Phase II screening trial of lithium carbonate in amyotrophic lateral sclerosis: examining a more efficient trial design.

OBJECTIVE: To use a historical placebo control design to determine whether lithium carbonate slows progression of amyotrophic lateral sclerosis (ALS). METHODS: A phase II trial was conducted at 10 sites in the Western ALS Study Group using similar dosages (300-450 mg/day), target blood levels (0.3-0.8 mEq/L), outcome measures, and trial duration (13 months) as the positive trial. However, taking riluzole was not a requirement for study entry. Placebo outcomes in patients matched for baseline features from a large database of recent clinical trials, showing stable rates of decline over the past 9 years, were used as historical controls. RESULTS: The mean rate of decline of the ALS Functional Rating Scale-Revised was greater in 107 patients taking lithium carbonate (-1.20/month, 95% confidence interval [CI] -1.41 to -0.98) than that in 249 control patients (-1.01/month, 95% CI -1.11 to -0.92, p = 0.04). There were no differences in secondary outcome measures (forced vital capacity, time to failure, and quality of life), but there were more adverse events in the treated group. CONCLUSIONS: The lack of therapeutic benefit and safety concerns, taken together with similar results from 2 other recent trials, weighs against the use of lithium carbonate in patients with ALS. The absence of drift over time and the availability of a large database of patients for selecting a matched historical control group suggest that use of historical controls may result in more efficient phase II trials for screening putative ALS therapeutic agents. CLASSIFICATION OF EVIDENCE: This study provided Class IV evidence that lithium carbonate does not slow the rate of decline of function in patients with ALS over 13 months.

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome.

An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.

The utility of presurgical CA125 to predict optimal tumor cytoreduction of epithelial ovarian cancer.

The objective of this study was to evaluate the ability of a preoperative serum CA125 to predict whether optimal debulking (OD) could be achieved for patients with stage III and IV epithelial ovarian cancer (EOC). The records of consecutive patients who underwent primary surgery for EOC at Indiana University Hospital between January 1997 and January 2003 were reviewed. Eligibility criteria included FIGO stage III/IV disease, surgery by gynecologic oncology faculty, preoperative CA125, and an operative note clearly defining volume of residual disease. The Medcalc software statistical package was used to generate a receiver-operating characteristic (ROC) curve. Two hundred and eighty-nine cases of stage III/IV EOC were identified, of which 164 met the eligibility criteria. Serum CA125 /=75% of the time. Conversely, OD was achieved in /=4500. The area under the ROC curve for CA125 was .670. The OD rate for those with and without ascites was 49% and 79%, respectively (P < 0.001). In a multivariate analysis using CA125, age, and ascites, the area under the curve was 0.686. We conclude that preoperative serum CA125 did not reliably predict OD in patients with stage III-IV EOC.

In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis.

Angiopoietin (Ang)-2, the natural antagonist of the Ang1/Tie2 receptor is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting [in the presence of vascular endothelial growth factor (VEGF)-A] and vessel regression (in the absence of VEGF-A). Based on the spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in placental angiogenesis. Further, to examine this tenet we have developed a novel murine model system in which in utero Ang2 gene delivery via a non-replicating adenoviral expression vector has the potential to manipulate the blood vessel phenotype in vivo during pregnancy. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular extracellular matrix, subsequently promoting plasticity of the maternal and fetal vessels, which appear honeycombed due to a 2-fold increase in blood vessel luminal area. High levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness with perivascular oedema, which increases placental weight. These observations suggest that the Ang2 overexpression may play a key role in placental vascular remodelling. Furthermore, we suggest a novel new model to study the pathobiology of placental vascularization and the effect of placental blood vessels on fetal phenotype.

The risk of nodal metastasis in early adenocarcinoma of the uterine cervix.

A functional and widely accepted definition of microinvasive cervical adenocarcinoma remains elusive. The purpose of this study was to determine at which depth of invasion the likelihood of lymph node metastasis or disease recurrence was so small that conservative surgery could be considered appropriate. Charts of patients with adenocarcinoma of the cervix (ACC) who underwent radical hysterectomy and pelvic lymphadenectomy (n = 98) at Indiana University Medical Center from 1987 to 1998 were retrospectively reviewed. Patients with stage IA1-IB1 lesions were included in the study. Patients treated with preoperative radiotherapy were excluded. Pathologic parameters evaluated included histologic type, depth of stromal invasion (DOI), and the presence of lymphatic vascular space invasion, or lymph node metastases. The patient median age was 39 years (20-65). The median time of follow-up was 30 months (4-124). Lymph node metastases were found in ten patients and 11 developed recurrences. The precise DOI could be measured in 84 patients. Of the 48 patients with cancers with a DOI 5 mm had nodal metastases (P = 0.00069). None of these 48 patients with a tumor DOI 5 mm developed recurrent disease (P = 0.0048). The risk of nodal metastases and recurrence is so low in patients with ACC and DOI

Diagnostic accuracy of fine-needle aspiration biopsy is determined by physician training in sampling technique.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) has been used with variable success as a diagnostic test for benign and malignant breast lesions. The goal of this study was to examine the effects of training physicians in the fine-needle aspiration sampling-technique on the diagnostic accuracy of FNAB of palpable breast masses. The settings for this study were private physicians' offices and university clinics of primary care physicians, surgeons, and cytopathologists. METHODS: We reviewed 1043 consecutive FNAB specimens of the breast obtained during 1 year (1992): 729 FNABs were performed by formally trained physicians (at least 150 FNABs performed previously under supervision during fellowship training or the equivalent) who had done at least 100 FNABs during the year; 314 FNABs were performed by physicians without formal training who had done a median of only 2 FNABs during the year (range, 1-43 FNABs). All FNAB specimens were reviewed microscopically and evaluated for cellularity and type of material present, for diagnostic accuracy, and for the rate of surgical intervention. A minimum of 2 years of follow-up was obtained by matching all cases to the population-based Northern California Cancer Registry. FNAB specimens were correlated with histologic specimens when they were available. RESULTS: Using FNAB, the formally trained physicians missed 2% of cancers, whereas the physicians without formal training missed 25%. Among the patients with benign lesions seen by the formally trained physicians, 8% went on to surgery, whereas 30% of those seen by physicians without formal training did so. Specimens obtained by the formally trained physicians were significantly more cellular and were significantly less likely to be nondiagnostic. CONCLUSIONS: FNAB, when performed by physicians who are well trained in the technique, is a highly accurate, cost-effective diagnostic method that carries minimal morbidity and could replace a large number of surgical biopsies. When performed by physicians without adequate training, FNAB is often misleading and potentially harmful.

Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers.

PURPOSE: erbB-2 and epidermal growth factor receptor (EGFR) may mediate motility via signaling that enables changes in the actin cytoskeleton. A physical basis for this motility may depend on the coexpression of gelsolin, a M(r) 80,000 actin-binding protein. EXPERIMENTAL DESIGN: The expression of erbB-2, EGFR, and gelsolin was analyzed in 790 archival invasive breast cancers. These data were compared with histological, clinical, and outcome data (median follow-up, 16.3 years). RESULTS: Protein overexpression was observed in overlapping subsets of breast cancers (38% of cases were erbB-2+; 15% of cases were EGFR+; and 56% of cases were gelsolin+). Tumor gelsolin was associated with overexpression of erbB-2 and EGFR, as well as with an aggressive tumor phenotype. By univariate and multivariate analyses, tumor gelsolin alone was not a prognostic factor. Overexpression of all three factors significantly predicted poor clinical outcome by univariate and multivariate analyses. For example, in node-positive patients, coexpression of all three markers was associated with a 3-year disease-specific survival (as compared with erbB-2+, EGFR+, gelsolin- patients, who had a median survival of 6 years). CONCLUSIONS: These data suggest that gelsolin coexpression may be an important additional prognostic factor in erbB-2+, EGFR+ breast cancer patients. We hypothesize that this is due to the role of gelsolin in mediating motility and invasion.

Phase II study of paclitaxel and valspodar (PSC 833) in refractory ovarian carcinoma: a gynecologic oncology group study.

PURPOSE: A phase II study was conducted to determine the efficacy of paclitaxel and valspodar (PSC 833) in patients with advanced epithelial ovarian cancer. Valspodar, a nonimmunosuppressive cyclosporine D analogue that reverses P-glycoprotein-mediated multidrug resistance, in combination with paclitaxel might be active in paclitaxel-resistant and refractory ovarian cancer. PATIENTS AND METHODS: Patients received valspodar 5 mg/kg orally qid x 12 doses. Paclitaxel (70 mg/m(2) intravenously for 3 hours) was administered on day 2, 2 hours after the fifth or sixth dose of valspodar. This treatment was repeated every 21 days. One blood sample was collected before the sixth dose of valspodar for the first three cycles to evaluate valspodar trough concentration. Tumor tissue was obtained from patients for immunohistochemical staining of P-glycoprotein. RESULTS: Of 60 patients entered, 58 were assessable for response. There were five partial responses (8.6%; 90% confidence interval [CI], 3.8 to 20.0; median duration of response, 5.0 months [range, 1.9 to 10.5 months]). Median progression-free survival was 1.5 months (90% CI, 1.4 to 2.4). Grade 3 or 4 toxicities observed were neutropenia, anemia, nausea and vomiting, peripheral neuropathy, and cerebellar ataxia. The trough concentrations of valspodar were > or = 1,000 ng/mL in all but two of 40 patients in the first cycle. Immunohistochemical staining for P-glycoprotein was positive for one of two responding patients. CONCLUSION: Valspodar in combination with paclitaxel has limited activity in patients with paclitaxel-resistant ovarian carcinoma. An international randomized clinical trial of paclitaxel and carboplatin with or without valspodar as first-line therapy in advanced ovarian cancer is underway.

Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer.

Our objective was to investigate the prognostic significance of cell turnover (apoptosis and proliferation) in breast cancer patients. Apoptosis was microscopically quantitated on histological sections from 791 breast cancer patients with long-term follow-up (median, 16.3 years). Apoptotic counts were also compared with proliferation data (mitotic counts and MIB-1 labeling); apoptosis data derived from terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay; and pathobiological variables, including p53, erbB-2, and estrogen receptor (ER). High apoptotic counts were associated with increased cellular proliferation, ER negativity, immunopositivity of erbB-2 and p53 (P < 0.0001), and shortened disease-specific survival (DSS; P = 0.0009) and disease-free survival (DFS; P = 0.0006). Other factors associated with shortened DFS and DSS by univariate analysis were high tumor grade, nodal metastases, and large tumor size (P < 0.0001 for each). Multivariate analysis of data for all of the patients demonstrated that tumor size, nodal status, ER, histological grade, and erbB-2 showed independent prognostic value. In node-negative patients, tumor size and mitotic rate per 1000 cells independently predicted DFS (P = 0.0055). Tumor grade was the only independent predictor of DSS. For node-positive patients, tumor size, nodal status, ER, and erbB-2 were independent prognostic factors. The number of mitoses per 1000 was independently associated with DFS (P = 0.043) but not with DSS. Apoptosis data did not provide independent prognostic value in any, node-positive or node-negative, breast cancer patients.

Persistence of aneuploid immature/primitive hemopoietic sub-populations in mice 8 months after benzene exposure in vivo.

Benzene (bz) is a common environmental contaminant associated with increased risk of myeloid leukemia. Chronic bz exposure in vivo increases the frequency of aneuploid circulating lymphocytes in humans. However, there is no information about persistence of bz-associated aneuploidy in immature/primitive cells, at risk of leukemic transformation, after bz exposure in vivo. We explored the relationship between the induction and persistence of aneuploidy in primitive hemopoietic cells from mice that received oral doses of bz in vivo. Short- and long-term persistence of aneuploidy were evaluated in immature/primitive sub-populations (Lin(-)c-kit(+)Sca-1(+)), as well as lymphoid and myeloid cells, 6 days and 2-8 months after exposure. Mice receiving bz in a corn oil carrier, or corn oil alone, both have increased aneuploidy frequencies (1-5%, compared to <1% in untreated controls) in all sub-populations, 6 days after exposure. However, unlike bz-induced aneuploidy, corn oil-induced aneusomies are transient, with frequencies returning to background levels in lymphoid and myeloid cells, 9 weeks after exposure. The frequency (5-9%) of aneuploid lymphocytes and myeloid cells is higher at 9 weeks than at 6 days, suggesting that bz disrupts chromosomal segregation in differentiated cells and/or progenitors. About 8 months after bz exposure, the Lin(-)c-kit(+)Sca-1(+) sub-population contains up to 14% aneuploid cells with numerical chromosomal aberrations affecting chromosomes 2 or 11. These data demonstrate that bz induces DNA copy number changes in immature/primitive cells, and that these changes persist for long periods. Although, initial exposures are not leukemogenic, subsequent exposures of cells to genotoxins or oxidative radicals that induce additional genetic hits may increase the risk of transformation. The contribution of bz-induced aneuploidy in immature/primitive cells to leukemogenesis remains to be determined.

Dermal benzene and trichloroethylene induce aneuploidy in immature hematopoietic subpopulations in vivo.

Accumulation of genetic damage in long-lived cell populations with proliferative capacity is implicated in tumorigenesis. Hematopoietic stem cells (hsc) maintain lifetime hematopoiesis, and recent studies demonstrate that hsc in leukemic patients are cytogenetically aberrant. We postulated that exposure to agents associated with increased leukemia risk would induce genomic changes in cells in the hsc compartment. Aneusomy involving chromosomes 2 and 11 in sorted hsc (Lin(-)c-kit(+)Sca-1(+)) and maturing lymphoid and myeloid cells from mice that received topical doses of benzene (bz) or trichloroethylene (TCE) was quantified using fluorescence in situ hybridization. Six days after bz or TCE exposure, aneuploid cells in the hsc compartment increase four- to eightfold in a dose- and schedule-independent manner. Aneuploid lymphoid and myeloid cells from bz- and TCE-treated mice approximate controls, except after repeated benzene exposures. Aneuploid cells are more frequent in the hsc compartment than in mature hematopoietic subpopulations. Hematotoxicity was also quantified in bz- and TCE-exposed hematopoietic subpopulations using two colony-forming assays: CFU-GM (colony-forming units/granulocyte-macrophage progenitors) and CAFC (cobblestone area-forming cells). Data indicate that bz is transiently cytotoxic (< or =1 week) to hsc subpopulations, and induces more persistent toxicity (>2 weeks) in maturing, committed progenitor subpopulations. TCE is not hematotoxic at the doses applied. In conclusion, we provide direct evidence for induction of aneuploidy in cells in the hsc compartment by topical exposure to bz and TCE. Disruption of genomic integrity and/or toxicity in hsc subpopulations may be one step in leukemic progression.

Preoperative chemo-radiation for carcinoma of the vulva with N2/N3 nodes: a gynecologic oncology group study.

PURPOSE: To determine if patients with carcinoma of the vulva, with N2/N3 lymph nodes, could undergo resection of the lymph nodes and primary tumor following preoperative chemo-radiation. METHODS AND MATERILAS: Fifty-two patients were entered in the study, but six patients did not meet the criteria of the protocol and were excluded. The remaining 46 patients are the subject of this report. Patients underwent a split course of radiation, 4760 cGy to the primary and lymph nodes, with concurrent chemotherapy, cisplatin/5-FU, followed by surgery. RESULTS: Four patients did not complete the chemo-radiation, because three expired and one refused to complete the treatment. Four patients who completed chemo-radiation did not undergo surgery, because two of them died of non-cancer-related causes, and in the other two patients, the nodes remained unresectable. Following chemo-radiation, the disease in the lymph nodes became resectable in 38/40 patients. Two patients who completed the course of chemo-radiation did not undergo surgery as per protocol because of pulmonary metastasis. One underwent radical vulvectomy and unilateral node dissection and the other radical vulvectomy only. The specimen of the lymph nodes was histologically negative in 15/37 patients. Nineteen patients developed recurrent and/or metastatic disease. The sites of failure were as follows: primary area only, 9; lymph node area only, 1; primary area and distant metastasis, 1; distant metastasis only, 8. Local control of the disease in the lymph nodes was achieved in 36/37 and in the primary area in 29/38 of the patients. Twenty patients are alive and disease-free, and five have expired without evidence of recurrence or metastasis. Two patients died of treatment-related complications. CONCLUSION: High resectability and local control rates of the lymph nodes were obtained in patients with carcinoma of the vulva with N2/N3 nodes treated preoperatively with chemo-radiation.

An approach to analysis of large-scale correlations between genome changes and clinical endpoints in ovarian cancer.

This report describes analyses of associations of genome copy number abnormalities in ovarian cancers with clinical features using genome-wide graphical and analytical procedures. These studies show that tumor grade is a better indicator of the extent of genomic progression than stage, that loss of chromosome 4 occurs preferentially in high-grade tumors, and that gains of 3q26-qter, 8q24-qter, and 20q13-qter occur frequently in low-grade and low-stage tumors and thus may be early events in ovarian cancer development. In addition, loss of chromosome 16q24 and a total number of independent genome copy number aberrations >7 are associated with reduced survival duration. The association of loss of 16q24 (D16S3026) with decreased survival duration was confirmed by quantitative PCR. Regions that frequently are abnormal and associated with altered survival duration are strong candidates for higher resolution analysis and gene discovery and may be useful markers for prediction of clinical outcome.

Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis.

This report describes the development and validation of quantitative microsatellite analysis (QuMA) for rapid measurement of relative DNA sequence copy number. In QuMA, the copy number of a test locus relative to a pooled reference is assessed using quantitative, real-time PCR amplification of loci carrying simple sequence repeats. Use of simple sequence repeats is advantageous because of the large numbers that are mapped precisely. In addition, all markers are informative because QuMA does not require that they be polymorphic. The utility of QuMA is demonstrated in assessment of the extent of deletions of chromosome 2 in leukemias arising in radiation-sensitive inbred SJL mice and in analysis of the association of increased copy number of the putative oncogene ZNF217 with reduced survival duration in ovarian cancer patients.

Radiation-induced translocations in mice: persistence, chromosome specificity, and influence of genetic background.

The translocation frequency response in the chromosomes of peripheral blood lymphocytes is widely used for radiation biomonitoring and dose estimation. However, this assay is based upon several assumptions that have not been rigorously tested. It is typically assumed that the translocation frequency in blood lymphocytes reflects the level of genomic damage in other hemopoietic tissues and is independent of the chromosome probe and genetic background. We conducted studies to evaluate these assumptions using mice with different genetic backgrounds. Six different whole-chromosome fluorescence in situ hybridization (FISH) probes were used to detect translocations in peripheral blood lymphocytes at multiple times after whole-body irradiation. Translocation frequencies were chromosome-independent at 6 and 16 weeks after exposure but were chromosome-dependent at 1. 5 years after exposure. Similar translocation frequencies were observed in blood, bone marrow and spleen at 1.5 years, supporting previous suggestions that genetically aberrant peripheral blood lymphocytes may derive from precursor populations in hemopoietic tissues. Translocations measured 66 h after irradiation differed among some strains. We conclude that the translocation frequency response is a complex phenotype that is influenced not only by exposure dose but also by genetic background, the choice of chromosome analyzed, and time after exposure. These results raise important considerations for the use of the FISH-based translocation frequency response for radiation dosimetry and biomonitoring.

p(21WAF1/CIP1) expression in breast cancers: associations with p53 and outcome.

p21(WAF1/CIP1) is transcriptionally activated by wt p53 and inhibits G1 associated cyclins, a major mechanism by which p53 inhibits cellular proliferation. Archival breast cancers (798) with a median follow-up of 16.3 years were used to explore the prognostic value of p21 immunohistochemical analyses. p21 immunostaining was detected in the majority (726/798: 91%) of breast cancers as well as adjacent in situ carcinomas (125/170: 74%), hyperplastic lesions (140/349: 40%) and normal breast epithelium adjacent to carcinoma (3/89: 3%). Complete immunonegativity was observed in only 9% of invasive cancers and was associated with p53 immunopositivity (p < 0.05). Univariate analysis of all patients showed that p21 negativity was associated with a longer disease specific survival (relative risk (RR) 1.5). Node positive p21- patients also showed a longer disease free and disease specific survival as compared to tumor p21+ patients. In node negative patients, p53 positivity but not p21 alone, was significantly associated with a shortened disease free survival (RR = 1.6). Node negative patients who were p53+ p21-, in particular had the shortest disease free survival compared to other p53, p21 subgroups (i.e., p21 negativity was associated with a worse outcome). Multivariate analysis of lymph node negative patients (n > 300) demonstrated that tumor size and tumor grade were independently predictive of outcome, whereas neither p53 nor p21 were significant. For node positive patients, p21 positivity (p = 0.05), p53 positivity (p = 0.03), a higher number of positive nodes, larger tumor size, steroid receptor negativity, high proliferation rate, and erbB-2 expression were each independently associated with poor outcome. In summary, p21 negativity was inversely correlated with p53 immunopositivity in the majority of cases. p21 negative tumor patients had an improved outcome if they were node positive, whereas p21 status was not significantly associated with survival in node negative patients. This observation may be due to the reported 'uncoupling of S phase and mitosis' associated with a loss of p21 expression which may result in enhanced sensitivity to chemotherapy.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67.

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices. With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Gene amplifications characterize acral melanoma and permit the detection of occult tumor cells in the surrounding skin.

Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each (P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11-13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary, thereby revealing one mechanism of local recurrence.

Angiogenesis, proliferation, and apoptosis in anal high-grade squamous intraepithelial lesions.

PURPOSE: Management of anal high-grade squamous intraepithelial lesions is controversial. Anal and cervical high-grade squamous intraepithelial lesions are similar in that they occur in transitional squamous epithelium, are associated with human papilloma virus infection, and have increased incidence in the immunocompromised population. Ablation of cervical high-grade squamous intraepithelial lesions is preferred, but similar ablation or excision of anal high-grade squamous intraepithelial lesions may compromise bowel control; thus, there is a need to define the malignant potential of anal high-grade squamous intraepithelial lesions. METHODS: We analyzed 50 paraffin sections of normal anoderm, anal low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and anal squamous-cell carcinoma. Microvessels were detected immunohistochemically with von Willebrand factor and counted manually along the epithelial-stromal junction. Proliferation and apoptosis were determined in the epithelial cells with MIB-1 antibody immunostaining and the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling, respectively. RESULTS: Microvascular density was significantly greater in anal high-grade squamous intraepithelial lesions (mean, 0.50 vessels/cm) vs. normal anoderm (mean, 0.21 vessels/cm; P = 0.0017, Mann-Whitney U test). The proliferative percentages were greater in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and squamous-cell carcinoma (mean, 20.4, 21.8, and 23.6 percent) vs. normal anoderm (mean, 14.4 percent), although not significantly (P = 0.06, Kruskal-Wallis statistic). Although the mean proliferative proportions were similar in low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions, the apoptotic proportion was lower for high-grade squamous intraepithelial lesions than low-grade squamous intraepithelial lesions (10.13 vs. 19.96 percent, respectively; P = NS, Mann-Whitney U test). CONCLUSIONS: Angiogenesis, increased proliferation, and decreased apoptosis occur in anal high-grade squamous intraepithelial lesions as they do in the cervix before the development of malignancy. These biologic markers support the importance of anal high-grade squamous intraepithelial lesions as a potential premalignant lesion warranting surgical intervention.

Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences.

BACKGROUND: Ductal carcinoma in situ (DCIS) recurs in the same breast following breast-conserving surgery in 5%-25% of patients, with the rate influenced by the presence or absence of involved surgical margins, tumor size and nuclear grade, and whether or not radiation therapy was performed. A recurrent lesion arising soon after excision of an initial DCIS may reflect residual disease, whereas in situ tumors arising after longer periods are sometimes considered to be second independent events. The purpose of this study was to determine the clonal relationship between initial DCIS lesions and their recurrences. METHODS: Comparative genomic hybridization (CGH) was used to compare chromosomal alterations in 18 initial DCIS lesions (presenting in the absence of invasive disease) and in their subsequent ipsilateral DCIS recurrences (detected from 16 months to 9.3 years later). RESULTS: Of the 18 tumor pairs, 17 showed a high concordance in their chromosomal alterations (median = 81%; range = 65%-100%), while one case showed no agreement between the paired samples (having two and 20 alterations, respectively). Morphologic characterization of the DCIS pairs showed clear similarities. The mean number of CGH changes was greater in the recurrent tumors than in the initial lesions (10.7 versus 8.8; P =.019). The most common changes in both the initial and the recurrent in situ lesions were gains involving chromosome 17q and losses involving chromosomes 8p and 17p. The degree of concordance was independent of the time interval before recurrence and of the presence of positive surgical margins. CONCLUSIONS: In this study, DCIS recurrences were clonally related to their primary lesions in most cases. This finding is consistent with treatment paradigms requiring wide surgical margins and/or postoperative radiation therapy.