Pulmonary mucormycosis diagnosed by bronchoalveolar lavage: a case report and review of the literature.
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Adolescent↗
Biomedical subjects
E J Wilkinson
Publications and source records attributed to E J Wilkinson.
Deep loop excision for prehysterectomy endocervical evaluation.
OBJECTIVE: Our purpose was to determine whether office deep loop excision should replace cone biopsy for frozen-section endocervical evaluation before planned hysterectomy. STUDY DESIGN: This cohort study comprised 31 patients who underwent office deep loop excision with frozen-section analysis followed by hysterectomy and 50 historic controls who underwent cone biopsy with frozen-section analysis followed by hysterectomy. Diagnostic accuracy, margin status, presence of residual disease, morbidity, and cost were compared. RESULTS: Loop excision frozen sections had sensitivity (ectocervical specimen, 96%; deepest endocervical specimen, 93%), specificity (100%, 86%), and positive (100%, 88%) and negative (75%, 92%) predictive values similar to those of frozen cone biopsy (95%, 80%, 98%, and 67%, respectively. No differences in margin status, presence of residual dysplasia, or morbidity were observed. The shorter operating room time for vaginal hysterectomy after loop excision (p < 0.01) resulted in an approximate $2000 savings. CONCLUSION: Office loop excision is a cost-effective option for endocervical evaluation before planned hysterectomy.
Adult↗
p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias: evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas.
Previous studies of vulvar carcinomas have shown two distinct subsets with respect to several clinicopathologic features. In younger women, the tumors are frequently human papillomavirus (HPV) positive, are usually of basaloid or warty histology, and are associated with vulvar intraepithelial neoplasia. In older women, the tumors are usually HPV negative, are typical keratinizing squamous carcinomas, and are associated with squamous hyperplasia--a lesion that has been purported to serve as a precursor to HPV-negative invasive carcinoma. In squamous carcinomas of the cervix, p53 inactivation (through gene mutation or interaction with the HPV E6 oncoprotein) occurs in most cases. Comparatively few studies have assessed p53 mutation and HPV status in vulvar carcinomas, and none has used molecular markers to evaluate squamous hyperplasias as direct precursors of HPV-negative invasive cancers. Of 18 invasive squamous carcinomas analyzed, seven (39%) were found to be HPV positive. Four p53 gene mutations were identified--all in HPV-negative tumors. DNA was subsequently prepared from microdissected archival tissues from all four specimens showing p53 gene mutations. DNA was separately isolated from normal squamous epithelium, invasive squamous carcinoma, and associated squamous hyperplasia. In each specimen, the p53 mutation was confirmed in the invasive tumor and absent in both normal and hyperplastic epithelium. To further investigate squamous hyperplasia as a potential precursor of HPV-negative invasive carcinoma, the authors determined the clonality of hyperplastic lesions adjacent to invasive carcinomas with p53 mutation. Clonality analyses were performed using a polymerase chain reaction (PCR)-based assay for X chromosome inactivation. Although all three informative carcinomas tested were monoclonal, corresponding normal epithelia and hyperplastic lesions were polyclonal. These findings underscore the heterogeneity of vulvar cancers with respect to loss of wild type p53 function either by interaction with the HPV E6 oncoprotein or somatic mutation of p53, and suggest that squamous hyperplasias do not serve as direct precursors of HPV-negative squamous carcinomas.
Adult↗
Prognostic factors in carcinoma of the vulva: a clinicopathologic and DNA flow cytometric study.
The clinical staging of carcinoma of the vulva is a predictor of patient survival; however, the significance of other prognostic factors remains somewhat controversial. Length of survival after diagnosis of invasive squamous cell carcinoma was determined for 39 clinically staged and surgically treated patients who were followed at our institution. Clinical stage, tumor type, use of radiotherapy (RT), histopathologic features (invasive pattern, depth of invasion, lymph node status, nuclear grade, adjacent dysplasia, desmoplasia, inflammation) and DNA ploidy (determined by flow cytometry from paraffin-embedded tissue) were evaluated as predictors of survival. Kaplan-Meier survival curves were generated for strata defined by each of the various predictors and compared using the log-rank test. Advanced stage (p = 0.0002), RT use (p = 0.0004), "spray" invasive pattern (p = 0.005), positive lymph node status (p = 0.001), increased positive lymph node number (p = 0.016), and greater depth of invasion (p = 0.039) were associated univariantly with decreased survival time. Spray invasive pattern (p = 0.018), positive lymph node status (p = 0.030), positive lymph node number (p = 0.040), and RT use (p = 0.045) continued to be associated with decreased survival time after controlling for stage. Of the significant factors, invasive pattern stands out as a qualitative feature that may have potential benefit in predicting survival independent of clinical stage in patients with vulvar carcinoma.
Adult↗
Role of the pathologist in biomarker studies.
Cancer chemoprevention is defined as intervention by chemical agents prior to invasion to inhibit or slow the carcinogenic process. Using surrogate endpoint biomarkers in chemoprevention studies may reduce the size, length and cost of clinical prospective randomized trials in high-risk populations. Intermediate biomarkers are measurable alterations in the tissues at risk and include differentiation, genetic composition, biochemical expression, and proliferation. Assessment is possible because invasive epithelial neoplasms are known to begin as intraepithelial proliferations with a spectrum of cellular abnormalities extending to carcinoma in situ. Genetic heterogeneity begins in the intraepithelial phase; a stochastic accumulation of genetic errors characterizes the progression of clonal evolution within the tumor through the process of invasion and metastasis. Pathologic features associated with this process include tumor classification as well as whether it is intraepithelial or invasive. If the process is intraepithelial, the grade and extent of the intraepithelial lesion are reported. If the neoplasm is invasive, tumor size, extent, degree of differentiation (histologic and nuclear grade), mitotic rate, vascular invasion, and lymph node involvement are evaluated. In assessing biomarkers relevant chemoprevention, and without complete regression of the neoplasm with the chemopreventive agent or agents, measurable parameters along with histopathologic features are applicable. Three methods readily applicable for this purpose that can be applied to paraffin-embedded, formalin-fixed tissue include quantitative pathology, immunohistochemistry, and molecular biologic applications. These methods require some consistency in handling and processing the tissues under study; results may deteriorate due to a number of processing variables, including time to fixation, time in fixative, and fixative type. Quantitative pathology, including static image analysis and flow cytometry, can determine total DNA content. Using static image analysis, very small tumors can be studied. In addition, adjacent intraepithelial and invasive components of a tumor may be studied from a single slide. Steroid receptors, oncogenes, and other proteins detectable through immunohistochemical or molecular biologic methods can be quantitated by this technique as well. Cell cycle synthetic function is assayable by both methods. Flow cytometry can calculate the total percentage of cells in S-phase, or the tumor cell S-phase fraction based on the percentage of cells detected between the G0, G1 peak and the G2 + M peak. A similar approach is generally not applicable with current image analysis equipment; however, cell cycle related proteins such as MIB-1 (Ki-67 associated) can be quantified. Immunohistochemical methods can employ a wide variety of monoclonal antibodies to detect oncogene related proteins, including HER-2/neu (c-erbB-2) and p53. Molecular biologic methods, including in situ hybridization, polymerase chain reaction, and in situ PCR, can have many applications when applied to paraffin-embedded tissues, including detection of viral DNA, identification and measurement of apoptosis, and defining gene deletions.
Biomarkers, Tumor↗
DNA tetraploidy in Feulgen-stained bladder washings assessed by image cytometry.
The prognostic utility of DNA cytometry has been demonstrated for irrigation specimens from bladder neoplasms. While the traditional method of measuring the DNA content of cells recovered by bladder irrigation is flow cytometry, image analysis has been applied increasingly, with successful results. In some cases, image analysis has been shown to detect DNA aneuploid populations missed by flow cytometry. The DNA aneuploid population most frequently missed by flow cytometry is in the DNA tetraploid range. The purpose of the present study was to review image cytometry data on bladder washings analyzed at the University of Florida Diagnostic Referral Laboratories during a one-year period, with special emphasis on the subset with DNA tetraploid histograms. Of the 205 cases reviewed, 127 (62%) were DNA diploid, 36 (18%) DNA aneuploid and 42 (20%) DNA tetraploid. Corresponding cytology was negative in 113/127 (89%) of DNA diploid, 3/36 (8%) of DNA aneuploid and 29/42 (69%) of DNA tetraploid cases. Within the DNA tetraploid group, 45% of cases had no clinical (cystoscopic) or pathologic (cytologic and histologic) evidence of neoplasia. None of these patients developed tumors during follow-up. The presence of DNA tetraploidy in cytologically negative cases should be interpreted cautiously.
Aneuploidy↗
How complete is influenza immunization coverage? A study in 75 nursing and residential homes for elderly people.
BACKGROUND: Elderly people in residential accommodation are particularly susceptible to outbreaks of influenza. Up to 70% of residents can become ill and many will develop complications or die. Immunization can prevent such outbreaks and is cost-effective. AIM: A study was undertaken to measure influenza immunization coverage in residential accommodation for elderly people and to identify factors that might influence uptake. METHOD: In March 1992, a questionnaire survey was conducted of all 113 registered nursing and residential homes for elderly people, in South Glamorgan. It asked about the demographic characteristics of people resident on 1 October 1991, their influenza immunization history and the homes' arrangements for administering immunizations. RESULTS: Questionnaires were returned by respondents from 75 homes (66%). Mean influenza vaccine uptake was 67%. Uptake was higher in nursing homes (mean of 82% in eight nursing homes) than in homes registered as both nursing and residential homes (mean of 76% in six homes) or in residential homes (mean of 65% in 61 homes). Nearly all of those immunized (94%) had been immunized by the end of November 1991. Residents who were reported to have underlying disease that increased their risk of complications if they contracted influenza were no more likely to have been immunized than those without risk factors. Immunization coverage varied considerably both between homes and between general practices. Most general practices in South Glamorgan had several elderly people in residential accommodation on their list, but only nine out of 64 practices had immunized all the elderly residents on their list and 12 practices had immunized fewer than half. Routine recording of immunization status in nursing and residential homes was variable, often as a consequence of poor communication between the primary health care team and staff at the home. Even where recorded, retrieval of the data was sometimes a problem. CONCLUSION: Influenza immunization coverage could be improved if general practices held a case register of all at-risk patients including elderly residents, and if nursing and residential homes were encouraged to keep better immunization records. These measures would facilitate year-on-year monitoring of influenza immunization coverage and the targeting of homes with low immunization coverage.
Adult↗
Vaginal columnar cell metaplasia. An acquired adenosis associated with topical 5-fluorouracil therapy.
Vaginal adenosis, with columnar epithelium of endocervical or endometrial type forming glands within the vagina, has been observed as a congenitally acquired process, associated with in utero exposure to diethylstilbestrol; approximately 20% of those women exposed in utero have adenosis. It has been observed that vaginal adenosis may occur in nonexposed women; the origin is thought to be acquired following puberty. This study analyzed eight patients who, following topical vaginal therapy with 5-fluorouracil for vaginal intraepithelial neoplasia, had unresolving, reddened areas in the upper one-third of the vagina. Biopsy of these areas demonstrated columnar epithelium on the vaginal surface, where stratified squamous epithelium would be expected. Superficial vaginal glands (adenosis) lined with epithelium identical to the surface columnar epithelium were also identified. This finding is consistent with a metaplastic process in which stratified squamous epithelium is replaced by columnar representing columnar cell metaplasia, or acquired vaginal adenosis.
Adult↗
Ki-67 expression in vulvar carcinoma.
The proliferative activity of invasive squamous cell carcinoma of the vulva was examined using a Ki-67 equivalent monoclonal antibody (MIB1), which gives a strong immunoreaction in paraffin-embedded tissue. Quantitation of Ki-67 immunostaining was accomplished by image analysis. Ki-67 immunostaining revealed two general patterns of reactivity in vulvar tumors: (a) a diffuse distribution of Ki-67 positive nuclei within the tumor mass and (b) a localized distribution of Ki-67 positive nuclei staining predominantly basilar components of tumor aggregates. The distribution of localized and diffuse patterns did not differ significantly between various clinicopathologic categories (age, histologic type and grade, FIGO stage, and lymph node status). However, the survival times for patients with a diffuse Ki-67 labelling pattern tended to be shorter than those for patients with a localized pattern. Survival curves based on the median positive nuclear area (PNA) calculated by image analysis did not differ significantly. Thus, the pattern of Ki-67 immunostaining, rather than the percentage of PNA, may have prognostic significance in vulvar squamous cell carcinoma.
Aged↗
Pathology of the vulva and vagina.
Extensive clinical, epidemiologic and pathologic research published in the past year has enhanced our understanding of the pathogenesis and natural history of human papillomavirus induced female genital tract lesions, and refined diagnostic and therapeutic modalities in management of these patients. Current experience with vulvar intraepithelial neoplasia as well as diagnosis and treatment of squamous cell carcinoma of the vagina and vulva are discussed. Advanced imaging techniques and immunohistochemical studies are employed in diagnosis and classification of less common neoplasms. New antibiotics and potent topical steroids are available for treatment of inflammatory vulvovaginitis, particularly in cases of recurrent candidiasis and lichen sclerosus. Persistent or resistant vulvovaginitis may benefit from an interdisciplinary approach to diagnosis and treatment.
Female↗
Quality control considerations for Ki-67 detection and quantitation in paraffin-embedded tissue.
Proliferative activity has prognostic significance in many solid tumors. Immunohistochemical analysis of tumor proliferation may be accomplished with the Ki-67 monoclonal antibody which recognizes a nuclear antigen expressed throughout the cell cycle. This antibody, however, cannot be used with formalin-fixed, paraffin-embedded tissue. Recently, a Ki-67 equivalent murine monoclonal antibody (MIB-a; AMAC, Inc., Westbrook, ME) was generated which can detect tumor proliferative activity in routinely processed tissue with microwave oven heating. Using quantiative image analysis, we assessed the effect of delay in fixation, total time of formalin fixation, and microwave heating time on the immunoreactivity of this antibody. The effect of time to fixation (0, 2, 4, 8, or 24 hours) on MIB-1 immunostaining was determined in various tumor tissues using image analysis. No significant difference in positive nuclear area was observed for tissues in which fixation was delayed for as long as 8 hours relative to controls. A 24-hour fixation delay resulted in a small decrease in positive nuclear area was observed for tissues in which fixation was delayed for as long as 8 hours relative to controls. A 24-hour fixation delay resulted in a small decrease in positive nuclear area relative to controls. The effect of fixation time (4, 24, or 48 hours) and microwave oven heating time on MIB-1 immunostaining was studied in tonsil tissue, and quantitated by image analysis. Good MIB-1 immunostaining was observed for all microwave oven heating times in tissue fixed for 4 hours.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
Ploidy results in prostatic carcinoma vary with sampling method and with cytometric technique.
To evaluate the effect of sampling method and cytometric method on DNA ploidy results, a comparison study was performed on 20 whole prostate glands removed at prostatectomy. Fresh sampling was by sampling fine-needle aspiration (FNA). Paraffin sampling was by microdissection and re-embedding of 3 to 13 (average 6) 5-mm foci of microscopically proven tumor. Analysis was by flow cytometry and by image cytometry (microscopically guided). In tumor negative cases, flow and image cytometry of the FNA was diploid in each case, and flow cytometry of paraffin-embedded tissue was diploid in 5/6 cases. In tumor positive cases, non-diploid tumor was detected by image cytometry of the FNA in 70%, by flow cytometry of the FNA in 29%, and by flow cytometry of paraffin-extracted nuclei in 21%. The most effective combination was sampling fine-needle aspiration and image analysis.
Adenocarcinoma↗
Fine needle aspiration of the breast for diagnosis of preinvasive neoplasia.
Fine needle aspiration (FNA) of the breast is a well-tolerated procedure used to evaluate palpable breast masses, has a reported mean specificity of 99%, and a reported sensitivity of 70-99%. The false positive rate varies from 0-0.4% in most larger series, with a reported false negative rate ranging from 0.7-22%; however, higher false negative rates have been reported in tumors under 2 cm in diameter. The FNA technique uses a fine, 20 gauge or less, needle and is not associated with a significant risk of tumor growing out the needle tract. FNA cytology is not effectively used if a breast mass cannot be palpated or distinguished from fibrous tissue within the breast. The procedure can be applied to nonpalpable masses detected by mammography by employing stereotactic techniques. The cytologic samples obtained from FNA can be used to distinguish atypical ductal hyperplasia from in situ or invasive ductal carcinoma; however, cytologic criteria to effectively distinguish ductal carcinoma in situ (DCIS) from invasive adenocarcinoma are not definitive in many cases, and are dependent on variables related to the type of intraductal tumor, the size and character of the cell groups, and the presence of single or disaggregated tumor cells. Employing current cytologic criteria, lobular carcinoma in situ (LCIS) may be distinguished from invasive lobular carcinoma in some cases; however, the individual LCIS cells are not morphologically distinct from lobular carcinoma cells. Atypical lobular hyperplasia has cellular features essentially the same as those seen in LCIS. Needle biopsy (NB) employs larger needles of 14-16 gauge.(ABSTRACT TRUNCATED AT 250 WORDS)
Biopsy, Needle↗
Tissue section image analysis of breast neoplasms. Evidence of false aneuploidy.
Two methods have emerged for measuring the DNA content of paraffin-embedded tissue using image cytometry: (1) analysis of thin sections, and (2) analysis of nuclei extracted from thick sections. These methods were evaluated using 31 breast tumors for which paraffin-embedded material was available. Cases selected represented 11 diploid, 11 tetraploid, and 9 aneuploid tumors. Results generated using image cytometry methods were compared with those obtained using flow cytometry. For thin sections, the tissue correction feature of the CAS 200 Image Cytometer was used to estimate the DNA content of whole nuclei from measurements made on sectioned nuclei. DNA histograms were generated from tissue sections cut at the same microtome setting (5 microns) before and after software corrections of 4.5 microns, 5.0 microns, 5.5 microns, 6.0 microns. 6.5 microns, 7.0 microns, and 7.5 microns. A comparison of flow cytometry and thin-section image analysis in the absence of tissue correction showed 90% concordance for diploid, 27% concordance for tetraploid, and 77% concordance for aneuploid tumors. The ploidy estimated on thin sections by at least one of the correction values was discordant in 72% of diploid, 91% of tetraploid, and 78% of aneuploid tumors. For cell nuclei extracted from paraffin, excellent agreement was found between flow and image cytometry (r = 0.933). It was concluded that in most cases, cell nuclei extracted from paraffin are preferable to tissue sections for ploidy analysis of breast tumors using image cytometry.
Aneuploidy↗
Vulvar vestibulitis is rarely associated with human papillomavirus infection types 6, 11, 16, or 18.
Vulvar vestibular biopsy specimens from 31 women with clinical and pathologic findings of vulvar vestibulitis were studied using polymerase chain reaction (PCR) for the identification of human papilloma virus (HPV). The PCR technique specifically probed for HPV types 6, 11, 16, and 18. Of the 31 subjects, three were found to have HPV within the biopsy specimens; two had HPV type 11 and one had HPV 16. Five of the 31 cases had histopathologic features of koilocytosis consistent with HPV effect; three of these five were found to have HPV. The findings support the hypothesis that HPV types 6, 11, 16, and 18 are rarely associated with vulvar vestibulitis. The frequencies identified were similar to those seen with control patients. True koilocytosis is the most useful pathologic feature distinguishing HPV-related cases; it is rarely identified in typical vulvar vestibulitis. Nonspecific changes in the vestibular epithelium associated with glycogen effect should not be interpreted as koilocytosis.
Adult↗
Comparison of two antibodies for evaluation of estrogen receptors in paraffin-embedded tumors.
Estrogen receptor (ER) content in breast cancer specimens is correlated with a prolonged disease free survival and increased likelihood of response to hormone therapy. Relatively few anti-ER antibodies are currently available for use in formalin-fixed, paraffin-embedded tissue. Recently, a new anti-ER monoclonal antibody (ERID5; AMAC, Westbrook, Maine) was generated which requires antigen retrieval by microwave oven heating for detection in routinely processed tissue. The specific aim of this study was to compare the ERID5 antibody with the commercially available rat monoclonal (ER-ICA; Abbott, Chicago, IL) which requires proteolytic enzyme digestion for detection in paraffin-embedded tissue. Sections from 20 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis were examined. Quantitation of ER antibody staining was performed without knowledge of the DCC values. Specimens containing > or = 20% specifically stained malignant cells were considered ER positive. The sensitivity and specificity of visual ER-ICA immunostaining were 57% and 83%, respectively. The sensitivity and specificity of visual ERID5 immunostaining were 93% and 50%, respectively. The predictive value of positive staining was 89% for the ER-ICA antibody and 81% for the ERID5 antibody. The predictive value of negative staining was 45% for the ER-ICA antibody and 75% for the ERID5 antibody. Previous studies have demonstrated a linear correlation between DCC values and the positive nuclear area (PNA) generated by image analysis for ER-ICA immunostaining. In the present study, a similar correlation between DCC value and ERID5 percentage PNA was observed (R = 0.670; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A method for detecting variability arising from errors in sample processing of paraffin-embedded tissue for DNA content analysis.
We present a method for controlling variability that may arise from inconsistencies in sample preparation for DNA content analysis of paraffin-embedded tissue. Human tonsil tissue obtained from routine surgical specimens was embedded in paraffin according to standard protocols. Fifty-micrometer sections were cut from the block and analyzed each day for 20 days to establish control ranges. One tonsil tissue section was processed in parallel with each run of clinical specimens. In this context, a run was defined as the simultaneous processing of 50-microns tissue sections for extraction of cell nuclei (dewaxing and rehydrating). If the tonsil G0/G1 peak coefficient of variation (CV) exceeded 2 SDs of the established mean, and optimum instrument performance and staining were verified, all samples prepared with the tonsil control were reprocessed. Instrument performance and staining were assessed by using the appropriate external controls. By using this rejection rule (12s), the frequency of sample reprocessing in our laboratory was approximately 6%. When the run was repeated and the tonsil control CV was within acceptable range, the G0/G1 peak CV of the corresponding clinical specimens improved 25% of the time. Because most investigators are willing to accept higher CVs for paraffin-embedded tissue than for fresh tissue, it is desirable to have a control to detect decreased peak resolution, resulting from errors in sample processing.
Cell Nucleus↗
Quantitative morphologic assessment of nuclei extracted from paraffin for DNA flow cytometry.
We present a method for quality control of flow cytometric DNA content studies using nuclei extracted from paraffin-embedded tissue. This method is based on a quantitative morphologic assessment of extracted nuclei. Cell nuclei prepared from 22 paraffin-embedded tumors known to contain discrete diploid and aneuploid stemlines were deposited onto poly-L-lysine-coated glass slides. Nuclei were stained with Diff-quic and examined by light microscopy. Two hundred nuclei were counted and classified based on morphologic appearance into tumor and nontumor groups. Classification criteria included differences in nuclear size, nuclear chromatin structure, the degree of nuclear chromatin condensation, and the presence of nucleoli. Excellent agreement was found between two independent observers (R = 0.989) on the classification of nuclei. The relative number of tumor nuclei on the morphologic preparation was compared with the relative number of aneuploid cells in the DNA histogram. Good agreement was observed (R = 0.975) in all but three cases in which the relative number of tumor nuclei was underrepresented by the percentage of aneuploid nuclei in the DNA histogram. In each case, further analysis by image cytometry demonstrated a diploid and aneuploid component of the tumor cell population. This quantitative method of morphologic examination of the preparation ultimately analyzed by flow cytometry offers several distinct advantages, including: (a) identification of peaks in the DNA histogram, (b) assessment for selective loss of cell nuclei in the extraction process, and (c) identification of biologic heterogeneity in tumor populations.
Cell Nucleus↗