Grading immunohistochemical markers p16INK4a and HPV E4 identifies productive and transforming lesions caused by low- and high-risk HPV within high-grade anal squamous intraepithelial lesions.

OBJECTIVES: Because current guidelines recognise high-grade anal squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK 4a (p16) and E4 in low-risk human papillomavirus (lrHPV) and high-risk (hr)HPV-associated SILs as a potential basis for more selective treatment. METHODS: Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV-positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10-PCR-DEIA-LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0-4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0-2) to mark HPV production and life cycle completion. RESULTS: There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. CONCLUSIONS: Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a 'wait and see' policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high-risk populations, all patients with high-grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high-risk HPV-associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

Low- and high-risk human papillomavirus genotype infections in intra-anal warts in HIV-positive men who have sex with men.

BACKGROUND: Anogenital warts are often presumed to represent nondysplastic or low-grade anal intraepithelial neoplasia (LGAIN). We previously demonstrated that up to 20% of intra-anal warts in HIV-positive men who have sex with men (MSM) contain regions of high-grade AIN (HGAIN). OBJECTIVES: To determine the causative human papillomavirus (HPV) types of low- and high- grade dysplastic areas in warts from HIV-positive MSM. METHODS: A total of 42 intra-anal warts from 41 HIV-positive MSM were graded as nondysplastic, LGAIN or HGAIN. Whole-tissue sections (WTS) were analysed with the SPF10 polymerase chain reaction/LiPA25 HPV genotyping system. If the WTS contained multiple HPV types, dysplastic regions were isolated by laser capture microdissection (LCM) for HPV genotyping. RESULTS: Overall, 38 of 42 (91%) WTS tested positive for HPV DNA. Of these, 23 (61%) contained a single HPV type and 15 (39%) contained multiple HPV types. All LCM-selected regions contained no more than one HPV type. Ten of 42 (24%) WTS contained HGAIN disease, of which six (60%) were associated with a high-risk HPV (hrHPV) genotype. Twenty-three of 42 WTS contained LGAIN disease, of which two (9%) were associated with hrHPV. AIN lesions containing hrHPV types were identified using p16 staining. CONCLUSIONS: LGAIN lesions can be caused by high-risk HPV genotypes and vice versa. We therefore recommend routine follow-up and treatment of all dysplastic intra-anal warts for HIV-positive MSM.

The increased detection of cervical intraepithelial neoplasia when using a second biopsy at colposcopy.

OBJECTIVE: It has been suggested that colposcopy can miss a significant percentage of high-grade cervical intraepithelial neoplasia (CIN2+). Improved disease ascertainment was evaluated by taking multiple lesion-directed biopsies. METHODS: In a cross-sectional multicenter study in the Netherlands and Spain, 610 women referred to colposcopy following abnormal cervical cytology results were included. Multiple directed biopsies were collected from lesions and ranked according to impression. A non-directed biopsy of normal-appearing tissue was added if fewer than four biopsies were collected. We evaluated the additional CIN2+ yield for one and two directed biopsies. Colposcopic images were reviewed for quality control. RESULTS: In women with at least two lesion-directed biopsies the yield for CIN2+ increased from 51.7% (95%CI; 45.7-57.7) for one directed biopsy to 60.4% (95%CI; 54.4-66.2, p<0.001) for two biopsies. The highest CIN2+ yield was observed in women who were HPV16-positive, had high-grade squamous intraepithelial lesion (HSIL) cytology, and high-grade colposcopy impression. The yield increased from 83.1% (95%CI; 71.5-90.5) with one directed biopsy to 93.2% (95%CI; 83.8-97.3) with two directed biopsies. Only 4.5% additional CIN2+ were detected in biopsies not targeting abnormal areas on the cervix. CONCLUSIONS: A second lesion-directed biopsy is associated with a significant increase in CIN2+ detection. Performing a second lesion-directed biopsy and using a low threshold for abnormality of any acetowhitening should become the standard clinical practice of colposcopy.

The impact of human papillomavirus genotype on colposcopic appearance: a cross-sectional analysis.

OBJECTIVE: To study colposcopic performance in diagnosing high-grade cervical intraepithelial neoplasia or cervical cancer (CIN2+ and CIN3+) using colposcopic characteristics and high-risk human papillomavirus (hrHPV) genotyping. DESIGN: Cross-sectional multicentre study. SETTING: Two colposcopy clinics in The Netherlands and Spain. POPULATION: Six hundred and ten women aged 17 years and older referred for colposcopy because of abnormal cytology. METHODS: A cervical smear was obtained. Colposcopists identified the worst lesion, graded their impression and scored the colposcopic characteristics of the lesions. Up to four biopsies were collected, including one biopsy from visually normal tissue. MAIN OUTCOME MEASURES: CIN2+ and CIN3+, positive for HPV16 or other high-risk HPV types (non-16 hrHPV-positive). RESULTS: The mean age in HPV16-positive CIN2+ women was 35.1 years compared with 39.1 years in women with other hrHPV types (P = 0.002). Sensitivity for colposcopy to detect CIN2+ was 87.9% (95%CI 83.2-91.5), using colposcopic cut-off of 'any abnormality'. The remaining CIN2+ were found by a biopsy from visually normal tissue or endocervical curettage (ECC). Detection of CIN2+ by lesion-targeted biopsies was not different between HPV16-positive women [119/135; 88.1% (95%CI 81.2-92.9)] and non-16 hrHPV-positive women [100/115; 87.0% (95%CI 79.1-92.3); P = 0.776]. In multivariate analysis, 'acetowhitening' [odds ratio (OR) 1.91, 95%CI 1.56-3.17], 'time of appearance' (OR 1.95, 95%CI 1.21-3.15) and 'lesion >25% of visible cervix' (OR 2.25, 95%CI 1.44-3.51) were associated with CIN2+. CONCLUSIONS: In this population following European screening practice, HPV16-related CIN2+ lesions were detected at younger age and showed similar colposcopic impression as non-16 hrHPV high-grade lesions. There was no relationship between any of the colposcopic characteristics and HPV16 status.

Penetration of idarubicin into malignant brain tumor tissue.

Anthracyclines are effective in breast cancer and have in vitro cytotoxicity in glioma. In patients with glioma anthracyclines are not effective possibly because the hydrophilic drugs do not reach cytotoxic levels in tumor tissue. Idarubicin is more lipophilic than the other anthracyclines and is more cytotoxic in glioma cell lines. The uptake of idarubicin and its major metabolite idarubicinol in brain tumor tissue were measured in a patient with a brain metastasis from breast cancer and in 4 patients with malignant glioma after an oral dose of idarubicin (45 mg/m2 in 1 patient; 25 mg/m2 in 4 patients), given 15-24 h before brain tumor resection. The concentrations of idarubicin and of idarubicinol in tumor tissue exceeded the concurrent plasma concentrations as well as the peak plasma concentrations in all cases. The median tumor:concurrent plasma ratio of idarubicinol was 5.7 (range 1.7-18). The concentration of idarubicinol in the marginal zone between brain and tumor tissue was lower than in central tumor tissue, but was still higher than the plasma concentration in 2 of the 3 examined cases. Bone marrow suppression (platelets CTC grade 2, granulocytes CTC grade 4) occurred after a single dose of 45 ml/m2. No toxicity was seen at a dose of 25 mg/m2. These results, the in vitro activity of idarubicin in glioma, the convenience of oral administration, and its toxicity profile make clinical studies with idarubicin in malignant glioma, and perhaps also in brain metastases from breast cancer worthwhile.

[Squamous cell carcinoma of the nail bed]. / Het plaveiselcelcarcinoom van het nagelbed.

A 85-year-old woman presented clinically with paronychia. Treatment for this was uneffective, however. Finally, the diagnosis was Bowen's disease of the nail bed with underlying invasive squamous cell carcinoma. Treatment consisted of a subcapital amputation through the middle phalanx. Subungual tumors often appear to be a diagnostic problem because of their benign presentation. Every longstanding nail bed disease that does not respond to therapy needs to be biopted.

[Visceral granulomas and pericardial effusion caused by a Bartonella henselae infection]. / Viscerale granulomen en pericardeffusie veroorzaakt door een infectie met Bartonella henselae.

A 57 year-old woman was seen after a three-week period of upper abdominal pain, nausea, fever, headache and exertional dyspnoea. Laboratory examination showed an elevated ESR and serum gamma-GT activity. The chest X-ray showed cardiomegaly resulting from a pericardial effusion as was demonstrated by echocardiography. An abdominal CT-scan disclosed multiple hypodense lesions in the liver and spleen and lymphadenopathy along the hepatoduodenal ligament. Liver biopsy showed a necrotising granulomatous hepatitis. A recent infection with Bartonella, presumably B. henselae, was demonstrated serologically. The patient was treated with clarithromycin and recovered.

Relation of CD30 expression to survival and morphology in large cell B cell lymphomas.

AIMS: To investigate whether CD30 expression is correlated with anaplastic morphology, and whether this correlated with a better survival in large cell B cell lymphomas, as has been described for T cell lymphomas. METHODS: CD30 expression was investigated using frozen sections in a series of 146 large cell B cell lymphomas. Clinical data and follow up information were collected from 25 lymphomas with strong CD30 expression, 30 lymphomas with partial CD30 expression, and a control group of 25 lymphomas which did not express CD30. RESULTS: Morphological distinction between anaplastic and non-anaplastic tumours was difficult. Of the cases with an anaplastic morphology, 50% were CD30 positive, as were 24% of the polymorphic centroblastic B cell lymphomas. Only 65% of the morphologically non-anaplastic tumours were completely CD30 negative. There was no difference in survival among patients with lymphomas expressing CD30 and those that did not. Patients with morphologically anaplastic B cell lymphomas did not differ in their survivals from those with other high grade B cell lymphomas. Clinical stage at presentation was the only variable that was significantly associated with survival. CONCLUSIONS: CD30 expression occurs frequently in large cell B cell lymphomas and is poorly related to anaplastic morphology. Morphological distinction between anaplastic and non-anaplastic tumours is difficult. In contrast to T cell lymphomas, CD30 positive B cell lymphomas do not show a relatively favourable clinical course. The results presented here raise serious doubts as to whether large cell B cell lymphoma, based on the expression of CD30 or anaplastic morphology, can really be termed a separate entity.

Noncutaneous T-cell lymphomas. Recognition of a lymphoma type (large cell anaplastic) with a relatively favorable prognosis.

BACKGROUND: The clinical relevance of the updated Kiel classification for T-cell lymphomas is discussed. Large series with long-term follow-up are needed to investigate the clinical relevance of a separation into high- and low-grade T-cell lymphomas, based on the Kiel classification. METHODS: The clinicopathologic data of 97 consecutive noncutaneous T-cell lymphomas, diagnosed in the Comprehensive Cancer Center Amsterdam, between July 1, 1985, and December 1, 1990, were reviewed and analyzed for their prognostic significance. RESULTS: Immunohistochemistry contributed substantially in the diagnosis of T-cell lymphoma. Using the updated Kiel classification, many difficulties occurred in classifying these lymphomas. Only large cell anaplastic lymphoma (LCAL) and lymphoblastic lymphoma (LBL) were classified with a high degree of confidence. Variables associated with prolonged survival were classified as LCAL and low stage of disease (Stage I and II) at clinical presentation. Multivariate survival analysis revealed that subtype (grouped as LCAL versus non-LCAL) was selected as the most significant variable, closely followed by stage of disease at clinical presentation (grouped as Stage I/II versus Stage III/IV). LCAL was associated with a significantly better survival than were all other types of high-grade T-cell lymphoma (P = 0.018) and tended to be associated with a better survival than low-grade T-cell lymphoma (P = 0.067). No significant differences in survival were found between the other types of T-cell lymphoma or between high- and low-grade T-cell lymphomas as classified according to the updated Kiel classification. Other variables, such as sex and age (younger than 60 years versus older than 60 years) had no significant influence on survival time. CONCLUSIONS: This study indicates that the clinical relevance of classifying primary noncutaneous T-cell lymphomas according to the updated Kiel classification is limited because only a diagnosis of LCAL has prognostic relevance in predicting survival.

Maternal death due to acute necrotizing colitis in homozygous sickle cell disease.

The case history is described of a 21-year-old female with homozygous sickle cell disease and fatal postpartum haemorrhage from the colon. At autopsy many sickled cells were found in the arterioles of the mucosa and submucosa in combination with necrotic lesions and ulcerations of the sigmoid colon. A causal association between occlusion of blood vessels by sickled cells and the occurrence of vascular necrosis of the sigmoid colon is suspected.

Flow cytometric DNA content of fresh tumor specimens using keratin-antibody as second stain for two-parameter analysis.

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium-iodide for assessing DNA content and, on the other, a monoclonal keratin-antibody for marking epithelial and tumor cells. Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid and 146 (46%) as DNA "euploid." By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (22%) DNA euploid. The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%. In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA-content assessment becomes more reliable.

Malignant lymphoma of the gastrointestinal tract and mesentery. A clinico-pathologic study of the significance of histologic classification. NHL Study Group of the Comprehensive Cancer Center West.

A series of 92 malignant lymphomas of the gastrointestinal tract and mesentery obtained from a population-based registry was studied to assess whether the newly defined concept of mucosa-associated lymphoma has clinical relevance. The cases were grouped according to localization; gastric, intestinal, and mesenteric lymphoma. All cases were reviewed histologically, graded according to the Working Formulation, and reclassified according to the Kiel classification, which was modified to include the categories low- and high-grade mucosa-associated lymphoma. Clinical data, as well as staging and follow-up data, were related to both the original diagnosis and the diagnosis after reclassification. The results showed that the distribution of the types of lymphoma is related to site: centroblastic lymphoma was predominant in the stomach, lymphoblastic in the bowel, and follicular centroblastic-centrocytic in the mesentery. Gastrointestinal lymphoma was disseminated in approximately 50% of the patients at presentation. Survival analysis revealed that classification according to the original Kiel classification and grading according to the Working Formulation provided important prognostic information, whereas introduction of mucosa-associated lymphoma as an entity did not. It was concluded that modification of current classifications to include a separate category for mucosa-associated lymphoma is not useful.

Reproducibility of flow cytometric assessment of follicular tumours of the thyroid.

The reproducibility of the DNA index of paraffin wax sections from 44 follicular tumours of the thyroid (18 follicular adenomas and 26 follicular carcinomas), which had been assessed by flow cytometry was analysed in two laboratories, using consecutive sections of the same specimens and two different commercially available flow cytometers. Two slightly different cell preparation and staining techniques were used in the two laboratories. Using strictly defined criteria the histograms were classified blind as diploid, peritetraploid, aneuploid, or inadequate and insufficient by two independent investigators. Both the concordance between the two different flow cytometers and the agreement of duplicate assessments within the same flow cytometers were assessed. The mean coefficient of variation of the G0/G1 peak of the diploid tumours in the PARTEC flow cytometer was 5.5 (range 2.3-9.8) and in the FACS flow cytometer 5.2 (range 3.7-8.3); this difference was not significant. There was concordance of classification between the two laboratories in 35 of 36 cases. In 25 cases (18 diploid, seven aneuploid) the intralaboratory variation showed a 100% concordance in histogram classification. It is concluded that flow cytometer DNA index assessment of follicular tumours of the thyroid is reproducible and can be used to evaluate the discriminating and prognostic value of this feature.

Immuno- and enzyme-histo/cytochemical analysis of resin sections and cell suspensions: a comprehensive diagnosis of bone marrow on a single aspiration sample.

A protocol is described in which a single bone marrow aspiration specimen is used to prepare resin sections and cell suspensions. Using this protocol, a full battery of morphological, enzyme-histochemical, immuno-histochemical and cyto-pathological techniques can be applied. This allows a definitive bone marrow diagnosis to be established without resorting to bone marrow biopsy in the majority of patients.