomarking in 25 patients using peroxidase-antiperoxidase (PAP) technique, with antiprotein monoclonal antibody MIB-1 (Immunotech, Marseilles, France) to study MIB-1 expression before BCG therapy. Consensus was obtained from three independent pathologists for all sections. The results were expressed in a percentage of marked nuclei. Ten percent increment thresholds were established from 10 to 60%. Contingency tables were established, chi2 (p1) and Fisher exact test (p2) were performed for each threshold of 10%. RESULTS: Median follow-up was 57.3 months (range 25-144). Of the 25 patients, 8 (32%) did not respond to BCG therapy, 17 (68%) responded positively. With a 20% threshold, there was a statistical difference (p1 = 0.03, p2 = 0.04) between responder (R) and nonresponder (NR) patients. All the 7 patients with less than 20% of nuclear activity positively responded to BCG. At this threshold level, sensitivity was high but specificity low (positive predictive value = 0.44). If we consider other reactivity thresholds there were no statistical differences between R and NR patients (10%) threshold p1 = 0.13, p2 = 0.19; 30% p1 and p2 = 0.20; 40% p1 = 0.82, p2 = 0.61; 50% p1 = 0.57, p2 = 0.55). CONCLUSION: Our study indicates that the proliferation rate, assessed by MIB-1 immunoreactivity in Bouin's solution-fixed primary tissue, could be a useful predictive marker of outcome in T1G3 bladder carcinoma. With a 20% reactivity cut-off, a negative MIB-1 immunomarking appears to predict a positive response to BCG instillations. However, on the other hand, MIB-1 is of limited clinical use because the low specificity of this test cannot predict failure and then select candidates for cystectomies.

[40 years of progress in breast imaging]. / Quarante ans de progrès en imagerie mammaire.

In the sixties, mammary diagnosis is just clinical, then the low contrast mammography, not very efficient, appears in the seventies. During the eighties, the ultrasound is set up while modern mammography with high contrast allows the non palpable breast lesions diagnosis. In the nineties years the mammography come before the clinical examination within the context of the breast cancer screening program. Some histological correlation are more specific about the ductal carcinoma in situ grading with microcalcifications, while new techniques (MRI, CT) are evaluated. At present the stereotactic large core breast biopsies benefit from the digital prone table, allow a histological diagnosis and avoid surgical excision of some indeterminate images. After the pernicious effects of imaging, we assess the progress according to the cancerous disease results. We also consider the problem of over-diagnosis and over-treatment of ductal carcinoma in situ.

[Breast cancer: prognostic value of a dissemination index based on 4 components of the urokinase-type plasminogen activator system]. / Cancer du sein: valeur pronostique d'un index de dissémination basé sur quatre composants du système activateur du plasminogène type urokinase.

Among the proteases involved in the tumor invasion process, components of the plasminogen activator system (plasminogen activator type-urokinase uPA, its membrane receptor uPAR and its two inhibitors PAI-1 and PAI-2) appear to define high risk patients in primary breast cancer. As individual analysis of each component of the plasminogen activator system does not reflect the complex interactions between the different components, we studied the prognostic impact of a dissemination risk index combining the four variables. We found that this index was the most powerful prognostic factor, particularly in node-negative patients.

[Study of the Ki-67 antibody immunolabeling of renal adenocarcinomas with or without renal vein thrombosis]. / Etude de l'immuno-marquage des adénocarcinomes rénaux, avec et sans thrombose de la veine rénale, par l'anticorps Ki67.

OBJECTIVES: The objective of this study was to compare the proliferation index (immunolabelling by monoclonal antibody Ki67 - MIB-1) of renal cell carcinomas as a function of the presence or absence of renal vein thrombus. Analysis of the numbers of Ki67-positive nuclei can be used to assess the degree of aggressiveness of the cell populations of these various carcinomas (tumour without thrombus, tumour with thrombus and neoplastic thrombus). METHODS: Twenty three renal cell carcinomas with renal vein thrombus were matched for Furhman grade (1st degree), tumour volume (2nd degree) and the patient's age (3rd degree) with 23 renal cell carcinomas not presenting any vascular embolus on histology. Monoclonal antibody MIB-1 immunolabelling was performed on 69 paraffin-embedded specimens: 23 tumours with thrombosis, the 23 corresponding neoplastic thrombi and 23 tumours without vascular embolus. RESULTS: A correlation between Furhman grade and the percentage of immunolabelled nuclei was observed (mean: 2.67% for low-grade tumours and 14.34% for high-grade tumours). No labelling difference was observed between the two populations of primary tumours (with thrombus/without embolus). Primary tumours presented significantly weaker Ki67 labelling than their corresponding neoplastic thrombus (mean of 2.47% versus 10.3%, p < 0.01). CONCLUSION: This study shows that there is no difference of the proliferation index between tumours with neoplastic venous thrombus and those with no histological vascular embolus. However, a difference of proliferation index was observed between the primary tumour and its corresponding thrombus, which presented a statistically higher immunolabelling. This finding suggests that the thrombus possesses more dividing cells than the primary tumour, i.e. has a shorter doubling time.

Dissemination risk index based on plasminogen activator system components in primary breast cancer.

PURPOSE: To study interactions between disease-free survival (DFS) and four components of the plasminogen activator system: urokinase-type plasminogen activator (uPA), its two inhibitors (PAI-1 and PAI-2), and its membrane receptor uPAR. PATIENTS AND METHODS: We conducted a retrospective study of 499 primary breast cancer patients (median follow-up, 6 years). uPA, PAI-1, and PAI-2 were determined on cytosols and uPAR on solubilized pellets, using enzyme-linked immunoadsorbent assay kits (American Diagnostica, Greenwich, CT). Classical univariate and multivariate statistical methods were used together with multiple correspondence analysis to graphically examine interactions between the variables and outcome. RESULTS: By univariate analysis, higher uPA and PAI-1 values were significantly related to shorter DFS (P =.002; P <.00002). PAI-2 was not significantly related to DFS, although patients with high and very low PAI-2 values had a longer DFS. Multiple correspondence analysis showed the parallel impact of uPA and PAI-1 on outcome, and the clearly different behavior of PAI-2 compared with PAI-1. The prognostic contribution of uPAR seemed weak by both methods. A dissemination risk index [uPA x PAI-1/(PAI-2 + 1)], taking into account the modulation of uPA proteolytic activity by the ratio of its two inhibitors, was then tested. Dissemination risk index was selected as an independent variable in the Cox model in the overall population (P <.000001) and in node-positive patients (P <.00001). It was the only variable selected in node-negative patients (P =. 003). CONCLUSION: A dissemination risk index determined on primary tumor and taking into account the different effects of PAI-1 and PAI-2 on uPA can be of major help in clinical management of breast cancer, particularly in node-negative patients.

Study of FHIT transcripts in normal and malignant breast tissue.

Cytogenetic and molecular studies have suggested that the 3p14.2 chromosome subband contains tumor suppressor genes involved in the pathogenesis of many types of human cancers. Recently, the FHIT (fragile histidine triad) gene was identified in this part of chromosome 3 as a candidate suppressor gene, and abnormal transcripts of this gene have been observed in various human tumors, including breast tumors. However, several investigators have challenged the involvement of FHIT in human cancers, especially because of discrepancies between data obtained with various PCR strategies and the observation that FHIT is alternatively spliced in normal tissues. We examined FHIT gene transcripts in a panel of normal (n = 27) and malignant (n = 33) breast tissue samples using single-stage PCR and two nested PCR strategies. In addition to a normal transcript, multiple variant transcripts were found at very low levels (<1% of the wild-type FHIT transcript) in the majority of the breast tumors, but also in adjacent normal breast tissues and normal breast tissue from women without cancer. These results do not support the involvement of the FHIT gene in breast tumorigenesis.

Overexpression of the stathmin gene in a subset of human breast cancer.

Stathmin is a highly conserved cytosolic phosphoprotein that destabilizes microtubules. Stathmin, which has been proposed as a relay protein integrating diverse cell signalling pathways, acts in vitro as a tubulin-sequestering protein, and its activity is dramatically reduced by phosphorylation. Interestingly, stathmin expression and phosphorylation are regulated during the control of cell growth and differentiation, and there is much evidence suggesting that in vivo stathmin plays a role in the control of microtubule dynamics during mitosis. Stathmin may thus be considered as one of the key regulators of cell division. We examined 50 human primary breast tumours for stathmin mRNA and protein expression and screened for abnormalities in the chromosome region harbouring the stathmin gene. Overexpression of stathmin was found in 15 tumours (30%). At the present stage, no clear correlation emerged between stathmin expression and several prognosis markers. Interestingly, perfect matching was observed between stathmin mRNA overexpression, protein overexpression and strong staining for stathmin on paraffin-embedded tumour sections when specimens were available. Furthermore, a tentative link between loss of heterozygosity (LOH) in the 1p32-1pter region and stathmin overexpression was observed. Our results suggest that stathmin might play a role in breast carcinogenesis and that stathmin-overexpressing tumours may represent a new subtype of breast cancer.

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods.

Urokinase-type plasminogen activator (uPA) is a potentially important prognostic factor in breast cancer for identifying patients at high risk of recurrence. This retrospective study assessed two enzyme-linked immunosorbent assay (ELISA) methods measuring uPA antigen levels in 499 primary breast cancer cytosols. Both uPA methods were applied to cytosols used routinely for oestrogen (ER) and progesterone (PgR) receptor assays. uPA was determined using a classical ELISA method (Imubind; American Diagnostica) and a novel automatic immunoluminometric assay (Lia; Sangtec Medical). The uPA Imubind method revealed about twice as much uPA antigen (median 0.75 ng mg(-1) protein) as the uPA Lia method (median 0.38 ng mg(-1) protein). The correlation coefficient between the two methods was acceptable (r = 0.81), but the two techniques are not interchangeable. Univariate analyses confirmed the poor outcome of patients whose tumours contained large amounts of uPA, regardless of the technique used. Multivariate analyses showed that uPA Imubind and uPA Lia values were both strong independent prognostic factors.