Analysis of loss of heterozygosity in atypical and negative bile duct brushing cytology specimens with malignant outcome: are "false-negative" cytologic findings a representation of morphologically subtle molecular alterations?

CONTEXT: Conventional cytologic evaluation of bile duct brushings for neoplasia has high specificity but relatively low sensitivity. OBJECTIVE: The aim of this pilot study was to examine whether K-ras mutations and loss of heterozygosity for multiple microsatellite markers in bile duct brushings would contribute to the detection of malignancy in cases initially reported as "negative" or "atypical." DESIGN: Bile duct brushing specimens with a negative or an atypical cytologic result (9 cases) had a benign result on the surgical pathology specimen, and 9 additional negative or atypical cases demonstrated adenocarcinoma on the resected surgical specimen. Cells from representative cytopathology and histology slides were microdissected and analyzed for K-ras mutations and for loss of heterozygosity with a panel of 15 polymorphic markers on chromosomes 1p, 3p, 5q, 9p, 9q, 10q, 17p, and 22q. RESULTS: Among cytology cases with malignant outcome, loss of heterozygosity or K-ras mutation was detected in 8 (88.8%) of 9 cases. In the corresponding 9 surgical pathology specimens with adenocarcinoma, K-ras mutations and/or allelic losses were detected in all (100%). Loss of heterozygosity or K-ras mutation was not detected in cytology cases that had a benign surgical outcome. The fractional allelic loss of these 9 cytology specimens ranged from 0 to 0.25 (mean, 0.14). This compared with the fractional allelic loss ranging from 0.15 to 0.42 (mean, 0.27) for the corresponding surgical specimens. CONCLUSIONS: This pilot study suggests that low-level fractional allelic loss or K-ras mutation in the negative/atypical cytology samples with malignant outcome is a representation of morphologically subtle molecular alterations.

Determination of sequential mutation accumulation in pancreas and bile duct brushing cytology.

Neoplastic progression is characterized by clonal expansion of tumor cells associated with accumulation of mutational damage. The timing of mutation acquisition could be of value in distinguishing preneoplastic conditions from early and advanced cancer as well as characterizing tumor aggressiveness and treatment response. Using quantitative methods applied to microdissected cell clusters selected according to cytomorphologic features, we sought to demonstrate the feasibility and efficacy for determining the time and course of mutation accumulation in pancreatobiliary cytology specimens. In all, 40 pancreatic duct and 21 biliary brushing cytology specimens were retrieved from the cytology database. Xylene-resistant markings were placed on the slide underside and coverslips removed. Clusters of benign, atypical and malignant cells were manually microdissected and DNA extracted. Mutations (allelic imbalance) (loss of heterozygosity) were quantitatively determined for a broad panel of 15 markers (1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q) as well as point mutation in K-ras-2 using PCR/capillary electrophoresis. Time course was based on earlier mutations having a higher proportion of mutant DNA for a particular marker. The descending frequency of detectable mutational involvement in pancreatic cytology was K-ras-2 point mutation (58%), 3p25-26 and 17q21 (35%), 5q23 (33%), 1p36 (28%), followed by the remaining molecular markers. The descending frequency of mutational content in bile duct cytology was 17p13, 1p36, 3p25-26, and 5q23 followed by remaining molecular markers. K-ras-2 point mutation was not seen in bile duct specimens. While there was overlap in the spectrum of mutational markers in pancreatic duct and biliary brushing cytology, the temporal profile was significantly different (P<0.001). Pancreatic and biliary neoplasia progression involves distinct subset of accumulated defined mutations. Determination of timing of the mutational damage in cytologic material could be incorporated in the work-up and help in making a more definitive diagnosis of malignancy in pancreatobiliary cytology specimens.

The role of pancreatic cyst fluid molecular analysis in predicting cyst pathology.

BACKGROUND & AIMS: Current methods to detect malignancy in mucinous cystic neoplasms of the pancreas remain inadequate. The role of detailed molecular analysis in this context was investigated. METHODS: Endoscopic ultrasound-guided pancreatic cyst aspirates were prospectively collected during a period of 19 months and studied for cytology, carcinoembryonic antigen level, and molecular analysis. Molecular evaluation incorporated DNA quantification (amount and quality), k-ras point mutation, and broad panel tumor suppressor linked microsatellite marker allelic loss analysis by using fluorescent capillary electrophoresis. The sequence of mutation acquisition was also calculated on the basis of a clonal expansion model, and comparison was made to the final pathology. RESULTS: Thirty-six cysts with confirmed histology were analyzed. There were 11 malignant, 15 premalignant, and 10 benign cysts. Malignant cysts could be differentiated from premalignant cysts on the basis of fluid carcinoembryonic antigen level (P=.034), DNA quality (P=.009), number of mutations (P=.002), and on the sequence of mutations acquired (P<.001). Early k-ras mutation followed by allelic loss was the most predictive of a malignant cyst (sensitivity, 91%; specificity, 93%). CONCLUSIONS: Malignant cyst fluid contains adequate DNA to allow mutational analysis. A first hit k-ras mutation followed by allelic loss is most predictive of the presence of malignancy in a pancreatic cyst. This approach should serve as an ancillary tool to the conventional work-up of pancreatic cysts. Cumulative amount and timing of detectable mutational damage can assist in diagnosis and clinical management.

Microdissection-based mutational genotyping of serous borderline tumors of the ovary.

Mutational changes in a number of genetic foci were studied in 12 serous borderline tumors (SBTs) of the ovary including 2 with a micropapillary pattern. The analysis was focused on chromosomal regions that have not been previously studied in these tumors. The findings were correlated with the morphology and the FIGO stage of the tumors. Six of the tumors were stage I, one was stage II, and five were stage III. Loss of heterozygosity analysis in each tumor was performed with a panel of 12 polymorphic markers on chromosomes 1p, 5q, 9p, 9q, 10q, and 17p. The ovarian tumors displayed allelic losses most frequently on 1p (83.3%), 9q (70%), and 17p (41.7%). In the extraovarian implants, allelic losses on 1p, 9q, and 17p were present in 66.7%, 75%, and 66.7% of cases respectively. In five of six cases, allelic losses were 88% concordant between multiple tumor sites. Only one case of stage III tumor displayed a discordant pattern of allelic loss at different tumor sites. Cumulative allelic losses did not show a statistically significant difference in stage I vs. higher stage disease. The pattern and cumulative allelic loss in the two cases with micropapillary architecture was similar to that of the other tumors. We report a high frequency of allelic loss on 1p and 9q that has not been previously reported in SBTs. Morphologically heterogenous areas including benign-appearing, typical borderline, and micropapillary areas had a similar pattern of allelic loss. Although the majority of SBTs seem to be monoclonal, a minor subset may be multiclonal in origin.

Genetic profile of cumulative mutational damage associated with early pulmonary adenocarcinoma: bronchioloalveolar carcinoma vs. stage I invasive adenocarcinoma.

To detect the possible genetic alterations characteristic of bronchioloalveolar carcinoma (BAC) and to study molecular genetic factors responsible for determining the biologic aggressiveness of pulmonary adenocarcinoma, comparative analysis of loss of heterozygosity (LOH) on 9 chromosomal regions was performed in 14 BACs and in 20 stage I adenocarcinomas (AD). The most frequently affected chromosome regions in BAC were 8q and 17p. In stage I AD, more than 60% of the cases showed LOH of 1p, 3p, 5q, 7q, 17p, and 18q loci, and LOH of 1p, 3p, 7q, and 18q was observed with greater frequency than in BAC (P < 0.05). Fractional allele loss (FAL) was significantly greater in stage I AD than in BAC (P < 0.001). In cases with microdissection of multiple sites, intratumoral heterogeneity of LOH status was observed in 73% of BAC and 94% of stage I AD, and homogeneous distribution of LOH of 9p was unique to BAC. The high FAL value was associated with a poor prognosis of BAC, but this trend did not reach statistical significance (P = 0.098). In stage I AD, no correlation was found between LOH of particular chromosomal region or FAL and clinical outcome. LOH of 1p, 3p, 7q, and 18q was associated with invasive properties of pulmonary AD and may be useful in identifying invasive adenocarcinoma when conventional histomorphological tools are not helpful.

Loss of heterozygosity reveals non-VHL allelic loss in hemangioblastomas at 22q13.

Hemangioblastomas (HBs) are low-grade (World Health Organization grade I/IV) central nervous system (CNS) tumors that frequently contain VHL (3p26) mutations. They occur sporadically and in von Hippel Lindau (VHL) disease. Encoded pVHL aids degradation of hypoxia-inducible factors (HIFs) in the presence of normal oxygen levels. HBs provide an in vivo view of HIF effects within a CNS tumor. Typically, HBs are cystic tumors containing a mural nodule formed by noninvasive, vacuolated stromal cells that are embedded in a network of capillaries. Nine HBs, consecutively resected from 8 patients at our institution during a recent 2-year time span, were evaluated for additional losses of tumor suppressor genes. Non-VHL microsatellites studied for loss of heterozygosity (LOH) are near tumor suppressor genes lost in gliomas, pituitary adenomas, several CNS tumors on 22q, neurofibromatosis 1, and colon carcinomas (13, 2, 2, 1, and 2 markers for each, respectively). LOH in the region of 3p21.3-3p26.3 occurred in 3 of 8 HBs informative for at least 1 marker (D3S1539, D3S2303, or D3S2373). By using 2 markers (D22S417 and D22S532) for 22q13.2, LOH was found in 5 of 8 informative HBs. All 3 HBs with allelic losses near VHL also showed LOH at 22q13.2. No consistent losses were found with markers for 1p34, LMYC, 5q21, 5q32, 9p21, 10q23, 17p13, and 19q13. LOH for the 22q13.2 region in HBs suggests that the loss of another tumor suppressor gene is involved in the pathogenesis of HBs in addition to VHL. Absence of LOH for glioma markers is consistent with the low-grade behavior of HBs.

Microdissection genotyping of mixed glial and primitive neuroectodermal central nervous system neoplasm.

A 22-year-old man with previous radiation treatment for childhood astrocytoma underwent resection of a right parietooccipital lesion. Histopathology revealed a malignant neoplasm with areas of astrocytic and primitive neuroectodermal components. To resolve the relationship and cellular origin, representative tissue was microdissected from several targets, obtaining a balanced mixture of each element. Nonneoplastic brain parenchyma was separately microdissected to determine polymorphic marker informativeness and to serve as an internal negative control. Despite the relatively small quantity of tissue removed for each microdissection target, sufficient material was available for reliable, balanced, polymerase chain reaction-format genotyping encompassing a panel of tumor suppressor genes and genetic loci associated with these forms of neoplasia. The findings revealed distinct discordant genotypic profiles for each of the neoplastic components. The efficacy of the approach used for molecular analysis of this complex neoplasm and the implication of the genotypic findings are discussed.

Loss of heterozygosity patterns of sclerosing hemangioma of the lung and bronchioloalveolar carcinoma indicate a similar molecular pathogenesis.

CONTEXT: The histogenesis and origin of sclerosing hemangioma (SH) of lung were uncertain for many years. Many immunohistochemical, ultrastructural, and recent molecular studies support the hypothesis that SH is a neoplasm originating from the cells of the terminal lobular unit, similar to the nonmucinous variant of bronchioloalveolar carcinoma (BAC). Most cases of SH are benign, but they can metastasize to the regional lymph nodes. OBJECTIVE: To compare the patterns of allelic loss of tumor suppressor genes in SH and BAC by microdissection-based genotypic analysis. DESIGN: Microdissection-based loss of heterozygosity analysis of 9 cases of SH and 14 cases of BAC, using a panel of 7 polymorphic microsatellite markers located on 1p, 5q, 9p, 10q, and 17p. Microsatellite marker and chromosomal arm-based fractional allelic loss (FAL) were calculated in each case. RESULTS: Our results showed similar patterns of allelic loss between the 2 groups of tumors on an individual case basis. Chromosomal arms 5q and 10q showed frequent allelic loss in SH (66.7% and 62.5%, respectively), whereas in BAC, chromosomal arm 17p (52.6%) was frequently affected. A statistically significant difference in allelic loss between SH and BAC was located only on chromosomal arm 5q (P =.04). Microsatellite marker D5S615 was significantly more frequently affected in SH than in BAC (66.7% vs 28.6%; P =.04). CONCLUSION: Our molecular data support the hypothesis of common origin of SH and BAC. A putative tumor suppressor gene that might play a role in tumorigenesis of SH may be located on the chromosomal arm 5q.

Microdissection genotyping of gliomas: therapeutic and prognostic considerations.

Molecular anatomic pathology represents the blend of traditional morphological methods and the multigene approach to determine cancer-related gene alterations for diagnostic and prognostic purposes. Microdissection genotyping was utilized to characterize 197 gliomas with targeted microdissection of 2-7 areas spanning the spectrum of histologic types and grades. The methodology described herein is complementary to the existing realities of pathology practice. The technique utilizes paraffin-embedded fixative-treated tissue of small sample size after the primary morphological examination by the pathologist. Molecular information derived from microdissection genotyping in combination with the traditional histological information, results in an enhanced understanding of glioma formation and biological progression leading to improvements in diagnosis and prediction of prognosis. In all, 100% or 32 of 32 cases with at least partial treatment response was observed in neoplasms possessing the 1p or 1p/19q loss. The 19q loss alone without coexisting 1p showed no improvement in treatment response. Gliomas lacking 1p loss with only allelic loss involving 3p, 5q, 9p, 10q and 17p showed unfavorable outcome of only 35%, or six of 17 cases with treatment response. In addition, the determination of fractional allelic loss (favorable/unfavorable), was a very good independent predictor of biological behavior. These findings emphasize the importance of determining the cumulative pattern of mutational damage on 16 distinct sites or more, especially in the presence of 1p loss which in isolation or in combination with 19q is a favorable prognostic factor for therapeutic response.

Allelic loss of tumor suppressor genes in ameloblastic tumors.

Ameloblastoma is an odontogenic tumor with a variety of histologic appearances and an unpredictable biologic behavior. Little is known about allelic losses of tumor suppressor genes in ameloblastomas. This study surveyed DNA damage in ameloblastomas and correlated this with histologic sub-type and clinical outcome. There were 12 ameloblastomas (two peripheral, eight solid, and two unicystic) and three ameloblastic carcinoma studied for loss of heterozygosity of tumor suppressor genes on chromosomes 1p, 3p, 9p,10q, and 17p (L-myc, hOGG1, p16, pten, and p53). The frequency of allelic loss and the intratumoral heterogeneity were calculated. L-myc (71% frequency of allelic loss) and pten (62% frequency of allelic loss) had the most frequent allelic losses. Overall frequency of allelic loss and intratumoral heterogeneity were higher in mandibular and in unicystic tumors and lower in tumors that recurred/metastasized. The rate of allelic loss in the three carcinomas was similar to that seen in benign tumors. The frequency of allelic loss and intratumoral heterogeneity did not correlate with age, gender, histologic subtype, or prognosis. Since tumors that behaved aggressively did not harbor more allelic losses, it is likely that DNA damage in ameloblastomas and ameloblastic carcinomas is sporadic and cumulative. We conclude that other genetic or epigenetic mechanisms may be responsible for malignant behavior in ameloblastic carcinomas.

Microdissection-based genotyping assists discrimination of reactive gliosis from glioma.

We used molecular anatomic pathology to determine the mutational status (loss of heterozygosity [LOH]) to make the distinction between reactive gliosis and glial neoplasia. LOH has been shown to be absent in reactive states and present in neoplastic cellular proliferations. Three groups of patient specimens were analyzed: group 1, reactive gliosis (n = 15); group 2, gliomas of varying histologic type and grade (n = 54); group 3, diagnostically challenging reactive gliosis vs glioma (n = 16). No group 1 cases (0/15 [0%]) showed allelic loss, whereas all high-grade glial neoplasms, a subset of group 2 (35/35 [100%]) manifested at least 1 allelic loss alteration, with most cases (33/35 [94%]) displaying 2 or more such changes. During a look forward at the group 3 patients, clinical history clarified the problematic diagnosis in a subset of 11 patients: 8 (73%) of 11 clinical outcomes were predicted correctly by our analysis. The molecular anatomic pathology approach outlined herein is designed for minute, formalin-fixed, paraffin-embedded specimens, which are encountered in everyday surgical pathology practice. Molecular anatomic pathology opens the possibilities of molecular analysis to everyday pathology practice.

Absence of Mycobacterium avium subsp. paratuberculosis in the microdissected granulomas of Crohn's disease.

The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.

Tumor suppressor gene allelic loss profiles of the variants of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTCa) is a relatively common, indolent tumor that usually has an excellent prognosis. While the diagnosis of conventional PTCa is relatively straightforward, encapsulated tumors with follicular growth pattern and unusual or incomplete cytologic features of papillary carcinoma can be diagnostically challenging. Encapsulated, noninvasive tumors are particularly controversial as the differential diagnosis includes a nonneoplastic nodule, a benign follicular adenoma, and papillary carcinoma. In this study, we performed molecular genotyping to identify loss of heterozygosity of tumor suppressor genes in conventional PTCa and in several different morphologic variants, including follicular variant, tall cell variant, and oncocytic variant. Our data demonstrate that conventional PTCas have the lowest frequency of allelic loss (7%), as compared with follicular, oncocytic, and tall cell variants (19%, 34%, and 20%, respectively). Frequency of allelic loss increased with increasing size of the tumors, but did not correlate with age, gender, extrathyroidal extension, or lymph node metastases. Though it is unlikely that these results will enable the distinction between different variants of papillary carcinoma, the finding of significant rates ofallelic loss in the variants of PTCa provides additional evidence of malignancy and may be useful in distinguishing encapsulated tumors from nonneoplastic or benign nodules.

Inflammatory fibroid polyps of the gastrointestinal tract: clinical, pathologic, and molecular characteristics.

Inflammatory fibroid polyp (IFP) of the gastrointestinal tract is an uncommon proliferative lesion. When sampled by biopsy, IFP can be mistaken for various lesions, from granulation tissue to high-grade sarcoma. We present an unusual case of IFP and review a large series of IFPs to characterize clinical, histologic, and molecular features of diagnostic value. A total of 42 IFPs were gathered from the pathology archives of the University of Pittsburgh Medical Center over the past 22 years. Clinical, histopathologic, and immunohistochemical features were collected. A random subset of IFPs (n = 12) underwent microdissection genotyping for a broad panel of tumor suppressor gene-associated mutations (loss of heterozygosity). IFPs occurred in both sexes (male, 17; female, 25) over a broad age range (29-85 years). IFPs varied in size from 0.2 to 8 cm. The stomach (n = 19) was the most common location, followed by large bowel (n = 13) and small bowel (n = 10). Most IFPs displayed typical morphologic features (eosinophils admixed with loose, mature fibrous tissue), and in 2 instances, sampled by biopsy, IFP was confused for sarcoma. All IFPs lacked c-kit staining. No mutations were identified in any IFPs examined. IFP is a clinically underrecognized entity with unique morphologic and immunohistochemical features. On biopsy alone, the differential diagnosis may include sarcoma and other malignancies. The absence of mutational change may help to exclude malignant lesions.

Microdissection genotyping of archival fixative treated tissue for Gaucher disease.

The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-microm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tis sue is available for molecular genotyping.

Molecular profiling of primary and metastatic neoplasms in the lung using cytologic material obtained by fine-needle aspiration: report of two cases.

Two cases are presented in which molecular analyses of cytologic material obtained by fine-needle aspiration were helpful in establishing relationships between morphologically similar neoplasms in the same patient. For appropriate clinical management, it is important to ascertain whether the tumors represent independent primaries or metastases. Alcohol-fixed cytologic material prepared as cell blocks and formalin-fixed paraffin-embedded tissue were microdissected and analyzed for allelic loss of heterozygosity at multiple preselected genetic loci. The first case illustrates a 69-yr-old man with multiple intrapulmonary nodules involving the upper and lower lobes of the left lung. Genomic analysis showed that the neoplasms in the left upper and lower lung lobes were independent primaries, because the loss of heterozygosity (LOH) patterns were substantially different. By contrast, the second case is that of a 58-yr-old man with a right thyroid nodule and multiple pulmonary tumors. LOH analysis confirmed that a sampled pulmonary tumor represented a metastasis from the thyroid primary, as similar LOH patterns involving locus D9S252 were observed on comparison of the thyroid and pulmonary neoplasms. These cases illustrate the practical diagnostic utility of genomic analysis using cytologic material in the assessment of primary and metastatic malignancies.

Pulmonary meningothelial-like nodules: a genotypic comparison with meningiomas.

BACKGROUND: Minute pulmonary meningothelial-like nodules (MPMNs) are incidental interstitial pulmonary nodules. They share histologic, ultrastructural, and immunohistochemical features with meningiomas (MGs). DESIGN: Sixteen cases yielding 33 separate MPMNs and 10 cases of benign MG were studied. Immunohistochemical studies and mutational analyses were performed on microdissected tissue using 20 polymorphic microsatellite markers targeting 11 genomic regions in an effort to identify genetic similarities of MPMN and MG. RESULTS: A total of 96.6% of MPMNs stained positive for vimentin, 33.3% for epithelial membrane antigen, 3% for S-100, and all were negative for cytokeratin and synaptophysin. Loss of heterozygosity (LOH) was identified in 25% of single MPMN affecting 3 genomic loci. No solitary MPMN had more than 1 LOH event. Multiple LOHs were seen only in MPMN-omatosis syndrome, where 33.3% of MPMNs showed LOH affecting 7 genomic loci. MG showed the highest frequency of LOH with major events seen at 22q (60%), 14q (42.8%), and 1p (44.4%) that were not shared by MPMN. CONCLUSION: Isolated MPMN lacks mutational damage, consistent with a reactive origin. MPMN-omatosis syndrome might represent the transition between a reactive and neoplastic proliferation. MPMNs are different from MG based on the major molecular genetic events seen in their formation and progression.

Molecular analysis to demonstrate that odontogenic keratocysts are neoplastic.

CONTEXT: Odontogenic keratocysts (OKCs) are unique odontogenic lesions that have the potential to behave aggressively, that can recur, and that can be associated with the nevoid basal cell carcinoma syndrome. Whether they are developmental or neoplastic continues to be debated. OBJECTIVES: To identify loss of heterozygosity of tumor suppressor genes in OKCs and to suggest a pathogenetic origin for these lesions. DESIGN: We examined 10 OKCs for loss of heterozygosity of tumor suppressor genes, using a microdissection and semiquantitative genotyping analysis. The genes analyzed included 10 common tumor suppressor genes, as well as the PTCH gene, which is mutated in nevoid basal cell carcinoma syndrome. RESULTS: Loss of heterozygosity was seen in 7 of 10 cases, with a frequency between 11% and 80% of the genes studied. The genes that exhibited the most frequent allelic losses were p16, p53, PTCH, and MCC (75%, 66%, 60%, and 60%, respectively). Daughter cysts were associated with a higher frequency of allelic loss (P =.02), but epithelial budding was not. CONCLUSIONS: Our study indicates that a significant number of OKCs show clonal loss of heterozygosity of common tumor suppressor genes. The finding of clonal deletion mutations of genomic DNA in these cysts supports the hypothesis that they are neoplastic rather than developmental in origin.

Molecular genotyping of medullary thyroid carcinoma can predict tumor recurrence.

Medullary thyroid carcinoma can have an aggressive behavior, and little is known about the molecular basis for clinical outcome. Defining risk of recurrent or metastatic disease is difficult, and it has been limited to clinical and pathologic features, such as advanced age, cervical lymph node metastases, and stage at presentation. Using microdissection and genotyping, we studied 11 cases of medullary carcinoma for allelic losses in a panel of known tumor suppressor genes. The tumor suppressor genes with the most frequent allelic losses were NF2, l-myc, and p53 (75%, 44%, and 44%, respectively). The average frequency of allelic loss across all tumors was 44% and was higher in tumors that recurred. A combination of previously described high-risk variables (increased patient age and cervical lymph node metastases) with the frequency of allelic loss yielded a high-risk group, in which 6 of 6 patients recurred, and a low-risk group, in which 0 of 5 patients recurred (P = 0.004). Frequency of allelic loss in tumor suppressor genes may provide a useful adjunctive prognostic test in medullary thyroid carcinoma.

Comparative molecular analysis of loss of heterozygosity in adenocarcinoma in bile duct brushings and corresponding surgical pathology specimens.

BACKGROUND: Bile duct brushing is the procedure of choice for the assessment of neoplasia of the biliary and pancreatic ducts. Conventional cytopathologic evaluation has been reported to have high specificity but relatively low sensitivity. Although a number of molecular studies regarding biliary tract tissue specimens have been performed, to the authors' knowledge their precise applicability to cytopathology specimens has not been critically analyzed. METHODS: Bile duct brushing specimens with the cytopathologic diagnosis of "suspicious" or "positive for malignant cells" along with corresponding surgical pathology specimens demonstrating adenocarcinoma were searched for in the files of UPMC-Presbyterian Hospital for the years 1990-1996. Tumor cells from representative cytopathology and histology slides were microdissected and analyzed for loss of heterozygosity (LOH) in a panel of microsatellite markers. The results obtained from cytopathologic and surgical pathology specimens were compared. RESULTS: Eight paired surgical and cytopathology cases of adenocarcinoma involving the biliary tract were identified. The fractional allelic loss (FAL) for the surgical specimens (FAL-S) ranged from 12.5-71.4% and the FAL for the cytopathology specimens (FAL-C) ranged from 25-71.4%. However, when evaluating the actual loci of LOH, the concordance rate of the surgical and cytopathology specimens ranged from 71.4-100% (mean, 88.6%). Only 3 of the 8 cases (37.5%) were found to have identical matching of the LOH loci. CONCLUSIONS: Although the overall concordance rate of LOH in biliary cytology and surgical specimens by molecular analysis is relatively high, the issue of molecular tumoral heterogeneity must be considered if clinical decisions are to be based exclusively on cytopathologic analysis.