Centromeric copy number of chromosome 7 is strongly correlated with tumor grade and labeling index in human bladder cancer.

The relationship between interphase cytogenetics and tumor grade, stage, and proliferative activity was investigated in 27 transitional cell carcinomas of the urinary bladder. Using fluorescence in situ hybridization with chromosome-specific DNA probes, the copy number of pericentromeric sequences on chromosomes 7, 9, and 11 was detected within interphase nuclei in touch preparations from tumor biopsies. Monosomy of chromosome 9 was detected in 9 of 22 cases (41%), while tetrasomy for chromosomes 7 and 11 was detected in 10 of 26 (38%) and 6 of 23 (26%) cases, respectively. Copy number of chromosome 7 was the most highly correlated with increasing tumor grade (r2 = 0.616, P less than 0.001, Spearman rank correlation) or increasing pathological stage (r2 = 0.356, P less than 0.002). Copy number for chromosome 9 did not correlate with either grade or stage (P greater than 0.05). Tumor labeling index (LI) was determined after in vitro 5-bromodeoxyuridine incorporation, while proliferating cell nuclear antigen LI was determined immunohistochemically. Increasing LI by either method correlated with increasing copy number for all three chromosomes tested (r2 = 0.473, P less than 0.002 for 7; r2 = 0.384, P less than 0.01 for 11; and r2 = 0.316, P less than 0.05 for 9). Since high tumor grade, stage, and LI are all indicative of more aggressive tumor behavior and worse prognosis, these findings suggest that polysomy, especially for chromosome 7, may be highly predictive for bladder tumor aggressiveness.

Heterogeneity of erbB-2 gene amplification in bladder cancer.

erbB-2 amplification and overexpression have been suggested as potentially useful prognostic markers in bladder cancer. We examined 141 bladder tumor specimens (45 fresh tissue samples and 96 formalin fixed tissue blocks) for erbB-2 amplification using fluorescence in situ hybridization. A dual labeling hybridization using a repetitive pericentromeric probe specific for chromosome 17 and a cosmid probe for the erbB-2 locus was performed to analyze the erbB-2 copy number in relation to chromosome 17 copy number on a cell by cell basis. Amplification (more than twice as many erbB-2 signals as centromere 17 signals per tumor) was found in 10 of 141 tumors. There was considerable heterogeneity in erbB-2 amplification. In a given tumor there was a wide range of erbB-2 copy number in amplified cells. The arrangement of erbB-2 signals in clusters in all amplified cases suggests that erbB-2 amplification occurs intrachromosomally in bladder cancer. Amplification was found only in tumors with aneusomy of chromosome 17 and was more frequent in pT2-T4 tumors than in pTa/T1 tumors. Overexpression was present without amplification in 51 tumors. All tumors with erbB-2 amplification showed erbB-2 overexpression. However, in 5 samples the proportion of cells with amplification was significantly lower than the fraction of cells with overexpression, indicating coexistence of two different mechanisms leading to overexpression in these tumors.

The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.

RHAMM is an oncogene that regulates signaling through ras and controls mitogen-activated protein kinase [extracellular signal-regulated protein kinase (ERK)] expression in embryonic murine fibroblasts. ERK is a dual-specificity kinase that controls expression of proteins relevant to tumorigenesis, proliferation, and motility. To assess whether RHAMM and ERK are involved in human breast tumor progression, we examined RHAMM, ras, and ERK expression in two cohorts of breast cancer patients using reverse transcription-PCR and immunocytochemistry. We show that overexpression of RHAMM in primary tumors of two patient cohorts was significantly prognostic of poor outcome in breast cancer progression. Furthermore, RHAMM overexpression occurred within subsets of tumor cells in the primary tumor, and this staining pattern was associated with lymph node metastases. The metastases exhibited a significantly higher level of staining for RHAMM than did the primary tumor. RHAMM expression strongly correlated with overexpression of both ras and ERK, although overexpression of either of these two signaling molecules was not by itself a prognostic indicator. These results identify a new parameter that is involved in lymph node metastasis of primary breast cancers and suggest that quantification of RHAMM overexpression may be a useful prognostic indicator for breast carcinoma progression.

Effect of skin temperature on selective vascular injury caused by pulsed laser irradiation.

The effect of skin temperature on vascular-specific injury caused by pulsed laser irradiation was examined. Ten healthy human volunteers were exposed to 1.5 microsecond pulses from a dye laser tuned to 577 nm. Compared to normothermic conditions (33 degrees C skin temperature) significantly more laser energy (p less than 0.01) was required to produce grossly visible purpura when the skin was cooled to 20 degrees C or heated to 40 degrees C. Histologically, laser-induced damage was confined to blood vessels at all three skin temperatures studied. At purpura threshold dose, there was intravascular agglutination without extravasation of red blood cells at 20 degrees C whereas at 33 degrees and 40 degrees C there was extravasation of red blood cells.

Effect of dye laser pulse duration on selective cutaneous vascular injury.

The pulsed dye laser at 577 nm, a wavelength well absorbed by oxyhemoglobin, causes highly selective thermal injury to cutaneous blood vessels. Confinement of thermal damage to microvessels is, in theory, related to the laser exposure time (pulsewidth) on selective vascular injury. This study investigates the effect of 577 nm dye laser pulsewidth on selective vascular injury. Nine Caucasian, normal volunteers received 577 nm dye laser exposures at pulsewidths of 1.5-350 microseconds to their skin. Clinical purpura threshold exposure doses were determined in each volunteer, and biopsies of threshold and suprathreshold doses were examined in each volunteer. The laser exposure dose required to produce purpura increased as pulsewidth increased in all 9 subjects (p less than 0.001). This finding corresponds to laser pulsewidths equal to or exceeding the thermal relaxation times for dermal blood vessels. Histologically, vessel damage was selectively, but qualitatively, different for short vs long pulsewidths. Pulsewidths shorter than 20 microseconds caused vessel wall fragmentation and hemorrhage, whereas longer pulsewidths caused no significant hemorrhage. The purpura noted clinically appears to be due to a coagulum of intralumenal denatured erythrocytes. At 24 h, there was marked vessel wall necrosis at all pulsewidths. The short pulsewidths may cause erythrocyte vaporization, rapid thermal expansion, and mechanical vessel rupture with hemorrhage. Long pulsewidths appear to cause thermal denaturation with less mechanical vessel damage. The selective, nonhemorrhagic, vascular necrosis caused by the long-pulsewidth dye laser may lead to a more desirable clinical outcome in the therapy of blood vessel disease processes.

The pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome.

Pulmonary toxoplasmosis, once considered a rare complication of human immunodeficiency virus infection, recently has been reported with increasing frequency in patients with the acquired immunodeficiency syndrome (AIDS). However, published descriptions of the pathologic changes have been scanty, involve mainly single cases, and appear to conflict. Four cases of pulmonary toxoplasmosis observed at autopsy in patients with AIDS were reviewed. Two of the patients presented with Toxoplasma pneumonia and in one the diagnosis was made antemortem by open lung biopsy. Two patterns of pulmonary lesions associated with Toxoplasma gondii pneumonia were identified and appeared to be related to the stage and intensity of the infection. A pattern of interstitial pneumonitis/diffuse alveolar damage with a fibrinous alveolar exudate appeared to antecede a necrotizing pneumonia characterized by large areas of parenchymal necrosis. In the former pattern T gondii tachyzoites were found in small numbers and were mainly intracellular, whereas in necrotic areas tachyzoites were numerous and were extracellular as well as intracellular. Histopathologic diagnosis of the infection required careful search for the organisms even when they were plentiful Immunohistochemistry identified far more organisms than could be appreciated with routine stains and confirmed the diagnosis.

Pulmonary aspergillosis in acquired immune deficiency syndrome: autopsy study of an emerging pulmonary complication of human immunodeficiency virus infection.

Pulmonary aspergillosis has recently been described as an emerging infection in patients with acquired immune deficiency syndrome (AIDS), but the pathological changes have not been well documented. In this autopsy study, 17 cases of AIDS-related pulmonary aspergillosis were identified from the files of two institutions. With the exception of hypersensitivity reactions, the entire spectrum of pulmonary aspergillosis was represented. Thirteen patients exhibited acute invasive aspergillosis, and seven patients had evidence of subacute or chronic invasive infection, four of whom also had areas of acute invasion. One patient had necrotizing bronchial aspergillosis as well as acute invasive infection, and one individual had saprophytic colonization of a cavity caused by previous Pneumocystis carinii pneumonia (PCP) without evidence of invasive aspergillosis. The same conditions known to predispose immunocompromised individuals without human immunodeficiency virus (HIV) infection to invasive pulmonary aspergillosis were also identified in these patients with AIDS and included neutropenia, steroid therapy, and underlying lung disease. Additional pulmonary conditions were identified in all but one case and consisted mainly of infection or some form of chronic lung disease. In particular, half of the cases were associated with pulmonary fibrosis related to prior PCP. All cases occurred in or after 1990, confirming the perception of the recent emergence of aspergillosis in AIDS. As suggested by this study, one reason for this may be that patients with AIDS are now living long enough to develop one or more of the predisposing conditions for pulmonary aspergillosis.

Comparison of five histopathologic methods to assess cellular proliferation in transitional cell carcinoma of the urinary bladder.

Transitional cell carcinomas of the urinary bladder vary in their biologic potential, which may be correlated with the grade and stage of the tumor. Cellular proliferation may prove to be another measure of predicting tumor biologic potential. We have compared five different methods to assess proliferation in 26 tumors and correlated these results with tumor grade and stage. A portion of each tumor was incubated in vitro with bromodeoxyuridine (BrdUrd). For each tumor this was compared with at least three of the following four other markers of proliferation: mitotic count, silver-stained nucleolar organizer regions, immunohistochemical staining with Ki67, and proliferating cell nuclear antigen. Statistical correlations were seen between tumor grade and stage and these markers. There were strong correlations between the BrdUrd labeling index (LI) and both the Ki67 LI and proliferating cell nuclear antigen LI. The correlation between the BrdUrd LI and mitotic count was more tenuous; no significant correlation was found between BrdUrd LI and silver-stained nucleolar organizer region count. The correlation between these measurements of proliferation and tumor grade and stage was less strong. Our data suggest that cellular proliferation of transitional cell carcinomas can be reliably assessed with several different markers and that most of these markers can be correlated with tumor grade but not with stage.

Cutaneous Cryptococcus infection and AIDS. Report of 12 cases and review of the literature.

BACKGROUND: Cryptococcal infections occur in 6% to 13% of patients with acquired immunodeficiency syndrome (AIDS), most commonly infecting the central nervous system. Cutaneous lesions have been described morphologically as umbilicated papules, nodules, and violaceous plaques and can mimic molluscum contagiosum and Kaposi's sarcoma. Cutaneous lesions can present months prior to other signs of systemic infection. OBSERVATIONS: Cases of infection with cutaneous Cryptococcus and AIDS were reviewed and compared with cases reported in the literature. Among patients with Cryptococcus infection and AIDS seen at our institutions, 5.9% had skin lesions. All patients with cutaneous lesions had systemic involvement. Women were less commonly infected than men. There was no apparent predisposition associated with age, race, or human immunodeficiency virus infection risk factors. The median CD4 helper T-cell count was 0.024 X 10(9)/L (24/microL), and 44% (16/36) of the patients had previous opportunistic infections. Lesions were most commonly seen on the head and neck (78% [36/46]) and often mimicked molluscum contagiosum (54% [25/46]). The median serum and cerebrospinal fluid cryptococcal antigen titers were 1:32,768 and 1:512, respectively. Patients in our group did well with therapy (one death at 6 weeks, compared with 38% [13/34] mortality in the literature). There was no correlation between onset of lesions, number of lesions, CD4 helper T-cell count, or histopathologic characteristics. CONCLUSIONS: Disseminated Cryptococcus infection in AIDS presents with cutaneous lesions in up to 6% of cases. Clinicians need to be aware of the varied morphologic characteristics, since cutaneous lesions may present well in advance of other signs of systemic infection.

p53 oncoprotein expression and proliferation index in keratoacanthoma and squamous cell carcinoma.

BACKGROUND AND DESIGN: Whether solitary keratoacanthoma (KA) is a malignant neoplasm despite its self-limited clinical behavior, and the distinction between KA and squamous cell carcinoma (SCC) are related aspects of a long-standing debate among dermatopathologists. Recent advances toward understanding the molecular basis of malignant transformation may allow this issue to be resolved. Mutant p53 tumor-suppressor protein has been shown to accumulate in cutaneous SCC and other tumors, and may be a relatively specific marker of malignancy. We studied 20SCCs, 20KAs, and an additional 10 regressing KAs (rKA) by immunohistochemistry for the expression of p53 protein. Since p53 is believed to play a pivotal role in the regulation of cell division, we also quantitated proliferation in the tumors by examining Ki-67 antigen expression. RESULTS: Sixteen (80%) of the KAs showed nuclear staining with anti-p53 antibody, distributed along the outermost layers of the aggregates of neoplastic cells, while 12 (60%) of the SCCs were p53 positive. Eight (80%) of the rKAs also showed p53 positivity. Mean Ki-67 proliferation fraction was higher for KA than for SCC (55% vs 46%), but this difference was not statistically significant. p53 Expression did not correlate with the grade of SCC. CONCLUSIONS: A majority of KA, rKA, and SCC contain stainable quantities of p53 protein, supporting the view that KA is a type of regressing SCC.

Cutaneous squamous cell carcinoma in human immunodeficiency virus-infected patients. A study of epidemiologic risk factors, human papillomavirus, and p53 expression.

OBJECTIVE: To examine risk factors for the development of cutaneous squamous cell carcinoma (SCC) in a group of human immunodeficiency virus (HIV)-infected patients, including evaluation and detection of epidemiologic risk factors of human papillomavirus (HPV) and p53 expression. DESIGN: Case-control study during a 3-year period. SETTING: Dermatologic referral center. PATIENTS: Thirty-three HIV-infected patients who had 97 SCCs were compared with 24 HIV-infected patients who had 70 basal cell carcinomas (BCCs). MAIN OUTCOME MEASURES: Age, skin type, amount of sun exposure, actinic damage, family history of skin cancer and history of smoking and warts. Specimens of SCC and BCC were examined for HPV using polymerase chain reaction. Presence of p53 was examined using immunohistochemical analysis. Specimens from tumor-free, non-sun-exposed areas from these same patients were used as controls. RESULTS: Risk factors for the development of both types of carcinoma included fair skin type and excessive sun exposure (> 6 h/d during the previous 10 years). The HIV-infected patients with SCCs tended to have outdoor occupations. The location of SCCs favored the head and neck; BCCs were located on the trunk. Patients with SCCs had later-stage HIV disease than did patients with BCCs. Half of the patients with SCC had a history of genital or nongenital warts. Seventy-one percent (17/24) had a smoking history. No statistical difference existed between patients with SCCs and BCCs for history of smoking or warts. Human papillomavirus was not found in most of our SCC, BCC, or control specimens. However, 92% (22/24) of the SCC specimens and 90% (18/20) of the BCC specimens stained for p53. Control specimens from non-sun-exposed skin of HIV-infected patients did not stain for p53. Epidermal staining was present in 95% (17/20) of tissue adjacent to SCCs and 47% (7/15) of tissue adjacent to BCCs. A significantly positive correlation existed between the amount of sun exposure and the amount of p53 staining seen in adjacent epidermal tissue (r = 0.07; P = .01). CONCLUSIONS: Risk factors for the development of SCCs and BCCs in HIV-infected patients are similar: fair skin type and excessive sun exposure. Our study does not support that HPV is an oncogenic factor in the development of these cutaneous tumors but provides evidence that p53 overexpression may play a role.

Cutaneous presentations of lymphoma in human immunodeficiency virus disease. Predominance of T cell lineage.

BACKGROUND AND DESIGN: Most non-Hodgkin's lymphomas in patients with human immunodeficiency virus infection are of B-cell lineage. Cutaneous lymphoma in the human immunodeficiency virus disease has not been systematically reviewed. We studied 25 patients with both human immunodeficiency virus infection and cutaneous presentations of lymphoma, using immunohistochemistry and in situ hybridization for Epstein-Barr virus. RESULTS: Two groups of patients were discerned: (1) those with conditions similar to mycosis fungoides or Sézary syndrome with an indolent course (n = 8) and (2) those with nodules or papules, greater immunosuppression, a rapid clinical course, and large cell lymphoma seen on biopsy specimens (n = 17). The epidermotropic lymphomas were T-cell lineage and CD30-. Thirteen of the large cell lymphomas were also of the T-cell type, and 71% were CD30+. Epstein-Barr virus was absent in the epidermotropic lymphomas, but it was present in 73% of the nonepidermotropic cases. CONCLUSIONS: Two forms of human immunodeficiency virus-associated cutaneous lymphoma were found: indolent disease resembling mycosis fungoides or Sézary syndrome and large cell lymphomas with a poor prognosis, whose cells often had a CD30+ T-cell phenotype and harbored the Epstein-Barr virus.

Normal vestibular function in chicks after partial exposure to microgravity during development.

Sixty-four fertilized chicken eggs, half at developmental Day 2 and half at Day 9, were exposed to micro-gravity for 5 days aboard the shuttle. Postflight examination showed that none of the Day 2 flight embryos had survived, whereas the Day 9 flight group and both groups of synchronous ground control embryos appeared viable. One-half of the Day 9 flight and ground control embryos were dissected and the temporal bones preserved in acetone for morphological examination. The other half was allowed to hatch to examine vestibularly related behavioral changes. Morphology of the lagenar otoconia was evaluated by scanning electron microscopy. Behavioral changes were accessed by a battery of reflex tests and recordings of spontaneous and vestibularly driven head movements. The results from both the morphological and behavioral studies showed no consistent difference between the flight and the control animals. Several hypotheses may account for this negative result. Because all the Day 2 embryos failed to survive, the remaining Day 9 chicks may have passed the critical developmental period of the chick's vestibular system. Also, the reexposure of the developing chick embryo to earth's 1-g environment may have masked any adverse behavioral effects that exposure to Microgravity may have caused.

Studies of the mechanism of nephrotoxicity of compound A in rats.

CO2 absorbents acting on sevoflurane produce compound A [CF2=C(CF3)OCH2F]. Rats breathing 25-50 ppm of compound A for 3-12 h demonstrate corticomedullary renal injury. Several halogenated alkenes also produce a well described corticomedullary lesion by conversion of glutathione conjugates of these alkenes to cysteine s-conjugates and subsequent metabolism by renal cysteine conjugate ß-lyase to nephrotoxic halothionoacetyl halides. We tested whether a similar mechanism explained the nephrotoxicity of compound A or whether an oxidative metabolism of compound A by cytochrome P-450 was required for the induction of nephrotoxicity. A closed rebreathing system was used and male Wistar rats were exposed for 1 h to: (1) oxygen alone; (2) 800 ppm compound A; (3) 800 ppm compound A after pretreatment with intraperitoneal aminooxyacetic acid (AOAA), 0.5 mmoles/kg, an inhibitor of renal cysteine conjugate ß-lyase; (4) 600 ppm compound A; (5) 600 ppm compound A after pretreatment with intraperitoneal AOAA, 0.50 mmoles/kg plus acivicin (AT-125), 0.25 mmoles/kg, an inhibitor of gamma glutamyl transpeptidase; (6) 600 ppm compound A after pretreatment with 1600 mg/kg piperonyl butoxide (PB) subcutaneously, and (7) 600 ppm compound A after pretreatment with 100 mg/kg 1-aminobenzotriazole (ABT) by intraperitoneal injection (both PB and ABT inhibit cytochrome P-450s). All rats were killed 24 h following exposure to compound A or oxygen, or to pretreatments without compound A, and the kidneys were collected for histological analysis. Pretreatments given without compound A did not cause renal injury. Necrosis was found in 20.9±16.7% (mean±SD) of corticomedullary tubule cells following exposure of Wistar rats to 600 ppm compound A. Pretreatment with AOAA plus AT-125 increased necrosis to 57.9±32.6%, (P<0.005). PB or ABT given prior to compound A increased corticomedullary injury to 39.0±31.4% (P<0.02) and 51.2±31.8% (P<0.025), respectively. In rats exposed to 800 ppm compound A, pretreatment with AOAA increased necrosis from 63.8±30.1% to 81.2±27.7% (P<0.1). Unlike many other halogenated alkenes, compound A does not appear to produce renal injury by conversion of a cysteine S-conjugate to a toxic thiol, nor does injury require metabolism mediaited by cytochrome P-450. Injury may result from direct toxicity of compound A or by an undetermined metabolic pathway.

Streptomycin in the chick embryo: post-hatching vestibular behavior and morphology.

Developing chick embryos were exposed to streptomycin injected on days 5 through 13 of the 21 day developmental period. Histological and behavioral abnormalities were found almost exclusively in chicks exposed after day 7. The nature of the behavioral deficits included abnormal head posture, head tremor, and inability to compensate for applied vestibular stimuli. Head movement measurements showed that the head tremor had frequencies of oscillations from 10-35 Hz. The amplitude of the tremor was a large as 10 degrees. Histology showed damage to the secretory dark cells of the membranous labyrinth in those chicks that showed behavior changes. Even with increased dosages chicks exposed prior to day 7 rarely showed abnormal vestibular behavior but instead experienced increased mortality. Further tests examining tissue levels of streptomycin showed little or no streptomycin in embryos until day 10. These results are discussed in terms of their utility as an alternative model to surgical manipulation of the vestibular system in developing embryos. Behavioral consequences are compared to other work with drugs and to the effects of weightlessness and unusual environments on vestibular orientation and behavior.

Lack of p53 protein immunoreactivity in bilateral diffuse uveal melanocytic proliferation.

BACKGROUND: Bilateral diffuse uveal melanocytic proliferation is a poorly understood disorder characterized by the progressive proliferation of uveal melanocytes associated with a systemic nonocular malignancy. Overexpression of p53 protein plays a role in the loss of regulatory control of normal cell proliferation, and p53 is the most commonly identified oncogenic protein in human malignancies. We tested the hypothesis that the aberrant cellular activity in bilateral diffuse uveal melanocytic proliferation involves the overexpression of p53 protein. METHODS: Eight eyes from four patients with bilateral diffuse uveal melanocytic proliferation were tested for p53 protein using an immunoperoxidase technique with an anti-p53 protein monoclonal antibody sensitive for normal and mutant p53 protein. RESULTS: The p53 protein could not be detected in any of the eight eyes. CONCLUSIONS: The proliferation of uveal melanocytes in bilateral diffuse uveal melanocytic proliferation does not depend on the overexpression of p53 protein. The loss of cellular regulatory control in bilateral diffuse uveal melanocytic proliferation is probably mediated through another mechanism.

The effect of epidermal pigmentation on selective vascular effects of pulsed laser.

The effect of epidermal pigmentation on the threshold exposure dose for inducing purpura with a tunable dye laser at 577 nm, 1.5 microseconds pulse duration, was studied in 21 human volunteers with varied genetically determined amounts of melanin. More laser energy was required to produce purpura as constitutive skin pigmentation increased. Histology showed that, in lighter skin, the laser threshold dose produced the most specific vascular injury with no disruption of surrounding structures. In more pigmented skin, damage occurred in the epidermal basal layer and very few changes were seen in blood vessels below.