Peter C.M. de Wilde

A panel of p16(INK4A), MIB1 and p53 proteins can distinguish between the 2 pathways leading to vulvar squamous cell carcinoma.

Two pathways leading to vulvar squamous cell carcinoma (SCC) exist. The expression of proliferation‐ and cell‐cycle‐related biomarkers and the presence of high‐risk (hr) HPV might be helpful to distinguish the premalignancies in both pathways. Seventy‐five differentiated vulvar intra‐epithelial neoplasia (VIN)‐lesions with adjacent SCC and 45 usual VIN‐lesions (32 solitary and 13 with adjacent SCC) were selected, and tested for hr‐HPV DNA, using a broad‐spectrum HPV detection/genotyping assay (SPF10‐LiPA), and the immunohistochemical expression of MIB1, p16INK4A and p53. All differentiated VIN‐lesions were hr‐HPV‐ and p16‐negative and in 96% MIB1‐expression was confined to the parabasal layers. Eighty‐four percent exhibited high p53 labeling indices, sometimes with parabasal extension. Eighty percent of all usual VIN‐lesions were hr‐HPV‐positive, p16‐positive, MIB1‐positive and p53‐negative. Five (of seven) HPV‐negative usual VIN lesions, had an expression pattern like the other HPV‐positive usual VIN lesions. In conclusion, both pathways leading to vulvar SCC have their own immunohistochemical profile, which can be used to distinguish the 2 types of VIN, but cannot explain differences in malignant potential.

Hue-Saturation-Density (HSD) Model for Stain Recognition in Digital Images From Transmitted Light Microscopy

BACKGROUNDnTransmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density.nnnMETHODSnThe usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels.nnnRESULTSnIn the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space.nnnCONCLUSIONSnIn the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose.

Intratumoral Recombinant Human Interleukin-12 Administration in Head and Neck Squamous Cell Carcinoma Patients Modifies Locoregional Lymph Node Architecture and Induces Natural Killer Cell Infiltration in the Primary Tumor

The objective of this study was to evaluate the histologic and immunohistopathologic effects of intratumorally given recombinant human interleukin-12 on the immune cells in the primary tumors and regional lymph nodes. Ten previously untreated patients with head and neck squamous cell carcinoma (HNSCC) were injected in the primary tumor twice to thrice, once weekly, at two dose levels of 100 or 300 ng/kg, before surgery. These patients were compared with 20 non-IL-12-treated control HNSCC patients. In the primary tumor, the number of CD56+ natural killer (NK) cells was increased in IL-12-treated patients compared with control patients. In some IL-12-treated patients, an impressive peritumoral invasion of CD20+ B cells was noticed. No differences were seen in the CD8+ or CD4+ T lymphocytes. Interestingly, major differences were apparent in the architecture of the enlarged lymph nodes of IL-12-treated patients; in particular, the distribution of B cells differed and fewer primary and secondary follicles with smaller germinal centers were observed. In addition, a decrease of dendritic cell lysosyme-associated membrane glycoprotein–positive cells in the paracortex was noted, resulting in a reduction of paracortical hyperplasia. In the lymph nodes, especially the CD56+ NK cells but also the CD8+ and CD4+ T lymphocytes, produced a high amount of IFN-γ. Patients, irrespectively of IL-12 treatment, with a high number of CD56+ cells in the primary tumor had a better overall survival than those with a low number. In conclusion, after i.t. IL-12 treatment in HNSCC patients, the largest effect was seen on the NK cells, with a higher number in the primary tumor and a high IFN-γ mRNA expression in the lymph nodes. Significant effects were noted on B cells, with altered lymph node architecture in every IL-12-treated patient and excessive peritumoral infiltration in some patients.

MIB1, A PROMISING MARKER FOR THE CLASSIFICATION OF CERVICAL INTRAEPITHELIAL NEOPLASIA

Formalin‐fixed and paraffin‐embedded tissue specimens of normal and dysplastic cervical epithelia (five CIN1, seven CIN2, five CIN3, and five normal) were assessed by an immunoperoxidase technique, using the monoclonal antibody MIB1, regonizing a formalin‐fixation‐resistant epitope on the cell proliferation‐associated Ki‐67 antigen. An image analysis system was used to measure four parameters associated with proliferative activity: the Ki‐67 labelling index (LI), the number of Ki‐67‐positive nuclei per unit length of basement membrane, and the maximum value and 90th percentile of the relative distances of Ki‐67‐positive nuclei from the basement membrane. All these four proliferation‐related parameters were highly correlated with the grade of dysplastic change in the epithelium (0·90

Intratumoral administration of recombinant human interleukin 12 in head and neck squamous cell carcinoma patients elicits a T-helper 1 profile in the locoregional lymph nodes.

The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary tumor, and peripheral blood. Ten previously untreated patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients. Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-γ and IL-10 increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections. A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed, due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells in the lymph nodes showed a 128-fold (mean) increase of IFN-γ mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.

An improved procedure to quantify tumour vascularity using true colour image analysis. Comparison with the manual hot-spot procedure in a human melanoma xenograft model

In a number of recent papers, the degree of tumour vascularization has been described as a promising new prognostic factor. Methods for the assessment of vascular density involve immunohistochemical staining of the vasculature, followed by counting the number of vessel profiles in the angiogenic hot spot. One of the problems of this procedure is the selection of the angiogenic hot spot, which has been described as being subject to inter‐observer variation. In this study, the value of true colour image analysis in reducing inter‐observer variation has been assessed. Highly (MV3) and poorly (M14) vascularized human melanoma xenografts were used to evaluate the image analysis procedure, and the image analysis results were compared with results from the conventional manual hot‐spot procedure. Assessment by image analysis was performed on measurement fields covering the entire tumour tissue specimens rather than on a single hot‐spot field. Also, by selecting the most densely vascularized area from all fields assessed by the semi‐automatic procedure, it was possible to objectify the hot spot selection (automated hot‐spot procedure). Manual assessment showed a good correlation between two independent observers for MV3 xenografts (r‐0·74, P‐0·014), but a poor correlation for M14 xenographs (r‐0·4, P>0·05). Automated assessment by different operators showed good correlations for both MV3 xenografts (r‐0·99, P<0·001) and M14 xenografts (r‐0·80, P‐0·006). It is concluded that although both manual vessel counting and semi‐automated image analysis can differentiate between the level of vascularization in the two types of xenograft (P<0·001 for both methods), the automated method is favourable in that it showed no significant inter‐observer effects. In M14 xenografts, the manual hot‐spot vessel densities did not correlate well with the automated hot‐spot densities (r‐0·27, P>0·05), indicating that selection of angiogenic hot spots in this tumour type is indeed subject to observer bias. The automated hot‐spot vessel densities were a reliable indicator of overall tumour vessel density in both tumour types. Image analysis allows analysis of vessel subclasses based on morphological criteria such as vessel profile area or diameter. In the model system used, the discrimination between MV3 and M14 xenografts was further enhanced by selectively examining vessels with diameters between 6 and 9 μm (P<0·0005). In conclusion, image analysis appears to offer an objective and more reproducible method to quantify tumour vascularity than manual counting of vessel profiles in the hot spot. Analysis of subclasses of vessels may further enhance the value of vessel density measurements in discriminating between tumour types differing in biological behaviour.

Effectiveness of routine follow-up of patients treated for T1–2N0 oral squamous cell carcinomas of the floor of mouth and tongue

The duration of follow‐up after treatment for head and neck cancer, the depth of the routine visits, and the diagnostic tools used are determined on the basis of common acceptance rather than evidence‐based practice. Patients with early‐stage tumors are more likely to benefit from follow‐up programs, because they have the best chance for a second curative treatment after recurrence. The purpose of this study was to determine the benefit of our 10‐year follow‐up program in patients with stage I and II squamous cell carcinoma (SCC) of the floor of mouth and tongue.

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells.

BACKGROUNDnAfter menopause, declining levels of estrogens may cause vaginal discomfort, or so-called vaginal atrophy. Evaluation of therapies for vaginal atrophy may be performed using the so-called maturation index. The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated.nnnMATERIALS AND METHODSnAutomated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs.nnnRESULTSnNo significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens.nnnCONCLUSIONSnIt was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.

Spectral imaging of multi-color chromogenic dyes in pathological specimens

We have investigated the use of spectral imaging for multi‐color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier‐transform spectroscopy and digital imaging. A pixel‐by‐pixel spectrum‐based color classification is presented of single‐, double‐, and triple‐color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki‐67 and TP53 in paraffin‐embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright‐field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi‐color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell‐ and tissue specimens. Figures on http://www.esacp.org/acp/2001/22‐3/macville.htm.

MIB1 expression in basal cell layer : a diagnostic tool to identify premalignancies of the vulva

Lichen sclerosus, high-grade usual vulvar intraepithelial neoplasia (VIN) and differentiated VIN have a different malignant potential. The objective of this study was to quantify the proliferative activity in the basal region of the epithelium of vulvar premalignancies. Furthermore, we investigated whether MIB1 expression in the basal region of vulvar epithelium can be helpful in diagnosing differentiated VIN, which may be hard to discern from normal epithelium. MIB1 was used to immunohistochemically visualise proliferating cells within formalin-fixed, paraffin-embedded, archival tissue sections of different vulvar premalignancies (N=48) and normal vulvar epithelium (N=16). Automatic digital image analysis software was developed to quantify the proliferating fraction in different parts of the epithelium (MIB1 positivity index). MIB1 expression differed among the various vulvar premalignancies; a MIB1-negative basal cell layer was a distinct feature of normal vulvar epithelium. No MIB1-negative basal cell layer was noted in differentiated VIN or other vulvar premalignancies. Owing to this negative cell layer, the MIB1 proliferation index in normal vulvar epithelium was significantly lower than in vulvar premalignancies. In conclusion, MIB1 expression can be a helpful tool in diagnosing a premalignancy and has additional value especially to distinguish differentiated VIN neoplasia from normal vulvar epithelium, but cannot explain the differences in malignant potential.