In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma.

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a rare but highly aggressive variant of endometrial cancer. Pertuzumab is a new humanised monoclonal antibody (mAb) targeting the epidermal growth factor type II receptor (HER2/neu). We evaluated pertuzumab activity separately or in combination with trastuzumab against primary USPC cell lines expressing different levels of HER2/neu. METHODS: Six USPC cell lines were assessed by immunohistochemistry (IHC), flow cytometry, and real-time PCR for HER2/neu expression. c-erbB2 gene amplification was evaluated using fluorescent in situ hybridisation (FISH). Sensitivity to pertuzumab and trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was evaluated in 5 h chromium release assays. Pertuzumab cytostatic activity was evaluated using proliferation-based assays. RESULTS: Three USPC cell lines stained heavily for HER2/neu by IHC and showed amplification of the c-erbB2 gene by FISH. The remaining FISH-negative USPCs expressed HER2/neu at 0/1+ levels. In cytotoxicity experiments against USPC with a high HER2/neu expression, pertuzumab and trastuzumab were similarly effective in inducing strong ADCC. The addition of complement-containing plasma and interleukin-2 increased the cytotoxic effect induced by both mAbs. In low HER2/neu USPC expressors, trastuzumab was more potent than pertuzumab in inducing ADCC. Importantly, in this setting, the combination of pertuzumab with trastuzumab significantly increased the ADCC effect induced by trastuzumab alone (P=0.02). Finally, pertuzumab induced a significant inhibition in the proliferation of all USPC cell lines tested, regardless of their HER-2/neu expression. CONCLUSION: Pertuzumab and trastuzumab induce equally strong ADCC and CDC in FISH-positive USPC cell lines. Pertuzumab significantly increases tratuzumab-induced ADCC against USPC with a low HER2/neu expression and may represent a new therapeutic agent in patients harbouring advanced/recurrent and/or refractory USPC.

Serum amyloid A (SAA): a novel biomarker for uterine serous papillary cancer.

BACKGROUND: Uterine serous papillary carcinoma (USPC) is a biologically aggressive variant of endometrial cancer. We investigated the expression of Serum Amyloid A (SAA) and evaluated its potential as a serum biomarker in USPC patients. METHODS: SAA gene and protein expression levels were evaluated in USPC and normal endometrial tissues (NEC) by real-time PCR, immunohistochemistry (IHC), flow cytometry and by a sensitive bead-based immunoassay. SAA concentration in 123 serum samples from 51 healthy women, 42 women with benign diseases, and 30 USPC patients were also studied. RESULTS: SAA gene expression levels were significantly higher in USPC when compared with NEC (mean copy number by RT-PCR=162 vs 2.21; P=0.0002). IHC revealed diffuse cytoplasmic SAA protein staining in USPC tissues. High intracellular levels of SAA were identified in primary USPC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (mug ml(-1)) had a median (95% CIs) of 6.0 (4.0-8.9) in normal healthy females and 6.0 (4.2-8.1) in patients with benign disease (P=0.92). In contrast, SAA values in the serum of USPC patients had a median (95% CI) of 15.6 (9.2-56.2), significantly higher than those in the healthy group (P=0.0005) and benign group (P=0.0006). Receiver operating characteristics (ROC) analysis of serum SAA to classify advanced- and early-stage USPC yielded an area under the ROC curve of 0.837 (P=0.0024). CONCLUSION: SAA is not only a liver-secreted protein but is also a USPC cell product. SAA may represent a novel biomarker for USPC to assist in staging patients preoperatively, and to monitor early-disease recurrence and response to therapy.

Lobular and ductal intraepithelial neoplasia.

Lobular and ductal intraepithelial neoplasias reflect proliferations of immunophenotypically variable, biologically and morphologically diverse cells with a potential, not always realized, for progression to carcinoma by breaking through the barriers of the myoepithelial cell layer and basement membrane, ultimately invading the stroma. Starting with the lobular and then the ductal proliferations, this review will address the evolution of our understanding of these lesions; the problems associated with the conventional terminology of ductal hyperplasia, atypical hyperplasia, and carcinoma in situ; and reasons for and advantages of the intraepithelial neoplasia terminology.

Remarkably high frequency of EGFR expression in breast carcinomas with squamous differentiation.

The human epidermal growth factor receptor (EGFR) is reportedly overexpressed in 15-20% of breast carcinomas. EGFR overexpression is associated with reduced survival and is inversely correlated with expression of estrogen receptor (ER). This study assessed EGFR expression in breast carcinomas with squamous differentiation. The immunohistochemical (IHC) expression of EGFR was evaluated in 39 breast carcinomas with squamous differentiation (30 pure squamous, 6 adenosquamous, 3 carcinosarcomas) by use of the pharmDx assay (clone 2-18C9, DakoCytomation). Cases were considered positive if at least 10% of the cells showed 1+ positivity in the squamous component. Squamous differentiation was confirmed with IHC for CK5-6 (clone D5/16B4, DakoCytomation). ER, PR, and HER2 status as well as clinical information regarding treatment and outcome were correlated. As a control, a tissue microarray comprising 280 lymph node positive breast carcinomas was evaluated with the same EGFR assay. The 39 patients ranged in age from 33 to 77 years (mean 52). The tumors measured 1.3-30 cm (mean 4.8). Sentinel or full axillary lymph node dissection was performed in 28 patients. Fourteen patients had positive lymph nodes. At the time of initial diagnosis, 3 patients had distant metastasis. Follow-up was available for 16 patients (mean 45 months). Disease-free survival at 3 years was 70%. Among the 39 tumors 87% (34) were positive for EGFR (p<0.0001). Sixty-nine percent (27 of 39) showed >50% 2+ EGFR staining. EGFR-positive tumor cells (showing squamous morphology) were also found in 1 bone, 1 lung, and 8 of 11 lymph node metastases available for evaluation. All 11 lymph nodes showed squamous differentiation. All but 1 of the EGFR+ tumors examined were ER and PR negative. Six EGFR-positive tumors were HER2 positive. No statistically significant differences in HER2 status, size, lymph node status and disease-free survival were observed between EGFR+ and EGFR- cases, but the number of EGFR-negative tumors was quite small. Nine of 280 (3%) of lymph node-positive invasive carcinomas on the tissue microarray were EGFR+; review of the initial diagnostic slides failed to reveal squamous features in all but 1 of the 9 EGFR+ tumors. Breast carcinomas with squamous differentiation are a distinct subgroup of breast tumors with a very high frequency of EGFR positivity. Breast carcinomas of this type would be ideal candidates for a trial with EGFR inhibitors.

Metastases to the breast.

AIM: The aim of this study was to report the clinico-pathological features of a series of patients with metastatic neoplasms to the breast. METHODS: A 10-year archive of surgical material was reviewed. A search was performed on all 10,650 breast neoplastic cases in the files of the Pathology Department from 1990 to 2000. RESULTS: There were 22 women and two men. The most common primary sites for solid tumours were cutaneous melanoma and ovarian carcinoma. Two of the 24 patients had no prior history of malignant disease. There was a solitary nodule in 17 cases; in seven cases there were multiple lesions in the same breast. Sixteen patients had a rapidly fulminating course and died of disease. Six patients are alive with disease and two patients were lost to follow-up. CONCLUSION: Recognition of these lesions as being metastatic may pre-empt radical surgery.

Central atypical papillomas of the breast: a clinicopathological study of 119 cases.

The clinicopathological features of central intraductal papillomas of the breast presenting with florid usual ductal hyperplasia or atypical ductal hyperplasia (ADH) were analyzed in a retrospective series of 119 patients, whose lesions were sent to the Armed Forces Institute of Pathology from 1976 to 1990. After histological review considering predefined morphological and quantitative criteria, the 119 central papillomas were classified into 22 papillomas with florid usual ductal hyperplasia (18%), 40 papillomas with focal atypia (34%), 24 atypical papillomas (20%) and 33 carcinomas arising in a papilloma (28%). After a median period of follow-up of 110 months, 16 recurrences (5 papillomas, 2 carcinomas arising in a papilloma, 4 ductal carcinomas in situ, 5 invasive carcinomas) occurred. No statistically significant difference was observed in relation to recurrence for the various categories of papillomas. The presence of epithelial hyperplasia, ADH or lobular neoplasia in the surrounding breast as well as infarction of the papilloma were significant predictive factors of recurrence ( P=0.02 and P=0.005, respectively, log-rank test). The main reason for the observed low rate of significant recurrences in this series was that epithelial atypia (whether comprising 20% or 60% of the papillary lesion) was, in most of the cases, localized in a confined lesion that was completely excised.

Papillary endothelial hyperplasia of the breast: the great impostor for angiosarcoma: a clinicopathologic review of 17 cases.

Seventeen cases of papillary endothelial hyperplasia (PEH, Masson's vegetant intravascular hemangioendothelioma) involving breast or mammary subcutaneous tissues are described. The mean patient age was 59; 14 (82%) were female and 12 (71%) presented with a mass. Nine women had mammographic evaluation, 3 of whom had microcalcifications. Five neoplasms were discovered by routine mammography. Sixteen cases were 2.7 cm or less in greatest dimension, and 8 (47%) were associated with a thrombus and/or cavernous hemangioma. Follow-up in 10 cases (up to nearly 8 years) showed no recurrences. Fifty-nine percent of the cases were received at AFIP for consultation with a working diagnosis of angiosarcoma. Features that help distinguish PEH from angiosarcoma include circumscription of the lesion, location in a vessel or association with thrombus, and papillary architecture without significant cytologic atypia or areas of solid growth. The recognition of the morphologic features of this lesion and its inclusion in the differential diagnosis of vascular mammary tumors will reduce the likelihood of its misdiagnosis as an angiosarcoma and avoid unnecessary and aggressive therapy.

Retiform Sertoli-Leydig cell tumours: clinical, morphological and immunohistochemical findings.

AIMS: To determine the morphological and immunohistochemical profile of retiform Sertoli-Leydig cell tumours and to compare the observed profile with that of adult rete ovarii. METHODS AND RESULTS: Nineteen retiform Sertoli-Leydig cell tumours were studied, eight by immunohistochemistry, and five examples of rete ovarii from adult females were also evaluated immunohistochemically. The patients ranged in age from 3 to 74 years with a mean age of 31 years. Four patients were virilized and had an abdominal mass; two were virilized with amenorrhoea and two had amenorrhoea alone. Eight presented with an abdominal mass and one patient was pregnant. Two tumours were incidental findings. Information on stage was available in 16 patients: 14 tumours were stage 1, one was stage 2, and one was stage 3. Fifteen tumours were of intermediate differentiation and four were poorly differentiated. Papillary structures were evident grossly in four cases. Microscopically, all cases had a retiform pattern in addition to varying quantities of sex cord, gonadal stromal and heterologous elements. Heterologous elements were present in 13 cases and consisted of hepatocytes (n = 7), mucinous epithelium (n = 7) and skeletal muscle (n = 2). Immunohistochemical evaluation of eight tumours showed a more intense positivity for keratin in the retiform areas, whereas the gonadal stromal component had a more intense expression of inhibin. Inhibin stains Leydig cells strongly and hepatocytes moderately. Rete ovarii epithelium was positive for keratin and vimentin in the five cases studied, and for inhibin in one case. Follow-up was available on 13 patients. Three tumours behaved in a malignant fashion: one each was stage 1, 2, and 3 at diagnosis. CONCLUSIONS: Immunohistochemistry is useful in distinguishing retiform Sertoli-Leydig cell tumours from other tumours that they may resemble. Inclusion of inhibin is essential in a panel of antibodies to evaluate these tumours. The clinical behaviour of these neoplasms cannot always be predicted from their morphology or clinical stage.

An improved method for DNA extraction from paraffin sections.

The acquisition of comparable quality and quantity of DNA extracts is the prerequisite to the success of comparative genetic analyses. Although several DNA extracting protocols on paraffin sections have been introduced, the importance of deparaffinization, the procedure for obtaining an adequate hematoxylin staining, the significance of the ratio of the cell number to the enzyme volume, and a practical means for monitoring the digestion process have not been sufficiently addressed. These, however, are the most important factors accountable for a failure of DNA extraction. To minimize the impact of these factors, we have developed several unique strategies, including: (1) incubating sections at 80 degrees C for 30-60 minutes prior to xylene treatment, (2) checking each section to insure the complete removal of paraffin; (3) treating hematoxylin stained sections or cells with de-staining solutions; (4) using a micrometer inserted into the eyepiece of a microscope to estimate the number of cells collected and adjusting the enzyme volume according to the cell number; and (5) monitoring the digestion process with a magnifier. With these strategies, we have been able to consistently obtain comparable quality and quantity of DNA extracts which yielded uniform PCR products regardless of variations in tissue embedding and processing.

Superficial cervicovaginal myofibroblastoma: fourteen cases of a distinctive mesenchymal tumor arising from the specialized subepithelial stroma of the lower female genital tract.

The clinicopathologic features and immunohistochemical profiles of 14 cases of a distinctive mesenchymal tumor that arises in the superficial lamina propria of the cervix and vagina and is histologically distinguishable from mesodermal (fibroepithelial) stromal polyp, including the cellular (pseudosarcomatous) variant, angiomyofibroblastoma, aggressive angiomyxoma, and other well-recognized lesions that occur in this location, are described. The lesions presented as a polypoid (n = 10) or nodular (n = 4) mass in the vagina (n = 12) or cervix (n = 2) of women ranging in age from 40 to 74 years (median, 58 years). The tumors were subepithelial in location, were well circumscribed, and ranged in size from 1 to 6.5 cm. (mean, 2.7 cm). Microscopically, the process was moderately to highly cellular and composed of relatively bland spindled and stellate-shaped mesenchymal cells embedded in a finely collagenous stroma that was punctuated by myxoid and edematous foci in 9 cases. The lesions characteristically had a multipatterned architecture with tumor cells focally assuming a lacelike/sievelike growth pattern in the more stroma-rich areas of the tumor and a vague fascicular growth pattern in the more cellular foci. Mitotic activity was minimal, and no atypical mitotic figures were identified. The tumors were immunoreactive (in decreasing order of relative strength) for vimentin (5 of 5 cases), estrogen (10 of 10 cases), and progesterone (10 of 10 cases) receptors, desmin (13 of 13 cases), CD34 (11 of 13 cases), alpha-smooth muscle actin (5 of 11 cases), and muscle-specific actin (2 of 8 cases). The desmin and CD34 antibodies highlighted the interconnecting, dendritic processes associated with many of the tumor cells. No immunoreactivity was detected for S100 protein, epithelial membrane antigen, or keratins. Follow-up data for 11 patients (range, 1 to 20 years; median, 4 years) showed no recurrence or metastasis after local excision. The term "superficial cervicovaginal myofibroblastoma" is proposed because it reflects the distinguishing features of this benign, relatively site-specific mesenchymal tumor. The process probably arises as a neoplastic proliferation of hormonally responsive mesenchymal cells native to the unique subepithelial stromal layer normally found through the endocervix and vulva of adult women.

Spindle cell ductal carcinoma in situ. An unusual variant of ductal intra-epithelial neoplasia that simulates ductal hyperplasia or a myoepithelial proliferation.

Seventeen examples of a variant of ductal carcinoma in situ (DCIS) composed exclusively or predominantly of spindle cells arranged in fascicles, whorls, and solid sheets are described. The fascicular arrangement of the spindle cells simulates the "streaming" phenomenon associated with ordinary intraductal epithelial hyperplasia (IDH). This process also resembles the myoid, solid form of intraductal myoepithelial proliferation. The women ranged in age from 38 years to 79 years with a mean age of 59.3 years. Five patients presented with a palpable mass. The remaining tumors were discovered using mammography. The radiological appearances of the lesions raised concern for carcinoma, but there were no distinctive mammographic findings to suggest an unusual variant of DCIS. Cytological preparations were suspicious for malignancy in two patients and were reported as malignant in another case. Sixteen patients were treated with wide local excision, and one woman had a partial mastectomy. The tumors measured from 3 mm to 15 mm (mean 8.65 mm). In three cases, minute foci of stromal invasion were associated with the spindle cell DCIS. In another specimen, a 2.7-cm invasive ductal carcinoma of no special type was identified in an area away from the foci of the spindle cell DCIS. None of the patients has experienced recurrence or metastasis during the relatively short mean follow-up period of 16.2 months (range 4-77 months). Spindle cell DCIS is distinguished from the streaming pattern of ordinary IDH by its solid growth pattern, lack of secondary spaces or peripheral fenestrations, uniformity of appearance and distribution of nuclei, cytological atypia in the range of low to intermediate-grade DCIS, and negative immunoreaction with CK-34betaE12 (HMW-CK903). When fenestrations are present, they are evident in areas of cribriform DCIS that merge with the solid, spindle cell areas in hybrid ducts harboring both patterns. This admixture, with conventional cribriform DCIS, and the association with foci of invasive ductal carcinoma in some cases further help recognition and confirmation of this lesion as in situ carcinoma. When there is no transition from the spindle cells to recognizable cribriform DCIS, distinction from intraductal myoepithelial hyperplasia (myoepitheliosis) requires immunostains for actin and S-100 protein. Recognition of this pattern of DCIS is important in order to avoid its frequent misclassification as a benign lesion.

Multiple use of slab gels in sequencing apparatus for separation of polymerase chain reaction products.

Attempting to assess whether a decrease of the electrophoresis temperature could prevent or reduce the extent of gel well deformations, and whether the utilization of native polyacrylamide gels (without urea) could speed up the separation of polymerase chain reaction (PCR)-amplified products with an automated 377 DNA sequencer, denatured PCR products were subjected to electrophoresis in 6% native gels under 45 degrees C. Results show that a decrease of the electrophoresis temperature from 51 degrees C (recommended by the User's Manual) to 45 degrees C substantially facilitates the preservation of gel wells, and that all PCR products tested migrate significantly faster in native than in denatured (with urea) gels of the same concentration. The combination of a 6% native gel and a lower (45 degrees C) electrophoresis temperature permits multiple uses of a given gel with consistent results, consequently reducing the electrophoresis time and reagent costs.

Mesonephric adenocarcinomas of the uterine cervix: a study of 11 cases with immunohistochemical findings.

Mesonephric adenocarcinoma is a rare variant of cervical carcinoma with relatively few, well-documented cases reported. We describe the clinicopathologic and immunohistochemical features of 11 examples of this neoplasm, which occurred in women between the ages of 35 and 72 years (mean, 52 years). Most (64%) patients had abnormal vaginal bleeding. Eight tumors were stage IB, and one each was stage IIB and IVB; in one, the stage was unknown. Microscopically, the carcinomas showed various morphologies, most commonly a small tubular pattern or a ductal pattern resembling endometrioid adenocarcinoma; one tumor had an associated malignant spindle cell component. Ten neoplasms were adjacent to hyperplastic mesonephric remnants. Follow-up in 10 cases showed six patients to be alive without evidence of recurrence after a mean of 4.8 years. The patients with stage IIB and IVB disease had local recurrences after 2.2 and 0.7 years and died of progressive disease at 3.2 and 0.8 years, respectively. In a patient with stage IB disease, a mediastinal metastasis and a malignant pleural effusion developed 5.6 years after diagnosis, and the patient died of disease at 6.2 years. Another patient with stage IB disease and a positive vaginal cuff margin that recurred locally after 1.7 years received chemotherapy and was alive and clinically free of disease at 2.5 years. Mesonephric adenocarcinomas were immunoreactive for epithelial markers (AE1/3; CK1, CAM 5.2, cytokeratin 7, and epithelial membrane antigen) (100%), calretinin (88%), vimentin (70%), androgen receptor (33%), and inhibin (30%, focal staining). No immunostaining was detected with cytokeratin 20, estrogen receptor, progesterone receptor, and monoclonal carcinoembryonic antigen. This staining profile is similar to that of mesonephric remnants and may be useful in the distinction of mesonephric carcinoma from mullerian endometrioid adenocarcinoma, with which it may be confused.

Allelic losses at 3p and 11p are detected in both epithelial and stromal components of cervical small-cell neuroendocrine carcinoma.

Microdissected epithelial and stromal cells from 15 cervical small-cell carcinoma patients and 9 healthy control subjects were assessed for loss of heterozygosity with polymorphic DNA markers at chromosomes 3p and 11p. Among malignant lesions assessed with 7 markers at 3p, 21 allelic losses were detected from 193 informative samples. Of losses, 20 were in epithelial and 1 was in normal-appearing stromal cells. Among losses in epithelial cells, 16 were from 44 samples informative for 3 markers within 3p21.2-p14.2 (0.36 loss/sample), whereas only 4 were from 54 samples informative for 4 markers outside the region (0.09 loss/sample), suggesting a "hot spot" of genetic alterations within 3p21.2-p14.2. Among malignant lesions assessed with 2 markers within 11p14-p12, 15 losses were seen in 52 informative samples. Of losses, 10 were in epithelial and 5 were in normal-appearing stromal cells. Of 10 epithelial samples showing losses within 11p14-p12, 8 also displayed losses within 3p21.2-p14.2, suggesting a concurrent involvement of these loci in tumor development or progression. The five losses in stromal cells were in four cases that showed no loss in epithelial cells with same markers, suggesting that stromal cells might play initiative roles in tumor development.

Follicle center lymphoma involving the female genital tract: a morphologic and molecular genetic study of three cases.

Primary female reproductive system lymphomas are distinctly uncommon, and are defined as lymphomas that present primarily as gynecologic tumors. We describe 3 cases which presented in this location, 1 primary ovarian disease and 2 others presenting the initial manifestations of disseminated lymphoma. Clinical history, follow-up information, and paraffin embedded archival tissue were available for all 3 cases. A panel of immunoperoxidase studies and molecular genetic studies were performed for each case. The primary ovarian follicle center lymphoma was a grade III/III (large cell) while the cases representing secondary involvement were grade I/III (small cell). Immunohistochemistry demonstrated reactivity of the malignant cells in each case with CD20 and bcl-2. In the grade I/III cases the cells were immunoreactive for CD45RA and CD10. Molecular genetic analysis demonstrated the t(14;18) translocation in the case of primary ovarian follicle center lymphoma. Follicle center lymphoma uncommonly presents in the female genital system, and may rarely be primary to this site. Immunoperoxidase and molecular studies in concert with the morphology are invaluable in rendering a correct diagnosis and ensuring correct treatment of the patient.

Glomus tumor arising in a mature teratoma of the ovary: report of a case simulating a metastasis from cervical squamous carcinoma.

Glomus tumors involving the female genital tract are rare. Herein we report the case of a glomus tumor constituting the major component of a mature teratoma of the ovary. This lesion represented an incidental finding in a 43-year-old woman who underwent bilateral salpingo-oophorectomy at the time of detection of locally recurrent squamous carcinoma of the cervix. The glomus tumor was initially interpreted as a metastasis due to its superficial morphologic resemblance to the recurrent carcinoma in the vagina. It was, however, morphologically similar to glomus tumors that classically arise in the extremities. The diagnosis was supported by immunohistochemistry and ultrastructural examination. To our knowledge, no similar tumor has been described in the ovary or in teratomas at any other site.

Mammary "comedo"-DCIS: apoptosis, oncosis, and necrosis: an electron microscopic examination of 8 cases.

The terms apoptosis and necrosis are commonly used to imply two distinct types of cell death. Apoptosis reflects a genetically mediated. ATP-dependent form of cell death. A passive form of cell death (oncosis) also occurs, often in response to some form of injury. Both pathways can lead to necrosis (postmortem autolytic cell changes). The nature of intraluminal necrosis in mammary ductal carcinoma in situ (DCIS) was evaluated using ultrastructural analysis on paraffin-embedded material of 8 cases with "comedo"-DCIS. In each case, intraepithelial proliferation zones and intraluminal zones (peripheral and central luminal zones) were examined. All cases with "comedo"-DCIS revealed abundant apoptosis, characterized by apoptotic cells showing chromatin condensation and margination with sharply circumscribed, uniformly dense crescents, as well as cytoplasmic condensation. Numerous membrane-bound apoptotic bodies with condensed cytoplasm (with or without nuclear fragments) were also observed. The central luminal zones of "comedo"-DCIS, however, revealed necrotic debris characterized by severe degradative changes, largely devoid of recognizable cell structures. In addition, two cases displayed features of oncosis, characterized by nuclear and cytoplasmic swelling, vacuolization of cytoplasm, and mitochondrial swelling with occasional dense bodies. The results indicate that necrosis (postmortem, secondary degradative cell changes) in "comedo"-DCIS is the end result of either apoptosis (programmed cell death) alone or a combination of apoptosis and oncosis (passive or "accidental" cell death).

Five useful approaches for generating more valid gel images of loss of heterozygosity and clonality analysis with an automated 377 DNA sequencer.

The recently introduced fluorescence-based gene scan system for assessment of loss of heterozygosity (LOH) and clonality with an automated DNA sequencer has several advantages over the traditional method. However, the production of gel images with this system is subjected to more technical challenges, including the interference of autofluorescence, weaker and less consistent signals that result from the restricted well size and difficulties in sample loading. To minimize the impact of these technical difficulties, several unique strategies were used, including the following: elimination of fabrics or paper towels in the cleaning of gel plates and containers; use of a modified loading buffer; use of more concentrated gels; use of an innovative apparatus to clean gel wells before and after the prerun; and covering the black printer cartridge with a sheet of scotch tape. With these strategies, the authors have been able to consistently obtain gel images that can be presented as either densitometric graphs or as band patterns for direct visual assessment.