Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs.

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.

Leptin expression in breast nipple aspirate fluid (NAF) and serum is influenced by body mass index (BMI) but not by the presence of breast cancer.

While obesity is a known risk factor for postmenopausal breast cancer, the molecular mechanisms involved are unclear. Systemic levels of leptin, the product of the ob (obesity) gene, are increased in obese individuals (body mass index, BMI, over 25) and are higher in women than men. Leptin has been found to stimulate the growth of breast cancer cells in vitro. Our goal was to determine whether leptin was 1) present in nipple aspirate fluid (NAF), and 2) whether NAF leptin levels were associated with a) levels in serum, b) obesity, and c) breast cancer. We collected and evaluated NAF specimens from 83 subjects and serum specimens from 49 subjects. NAF leptin was detectable in 16/41 (39 %) of premenopausal and 21/42 (50 %) postmenopausal subjects. NAF leptin was significantly lower (p = 0.042) in premenopausal than postmenopausal women with a BMI < 25, but not in those with a higher BMI. NAF leptin was significantly associated with BMI in premenopausal (p = 0.011) but not in postmenopausal women. Serum leptin was associated with BMI in both premenopausal and postmenopausal women (p = 0.0001 for both). NAF and serum leptin were associated in premenopausal (p = 0.02) but not postmenopausal women. Neither NAF nor serum leptin was associated with premenopausal or postmenopausal breast cancer. Our findings include that 1) leptin is present in the breast and detectable in a subset of NAF samples, 2) NAF leptin in premenopausal but not postmenopausal women parallels serum leptin levels, and 3) neither NAF nor serum levels of leptin were associated with premenopausal or postmenopausal breast cancer.

Congenital posterolateral diaphragmatic hernia in an adult.

A Bochdalek hernia (BH) occurs when abdominal contents herniate through the posterolateral segment of the diaphragm. Most BHs present with life-threatening cardiorespiratory distress in the neonatal period. Rarely, hernias that remain clinically silent until adulthood present as life-threatening surgical emergencies. Our recent experience with a life-threatening emergency due to a BH in a 29-year-old male patient prompted us to reinforce that this entity does exist in adults and should be considered in the differential of acute abdominal pain.

99mTc-peptide-peptide nucleic acid probes for imaging oncogene mRNAs in tumours.

Imaging oncogene mRNA in tumours would provide a powerful tool for the early detection of occult malignant lesions. The goal was to prepare a chimera consisting of a dodecamer antisense peptide nucleic acid (PNA) specific for c-MYC oncogene overexpressed in human breast cancer cells and a chelating moiety that facilitates quantitative radiolabelling with 99mTc and evaluate it for hybridization and tissue distribution in laboratory animals. The pentapeptide chelator-PNA dodecamer specific for c-MYC mRNA was extended from a solid support by 9-fluorenylmethyloxycarbonyl (Fmoc) coupling. Similarly, a chelator-PNA chimera with four central mismatches was also prepared which served as a control. The chimeras were purified, characterized and evaluated for hybridization to c-MYC mRNA by fluorescent, real-time polymerase chain reaction (RT-PCR). The chimeras were labelled with 99mTc and their tissue distribution was examined in athymic nude mice bearing experimental human breast tumours. 99mTc radiolabelling was quantitative and presented a single peak in reversed phase liquid chromatography. Fluorescent real-time polymerase chain reactions using primer and fluorescent probe sets previously calculated for c-MYC mRNA demonstrated inhibition of reverse transcription by the c-MYC specific chimera as compared to that of the control. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver, and appreciable levels in tumours. These observations suggest that 99mTc-peptide-PNA probes might be useful for imaging gene expression in tumours, and the approach is worthy of further investigation.

Nipple aspirate cytology and pathologic parameters predict residual cancer and nodal involvement after excisional breast biopsy.

We previously demonstrated that abnormal nipple aspirate fluid (NAF) cytology predicted residual breast cancer (RC) and tumour size after excisional biopsy (EB), although normal NAF cytology did not exclude RC. Tumour size correlates with the risk of lymph node (LN) metastases. LN metastases provide prognostic information allowing medical and radiation oncologists to determine the need for adjuvant therapy. We hypothesized that pathologic factors known after EB, combined with NAF cytology, would predict with a high degree of accuracy the presence of RC and LN spread. NAF cytology and pathologic parameters: tumour distance from biopsy margins, multifocal and multicentric disease, sub-type of ductal carcinoma in situ (DCIS) or invasive cancer (IC), grade of DCIS or IC, tumour and specimen size, tumour and biopsy cavity location, presence or absence of extensive DCIS, and biopsy scar distance from the nipple were evaluated bivariately and then by logistic regression (LR) for their association with RC and involved LN (> or = 1 (+) LN, useful to determine chemotherapy need, and > or = 4 (+) LN, useful to determine radiation need to the chest and axilla). Data were analysed using NAF cytology alone, pathologic parameters alone, and NAF cytology and pathologic parameters combined. The combined LR model was superior in predicting residual cancer (94%) to LR models using NAF cytology (36%) or pathologic parameters (75%) alone. When only subjects with normal NAF cytology were evaluated by LR, the model was 92% sensitive in predicting RC. Tumour size and NAF cytology predicted which patients had > or = 1 (+) LN, whereas tumour and specimen size predicted which patients had > or = 4 (+) LN. We propose an algorithm which, if confirmed in a larger study, may allow clinicians to be more selective in their recommendations of re-excision breast biopsy or mastectomy.

Fibroblast growth factor-binding protein expression changes with disease progression in clinical and experimental human squamous epithelium.

Basic fibroblast growth factor (bFGF) is synthesized by a wide variety of normal and malignant cells. However, bFGF cannot exert its effects unless it gets outside of the cell. Since it lacks a signal sequence to direct secretion, the method by which cells release it remains unclear. A 17 kDa secreted binding protein for bFGF (FGF-BP, HBp-17) is expressed at high levels in squamous cell carcinoma (SCC) and transformed keratinocytes and may act as a chaperone to transport bFGF outside of the cell. In our study, FGF-BP mRNA expression in normal keratinocytes was higher than in 5/5 SCCs. Using a new monoclonal antibody, we demonstrate that FGF-BP can dimerize. Immunoassays demonstrate that normal keratinocytes have a higher level of FGF-BP than SCCs. In normal human squamous epithelium, we observed diffuse, moderate to intense cytoplasmic and membranous expression of FGF-BP. Expression decreased and became focal with disease progression to invasive cancer. Injection of immortalized but non-tumorigenic HaCaT cells transduced with FGF-BP into normal human skin xenografts failed to result in tumors. Transfection of FGF-BP into the SCCs Det 562 and FaDu did not promote tumor growth more than controls, and peri-tumoral microvessel density was lower in FGF-BP-transfected than in control tumors. Taken together, these data suggest that FGF-BP expression in squamous epithelium does not play an important role in progression to invasive carcinoma.

Endogenous p53 gene status predicts the response of human squamous cell carcinomas to wild-type p53.

Prior reports suggest that p53 protein status may influence the response to gene transduction with wild-type (wt) p53. Adenoviral vectors containing the p53 gene were administered to normal keratinocytes, to squamous cell carcinoma (SCC) lines with varied p53 protein status (absent, mutant, wt, or degraded by papillomavirus), as well as to tumors formed in severe combined immunodeficient mice. The percentage of cells undergoing apoptosis, G1 growth arrest, WAF1/p21 induction, and in vivo tumor progression were studied after wt p53 gene transduction. Apoptosis developed first in normal keratinocytes, next in SCCs lacking p53 protein, and last in SCCs with mutant or degraded p53 protein. All of the cell lines studied demonstrated an increase in WAF1/p21 protein, but only those lacking p53 protein showed G1 arrest. Tumors lacking p53 protein were more susceptible to p53 overexpression than those containing mutant or degraded p53 protein. The endogenous p53 protein status of SCCs appears to influence the outcome of p53 gene transduction.

Prolonged response to antisense cyclin D1 in a human squamous cancer xenograft model.

Local recurrence of squamous cell cancer (SCC) causes high morbidity and is often readily accessible, making such patients potential candidates for gene therapy. Cyclin D1 (CD1), critical in the G1-S transition in the cell cycle, is amplified in 20-50% and overexpressed in up to 80% of head and neck SCC. Our earlier studies indicated that CD1 expression increased with progression from low grade to high grade dysplasia, and that treatment of established tumors with antisense cyclin D1 (AS-cyclin D1) led to tumor regression during a one week evaluation period. We hypothesized that: 1) CD1 expression increases with disease progression to advanced SCC, and 2) AS-cyclin D1 therapy would lead to prolonged tumor regression in a xenograft model of human SCC. CD1 expression, evaluated by immunostain in 30 stage III/IV head and neck SCC, increased in the basal layer from normal-dysplasia (P = 0.06) and from dysplasia-carcinoma (P = 0.004). In the germinative layer CD1 expression increased from dysplasia-carcinoma (P = 0.002) but not from normal-dysplasia. Western blotting of eight SCC and two transformed keratinocyte cell lines demonstrated CD1 overexpression in 8/10 (80%) lines. An 11th cell line (A431) had previously been shown to overexpress cyclin D1. 8/9 (89%) cell lines overexpressing CD1 formed tumors in immunodeficient mice, whereas 0/2 cell lines without CD1 overexpression formed a tumor. Three established SCCs, one fast growing, one with moderate growth rate (with CD1 overexpression) and one slow growing (without increased CD1), shrank significantly for 2-4 weeks after AS-cyclin D1 treatment, while tumors transduced with control vector grew. Cyclin D1 expression increases in frequency with disease progression, and antisense cyclin D1 was effective in a xenograft model of human cancer, independent of tumor growth rate.

Expression of a prostate-associated protein, human glandular kallikrein (hK2), in breast tumours and in normal breast secretions.

The recent demonstration of human glandular kallikrein (hK2) expression in a breast carcinoma cell line has suggested that this putatively prostate-restricted, steroid hormone-regulated protease may also be expressed in breast epithelium in vivo and secreted into the mammary duct system. Given that the only substrate yet identified for hK2 activity is the precursor of prostate-specific antigen (PSA), the expression of which in breast carcinomas may be associated with favourable prognosis, our purpose was to examine the expression pattern of both hK2 and PSA in breast tumour tissues. Cytosolic extracts of 336 primary breast carcinomas prepared for routine oestrogen receptor (ER) and progesterone receptor (PR) analysis, as well as 31 nipple aspirates from six women with non-diseased mammary glands, were assayed for hK2 and PSA using immunofluorometric assays developed by the authors. In the tumour extracts, measurable hK2 and PSA concentrations were detected in 53% and 73% of cases respectively, and were positively correlated to each other (r = 0.59, P = 0.0001). Higher concentrations of PSA and hK2 were found in tumours expressing steroid hormone receptors (P = 0.0001 for PSA and P = 0.0001 for hK2, by Wilcoxon tests for both ER and PR), and both PSA (r = 0.25, P = 0.0001) and hK2 (r = 0.22, P = 0.0001) correlated directly with PR levels. A negative correlation between patient age and PSA (r = -0.12, P = 0.03) was also found. Both proteins were present in nipple aspirate fluid at relatively high concentrations which were positively correlated (r = 0.53, P = 0.002). The molecular weights of the immunoreactive species quantified by the hK2 and PSA assays were established by high-performance liquid chromatography (HPLC) and were consistent with the known molecular weights of hK2 and PSA. Together these data provide the first evidence, to our knowledge, that both malignant breast tissue and normal breast secretion contain measurable quantities of hK2, and that the degree of hK2 expression or secretion is directly proportional to the expression of PSA and steroid hormone receptors. hK2 expression may therefore be a marker of steroid hormone action in breast tissue.

Biological markers of risk in nipple aspirate fluid are associated with residual cancer and tumour size.

We previously demonstrated that nipple aspirate fluid (NAF) can be obtained from virtually all non-Asian women between the ages of 30 and 72. The focus of this report is to (1) determine the association of candidate markers of breast cancer risk in NAF obtained from fresh mastectomy specimens with residual breast carcinoma, and (2) evaluate the association of the markers with breast tumour progression. Nipple aspiration was performed on 97 specimens. Cytology, DNA index (including % hypertetraploid cells), cell cycle parameters (S phase fraction, % cells in G2/M), prostate-specific antigen (PSA), epidermal growth factor (EGF), testosterone, carcinoembryonic antigen (CEA) and prostaglandin D synthase (PGDS) were evaluated in NAF for their association with (1) residual ductal carcinoma in situ (DCIS) or invasive cancer, and (2) pathologic tumour size. NAF was obtained from 99% (96/97) of specimens. Atypical and malignant NAF cytology were significantly associated with residual DCIS or invasive cancer (P = 0.001) and with larger tumours (P = 0.004). One hundred per cent and 88% of subjects with malignant and atypical NAF cytology, respectively, had residual carcinoma. The percentage of cells in G2/M and DNA index were associated both with risk of residual carcinoma (P = 0.01 for each) and larger tumour size (DNA index, P = 0.03; G2/M, P = 0.05), although neither biomarker improved the ability of NAF cytology, to predict residual breast cancer. Higher DNA index was associated with atypical cytology (P = 0.0001). In summary, atypical and malignant NAF cytology are associated with larger tumour size, and are highly predictive of residual carcinoma after needle or excisional biopsy of the breast.

Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa.

OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.

Antisense cyclin D1 induces apoptosis and tumor shrinkage in human squamous carcinomas.

Cyclin D1 plays an essential regulatory role in the G1 phase of the cell cycle. The cyclin D1 gene is amplified in 20-50% of squamous cell carcinomas (SCCs), and the protein is overexpressed in up to 80% of SCCs. Our hypothesis was that gene transduction of antisense (AS) cyclin D1 in human SCCs in vivo would result in tumor reduction. A cyclin D1 cDNA was inserted into an E1/E3-deficient serotype 5 adenovirus (AS cyclin D1) in an AS orientation using homologous recombination. AS cyclin D1 transduction suppressed cyclin D1 protein expression in both cultured cells and tumors. AS cyclin D1 significantly inhibited cell proliferation by both [3H]thymidine incorporation in six SCC cell lines (P = 0.01-0.001) and the conversion of tetrazolium salt to formazan in four SCC cell lines (P = 0.01-0.004). Apoptosis detected in >25% of cells in each cell line 48 h after AS cyclin D1 transduction paralleled the reduction in cyclin D1 protein. Preformed SCCs transduced with AS cyclin D1 were significantly inhibited (P = 0.002-0.005), and apoptosis was prominent in the AS cyclin D1-treated tumors, but not in tumors treated with the control vector. These data extend prior in vitro and ex vivo results and indicate that AS cyclin D1 suppresses SCC growth both in vitro and in vivo through suppression of cyclin D1 protein expression, leading to cellular apoptosis. Our findings suggest that cyclin D1 may have a role in cell survival and that cyclin D1 AS therapy may be useful as an adjunct to standard treatment for SCC.

Vascular endothelial growth factor is a marker of tumor invasion and metastasis in squamous cell carcinomas of the head and neck.

Angiogenesis has been linked to increased metastasis formation and decreased overall survival in patients with various tumors, including and neck squamous cell carcinomas (HNSCC). Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. In the present study, we evaluated VEGF expression and microvessel density (MVD), a quantitative means of angiogenesis, in both experimental and clinical models of HNSCC. Analysis of VEGF RNA expression in cell lines of keratinocyte origin [HNSCC, facial skin squamous cell carcinoma (SCC), and transformed but nontumorigenic keratinocytes] and normal skin keratinocytes revealed two VEGF transcripts corresponding to proteins of 165 and 121 amino acids in length, with the transcript for the 165-amino acid species predominating. Six of eight SCC cell lines showed increased levels of one or both transcripts, and seven SCC cell lines and the transformed keratinocyte cell line showed increased protein expression. We then evaluated VEGF protein expression in human head and neck specimens containing normal epithelium (n = 10), dysplasia or carcinoma in situ (CIS; n = 15), early invasive SCCs (n = 9), advanced primary SCCs (n = 10), lymph node metastases (n = 3), and s.c. tumors or cysts (n = 7) formed in severe combined immunodeficient mice. Intense VEGF staining was found in the majority of advanced primary SCCs, lymph node metastases, and human SCCs in severe combined immunodeficient mice, whereas no dysplasia, CIS, or early SCCs showed intense immunostain. A highly significant increase (P = 0.0001) in VEGF expression was seen in the advanced SCC versus dysplasias and CIS lesions, as was the difference between SCC versus normal epithelium from nonsmokers (P = 0.01). VEGF expression in advanced primary cancers was greater (P = 0.002) and, in early cancers, marginally greater (P = 0.05) than adjacent normal mucosa. MVD increased with the progression of preinvasive disease (P = 0.04). VEGF expression and MVD (both, P = 0.003) were directly associated with tumor aggressiveness in experimental tumors. These findings suggest a role for VEGF in both clinical and experimental HNSCC.

Overexpression of p53 protein and outcome of patients treated with chemoradiation for carcinoma of the anal canal: a report of randomized trial RTOG 87-04. Radiation Therapy Oncology Group.

BACKGROUND: Despite encouraging results with chemoradiation as the primary means of managing carcinoma of the anal canal, approximately 20% of patients will develop a local recurrence. This study examined the prognostic significance of p53 nuclear protein overexpression in the pretreatment biopsies of patients treated with chemoradiation for epidermoid carcinoma of the anal canal. METHODS: All patients were treated in a prospective, randomized Radiation Therapy Oncology Group trial (RTOG 87-04) in which radiotherapy to the pelvis was compared with concurrent 5-fluorouracil (5-FU) or 5-FU and mitomycin-C. Formalin fixed, paraffin embedded blocks or unstained slides from the pretreatment biopsies of 64 patients were obtained from referring institutions and evaluated immunohistochemically with the polyclonal p53 antibody CM-1. A multivariate analysis was conducted to analyze overexpression of p53 in terms of locoregional control, no evidence of disease (NED), and overall survival. RESULTS: p53 protein was overexpressed in 48.4% of the cases. Although not statistically significant, there was a trend for patients whose tumors overexpressed p53 to have inferior locoregional control (52% vs. 72%, P = 0.13), NED survival (52% vs. 68%, P = 0.27), and absolute survival (58% vs. 78%, P = 0.14). Of all the pretreatment factors analyzed, only International Union Against Cancer stage was predictive of outcome in multivariate analysis. Among those patients whose tumors overexpressed p53, there was a trend toward improved outcome in the arm that received 5-FU and mitomycin-C compared with the arm that received 5-FU only. CONCLUSIONS: Overexpression of the p53 protein may be associated with inferior outcome for patients managed with definitive chemoradiation for epidermoid carcinoma of the anal canal.

Molecular biology of human melanoma development and progression.

In the United States, Australia, Northern Europe, and Canada, malignant melanoma is increasing at a faster rate than any other cancer, with the exception of lung cancer in women. Major advances have been made in the molecular biology and immunology of melanoma. These advances in basic science have led to more rational approaches to specifically targeting melanoma cells, with promising results in the clinic. An increased understanding of how melanoma spreads has led to more selective, less invasive surgical procedures that do not compromise patient health. Combinations of chemotherapy and immunotherapy are now available for patients with advanced melanoma that affect both the length and quality of the patients' lives. This review of the molecular biology of melanoma development and progression discusses the disease's etiology, molecular genetics, cell-surface antigens, experimental models, biological markers, and new forms of treatment. As we continue to learn more about malignant melanoma, we will be able to devise more specific and effective treatments that will give patients with this potentially deadly disease longer and more productive lives.

Ultraviolet B-induced squamous epithelial and melanocytic cell changes in a xenograft model of cancer development in human skin.

We previously demonstrated that precancers (actinic keratoses and dysplasias) and squamous cell carcinomas (SCCs) develop in one quarter of human neonatal foreskins grafted onto recombinase-activating gene-1-knockout mice treated once with 7,12-dimethylbenz[a]anthracene (DMBA) followed by chronic intermediate-range ultraviolet (UV) B light irradiation. The goals of this study were to determine if a longer UVB exposure followed by further observation would increase the number of precancers and invasive cancers and to evaluate whether this model results in changes in p53 expression and cell proliferation similar to those seen in sun-damaged normal skin, actinic keratoses, and SCCs. The treatment consisted of a single dose of DMBA followed by 500 J/m2 UVB radiation administered three times weekly for at least 5 mo. Histologic changes (cysts, hyperplasias, precancers, and/or invasive cancers) were seen in 24 of 25 treated xenografts but not in controls. Ten of 25 grafts (40%) had two or more histological changes, and two human SCCs developed. After seven or more months of UV exposure and a total time from DMBA treatment to killing of 12-18 mo, 83% (15 of 18) of specimens developed squamous precancer or SCC of human origin, and 44% (eight of 18) developed melanocytic hyperplasia or melanoma. The change from moderate dysplasias to SCC required longer UV exposure (median, 11 mo), and 5 mo more observation than did the development of mild dysplasias (median UV exposure, 7 mo; median DMBA to death time, 12 mo). There was a direct correlation between both p53 expression and cell proliferation and the degree of histologic alteration both in squamous epithelial and melanocytic cells.

Markers of cell proliferation in normal epithelia and dysplastic leukoplakias of the oral cavity.

The expression of several markers of epithelial cell proliferation was analyzed to establish baseline data for future chemoprevention studies of oral premalignant lesions. Punch biopsies (n = 60) from three different sites of oral mucosa (bucca, lateral tongue, and the floor of the mouth) were obtained from 20 normal donors of both sexes. After formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect the proliferation markers Mib-1, cyclin D1, and centromere-associated protein CENP-F. Analysis of sections stained for the three markers showed similar patterns, i.e., a low labeling index (LI) in the basal layer and a high LI in the parabasal layer at all three intraoral sites. No proliferative activity was seen above the parabasal layer (superficial layer). All sites showed similar Mib-1 LI values for the proliferative markers. The tongue epithelium exhibited higher parabasal LIs of cyclin D1 and CENP-F than did the other two sites. No significant differences were detected between smokers and nonsmokers. The data from normal mucosa were compared with those from low (n = 30)- and high (n = 17)-grade dysplastic leukoplakias. The Mib-1 LI showed a very significant change, with a 9-fold increase in the basal layer LI in dysplastic leukoplakias. Cyclin D1 and CENP-F showed similar trends with increments of up to 7-fold in the basal layer of high-grade dysplasia. Although the proliferative activity of the parabasal layer was similar in normal and leukoplakic epithelia, the superficial layer showed a significant increment in proliferative activity mainly in high-grade leukoplakia. These studies suggest that proliferation markers in the basal and superficial cells of premalignant lesions may serve as surrogate end point biomarkers for chemoprevention trials.

Prostate-specific antigen production in the female breast: association with progesterone.

Prostate-specific antigen (PSA) is produced by the female breast. Prior in vitro evidence suggests that PSA expression in breast epithelial cells is regulated by androgens and progestins but not estrogens. The purpose of this study was to determine whether (a) PSA expression in breast nipple aspirate fluid (NAF) and in serum is influenced by progesterone (PG); (b) the ability to obtain NAF decreases with repeated breast aspirations; and (c) PSA in NAF correlates with abnormal NAF cytology. Eight pre- and three postmenopausal women with no breast cancer risk factors were enrolled in a pilot study and had NAF and serum collected every 3-4 days for a month to evaluate the influence of serum PG, luteinizing hormone, estradiol, and follicle-stimulating hormone on PSA in serum and in NAF. NAF was obtained in 99% (112 of 113) of aspiration visits. Median, mean, and peak NAF but not serum PSA levels were higher in pre- than in postmenopausal subjects. NAF PSA levels were associated with the rise or peak in serum PG in seven of eight premenopausal women (seven of seven with a PG surge) and in zero of three postmenopausal women. Considering all 11 women, there was an association between NAF PSA and PG (P = 0.005) but not luteinizing hormone, estradiol, or follicle-stimulating hormone. NAF volume did not significantly change over time. Atypical hyperplasia (9%) and hyperplasia without atypia (36%) were identified in the NAF of a subset of the subjects. Median, mean, and peak levels of NAF PSA (P = 0.05, 0.05, and 0.10, respectively) were higher in subjects with normal versus hyperplastic cytology. PSA production in the breast increases in association with PG. With aspiration every 3-4 days, NAF volume does not significantly decrease over time. NAF cytology and PSA levels in NAF may help identify women at increased breast cancer risk. Changes in biomarkers of breast cancer risk in NAF (including PSA and cytology) may predate mammographic abnormalities. NAF may, therefore, be useful as a breast cancer screening tool for young women who are not recommended to undergo mammography and as an adjunct to screen women who have mammograms performed.