Methylation markers differentiate thyroid cancer from benign nodules.

PURPOSE: The incidence of thyroid cancer (TC) is increasing. Cytology by itself cannot distinguish TC from some benign nodules especially in certain subtypes of TC. Our immediate goal is to identify DNA methylation markers for early detection of TC and to molecularly differentiate TC subtypes from benign nodules. METHODS: Promoter methylation status of 21 candidate genes was examined on formalin-fixed paraffin-embedded tissue (FFPE) utilizing quantitative methylation-specific polymerase chain reaction (QMSP) in a retrospective cohort of 329 patients (56% white, 29% African American, 61% female) comprising 71 normal thyroid, 83 benign nodules [follicular adenomas (FA)], 90 follicular TC (FTC) and 85 papillary TC (PTC). All genes were analyzed individually (Kruskal-Wallis and Wilcoxon rank sum tests) and in combination (logistic regression models) to identify genes whose methylation levels might best separate groups. RESULTS: Combination gene panels TPO and UCHL1 (ROC = 0.607, sensitivity 78%) discriminated FTC from FA, and RASSF1 and TPO (ROC = 0.881, sensitivity 78%) discriminated FTC from normal. Methylation of TSHR distinguished PTC from FTC (ROC = 0.701, sensitivity 84%) and PTC from FA (ROC = 0.685, sensitivity 70%). The six gene panel of TIMP3, RARB2, SERPINB5, RASSF1, TPO and TSHR, which differentiates PTC from normal thyroid, had the best combination sensitivity (91%) and specificity (81%) of the panels addressing discrimination of cancer tissue. CONCLUSIONS: Aberrant gene methylation used in combination panels may be useful clinically in differentiating FTC and PTC from benign nodules. If confirmed in additional studies, these findings could help reduce the over diagnosis of thyroid cancer and surgeries related to over diagnosis.

Targeted Theranostic Approach for Glioma Using Dendrimer-Based Curcumin Nanoparticle.

The delivery of anti-cancer agents to brain tumors represent a challenge because the blood-brain tumor barrier (BBTB) effectively limits the delivery of many agents. A new generation 3 (G3) dendrimer-based curcumin (Curc) conjugate was synthesized. The synthesized G3-Curc conjugate demonstrated full solubility in aqueous media. The in vitro study revealed that G3-Curc nanoparticles were internalized into glioma U-251 cells. Systemic delivery of G3-Curc conjugate led to preferentially accumulation in an orthotopic preclinical glioma model minimizing systemic toxic effect. Multicolor microscopy images of the tumor tissue showed that G3-Curc particles were internalized inside tumor cells selectively and further localized within nuclei. Enhanced bioavailability of G3-Curc conjugate was also observed with improved therapeutic efficacy against different cancers cells.

Malignant papillary renal tumors with extensive clear cell change: a molecular analysis by microsatellite analysis and fluorescence in situ hybridization.

CONTEXT: Histologic subtyping of renal cell carcinomas (RCCs) is based not only on cytoarchitectural pattern but also on distinct cytogenetic abnormalities. Some renal tumors demonstrate overlapping morphologic features, rendering histologic subtyping difficult. One such group of tumors is papillary renal neoplasms with extensive clear cell change. Because histologic subtyping has been shown to be of prognostic value, it is important that malignant epithelial renal tumors be accurately subtyped. It is not known if these tumors should be classified as papillary RCC (PRCC) or as conventional/(clear cell) RCC (CRCC). OBJECTIVE: To ascertain if this subgroup of renal neoplasms demonstrates the cytogenetic abnormalities seen typically in PRCC, that is, trisomy 7 and 17 or CRCC, that is, loss of 3p, using microsatellite analysis for loss of heterozygosity (LOH), and fluorescence in situ hybridization (FISH) for trisomies. DESIGN: Seven RCCs from 6 patients that showed more than 75% papillary architecture and more than 75% clear cell change were included in the study. Tumor size ranged from 2.5 to 7.0 cm (mean 4.7 cm) and all were confined to the kidney (stage I). DNA was extracted from formalin-fixed paraffin-embedded tissue. FISH was done using In Situ Kits for centromere probes for chromosomes 7 and 17. For LOH, microsatellite analysis using labeled primers for 4 markers in the 3p13 through 3p24.2 region were used. The amplified polymerase chain reaction products were analyzed using an automated DNA sequencer. As compared with normal DNA, LOH in tumor was recognized as a loss of 1 allele, and microsatellite instability as the addition of an extra allele. RESULTS: LOH in at least 1 of the markers spanning for 3pl3 through 3p24.2 was detected in 6 of 7 specimens (86%), of which 1 also showed concomitant microsatellite instability. FISH did not demonstrate trisomy for either chromosome 7 or 17. Instead, monosomy 7 was observed in 4 of 6 tumors (67%) and monosomy 17 in all tumors (100%). CONCLUSION: Because malignant papillary renal tumors with extensive clear cell change show molecular changes identical to CRCC, this subgroup of tumors may have to be classified as CRCC. This study underscores the utility of molecular studies in refining light-microscopic criteria in accurate histologic subtyping of RCCs.

Loss of sequences on the short arm of chromosome 17 is a late event in squamous carcinoma of the cervix.

AIMS: To determine by fluorescence in situ hybridisation (FISH) whether deletion of D17S34, a subtelomeric probe for 17p, occurs in invasive squamous carcinoma of the cervix, and to determine the extent of such loss by analysis of the p53 and HER2/NEU genes. METHODS: Fourteen invasive squamous cell carcinomas of the cervix were investigated by FISH for D17S34, p53, and HER2/NEU. Dual hybridisation of each probe with the chromosome 17 alpha satellite (D17Z1) probe was performed on paraffin wax embedded sections, and the fluorescence ratios of the paired signals were determined. Broad spectrum human papillomavirus (HPV) typing by ISH and GP5+/6+ polymerase chain reaction was also performed. RESULTS: Twelve tumours were HPV positive, nine with HPV-16, and one each with types 18, 31, and 39. Loss of D17S34 was identified in four tumours, one of which was HPV negative. In one tumour, D17S34 loss was accompanied by loss of p53 only, suggesting that deletion was limited to the p arm. A second tumour showed simultaneous losses of all probes, indicative of whole chromosome 17 loss during tumour growth. The two remaining specimens showed loss of D17S34 only, diffuse in one, and localised within the tumour in the other. Aberrations of p53 or HER2/NEU were not seen independently of D17S34 loss, and loss did not correlate with HPV presence or type. CONCLUSIONS: These data show that D17S34 loss is prevalent, marking 28% of the invasive squamous carcinomas in this study. The observed intratumoral heterogeneity indicates that, at least in some cases, this loss occurs after invasion and is therefore a late event in the path of cervical carcinogenesis.

Loss of nm23 expression predicts distal metastases and poorer survival for breast cancer.

The nm23 gene, originally identified by differential hybridization of metastatic murine melanoma cell lines has been associated with decreased metastatic potential. In this study, we evaluated the utility of nm23 expression levels as a predictive and a prognostic biomarker for distal metastases and poor survival in a Chinese cohort of 168 breast cancer patients. Our study indicates that high nm23 expression is associated with older age (older than 35 years) and smaller tumor size. There is no statistically significant association between nm23 expression and pathologic type or axillary metastasis. High nm23 expression is associated with the absence of distal metastases. Nearly 80% of women with high nm23 expression are alive after 10 years compared to 25% with low expression; at five years the cumulative survival proportions are 86% and 46%, respectively. The Cox model for survival indicates that controlling for tumor size and presence of axillary metastases at diagnosis, the hazard for women with low nm23 expression is nearly 4 times that of women with high nm23 expression. Low nm23 expression is predictive of distal metastases and appears to be a risk factor that is independent of the presence or absence of positive axillary nodes at diagnosis.

Detection of a single base substitution in a single cell using the LightCycler.

For known mutations, real time polymerase chain reaction followed by melting curve analysis, using hybridization probes, is highly sensitive, rapid and an efficient approach to mutation detection. We have used this approach on the LightCycler for the detection of single base mutations in a single cell, without nested PCR. Hybridization probes were designed for two sequences in the BRCA1 gene containing a single base substitution and deletion, respectively. Polymerase chain reactions of small fragments (100-200 bp) containing the probe sequences were optimized using SYBR Green1, before using hybridization probes. The 5'-probes were 3'-labeled with FITC, whereas the 3'-probes, covering the mutation, were 5'-labeled with LC-Red640 (wild type probes) or LC-Red705 (mutant probes). Dual color detection of wild type and mutant sequences in a single tube was tested on single cells. The reaction mix was prepared in reaction capillaries and a single cell, picked by micromanipulation, was added to this mix. The DNA from the cell is released during the 5-min preheating step of the PCR, using the FastStart hybridization kit (Roche). Reproducible results were obtained, without the need of nested PCR. The technique is useful for microdissected tumors and, with other genes, has great potential for pre-implantation diagnosis in IVF and analysis of residual disease in cancer.

Significance of p53 mutations in patients with chronic lymphocytic leukemia: a sequential study of 30 patients.

BACKGROUND: In patients with B-cell chronic lymphocytic leukemia (CLL), considerable disease heterogeneity within clinical stages necessitates the search for relevant prognostic indicators, particularly those that may help to determine the need for early therapeutic intervention. In the current study, the authors investigated the role of p53 mutations and chromosomal abnormalities in 30 patients with CLL. METHODS: Thirty patients were screened for p53 mutations. Half of the group had aggressive disease characterized by leucocytosis, lymph node enlargement, organomegaly, and shortened tumor doubling time. Because 95% of p53 mutations reside in "hot-spot" regions of exons 5-9 of the p53 gene, the authors sequenced these exons completely for mutation detection. RESULTS: Sequence analysis identified p53 mutations in 14 of 30 patients that were distributed equally among patients with aggressive disease and nonaggressive disease. There were six mutations in exon 7, five mutations in exon 5, and one mutation each in exons 6 and 8. Five of 15 patients with clinically aggressive disease had mutations in exon 7. Only one patient with nonaggressive disease had an exon 7 mutation. Abnormal cytogenetics were present in 22 of 30 patients (73%). Most patients with the p53 mutation (13 of 14 patients; 93%) displayed abnormal cytogenetics. Twelve of 15 patients with aggressive disease and 9 of 15 patients with average disease exhibited abnormal karyotypes. CONCLUSIONS: The presence of p53 mutations did not predict clinical behavior or disease outcome, although the frequency of mutations appears to be higher than reported previously. In this study, mutations of exon 7 (5 of 6 patients) occurred in patients with clinically aggressive disease. The significance of this observation warrants further examination.

Molecular approaches to identification of tissue contamination in surgical pathology sections.

The finding of possibly contaminant tissues or cells in surgical or cytology case material can be a challenging problem in diagnostic anatomical pathology samples. The reported rates of occurrence have ranged from 0 to 8.8% (including prospective and retrospective cases). A diagnostically dissimilar tissue fragment, whether contiguous with other tissue or among other fragments within a paraffin section, and which is not incompatible with the case tissue, often requires a rigorous investigation to confirm or deny its relevance to the case. Fluorescence in situ hybridization using dual red and green DNA probes to regions of the X and Y chromosomes, respectively, were used in one case where the potential contaminant was suspected to have originated from a male patient. The putative contaminant tissue fragment was confirmed as male, with cells having one X and one Y chromosome, unlike the other tissue fragments on the slide with two X chromosomes. In a second case, DNA polymorphisms were used to compare allelic patterns that were informative not only in proving the extraneous tissue as a contaminant, but in addition, could be used to trace the latter to its original tissue source. The molecular tools of fluorescence in situ hybridization in sex-mismatched cases and of DNA microsatellite probes that are applicable to paraffin sections can provide definitive identifiers of tissues and individual cells. They are important adjuncts to histology for the anatomical pathologist when faced with the diagnostic problems of tissue contamination encountered in routine practice.

Patterns of allelic loss at the BRCA1 locus in Arabic women with breast cancer.

Loss of heterozygosity (LOH) of BRCA1, a tumor suppressor gene, is one mechanism of genetic inactivation in both sporadic and familial forms of breast cancer. Studies reported in breast cancers from women of Northern European descent have shown LOH in 30-50% of sporadic tumors. Microsatellite instability (MSI) has served as evidence for involvement of DNA repair genes. This study investigates the extent of allelic imbalance at the BRCA1 region in Arabic women with breast cancer. Paired normal and tumor tissue were available for DNA analysis in 13 cases. Results using fluorescent tagged primers to microsatellite markers D17S1323, D17S1325 and D17S855 intragenic to BRCA1 were analyzed using an ABI 310 DNA sequencer. As compared to normal DNA, MSI and LOH were recognized as a gain and a loss, respectively, of one signal in one allele in the tumor DNA. Microsatellite analyses showed 12 of 13 (92%) cases with LOH or MSI or both. Three cases demonstrated LOH alone, 3 cases with MSI alone. Six cases indicated both LOH and MSI; 2 cases with either LOH or MSI in separate markers. The combined finding of LOH and MSI in the same marker was detected only with D17S1325 in 4/6 cases. The proportion of aberrant findings of the BRCA1 locus in breast cancer appears to be higher in Arabic women than in other populations studied to date.

A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analyses, using BRCA1 as an example.

BACKGROUND: The detection of 2 recurrent mutations in the BRCA1 gene (Ashkenazi Jewish and Dutch populations) was studied with real-time polymerase chain reaction (PCR) and melting curve analyses. METHODS: PCR products were designed around the 185delAG in exon 2 and the single- base substitution 2841G>T in exon 11. Hybridization probe sets were designed for both PCR products, with each probe overlapping the specific mutation. The exon 11 probe set also covered another mutation, the 2814insA. The 39 end of the 59 probe was labeled with fluorescein isothiocyanate and the 59 end of the 39 probe with LightCycler Red 640 (Roche Diagnostics, Indianapolis, IN). RESULTS: The 185del and 2841G mutations were easily detected with the hybridization probes, resulting in dual peaks for heterozygotes in melting curve analyses. The differences in melting characteristics of the heteroduplexes in heterozygotes were not detectable with SYBR Green I. CONCLUSION: For known mutations, melting curve analyses using hybridization-specific probes provide a sensitive, rapid, and efficient approach to mutation detection.

Race and differences in breast cancer survival in a managed care population.

BACKGROUND: African-American women with breast cancer have poorer survival than European-American women. After adjustment for socioeconomic variables, survival differences diminish but do not disappear, possibly because of residual differences in health care access, biology, or behavior. This study compared breast cancer survival in African-American and European-American women with similar health care access. METHODS: We measured survival in women with breast cancer who are served by a large medical group and a metropolitan Detroit health maintenance organization where screening, diagnosis, treatment, and follow-up are based on standard practices and mammography is a covered benefit. We abstracted data on African-American and European-American women who had been diagnosed with breast cancer from January 1986 through April 1996 (n = 886) and followed these women for survival through April 1997 (137 deaths). RESULTS: African-American women were diagnosed at a later stage than were European-American women. Median follow-up was 50 months. Five-year survival was 77% for African-American and 84% for European-American women. The crude hazard ratio for African-American women relative to European-American women was 1.6 (95% confidence interval [CI] = 1.1-2.2). Adjusting only for stage, the hazard ratio was 1.3 (95% CI = 0.9-1.9). Adjusting only for sociodemographic factors (age, marital status, and income), the hazard ratio was 1.2 (95% CI = 0.8-1.9). After adjusting for age, marital status, income, and stage, the hazard ratio was 1.0 (95% CI = 0.7-1.5). CONCLUSION: Among women with similar medical care access since before their diagnoses, we found ethnic differences in stage of breast cancer at diagnosis. Adjustment for this difference and for income, age, and marital status resulted in a negligible effect of race on survival.

Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by fluorescence in situ hybridisation.

AIMS: Chromosomal aberrations in tumour cells are often not discernable by direct analysis. Although cell culture allows qualitative analysis of the karyotype, potential selection and evolution during growth in vitro may yield misleading data. To determine whether aberrations observed in vitro are representative of the original lesion, chromosomal aberrations found after prolonged growth in vitro of two squamous cell carcinomas of the head and neck (SSCHN) were evaluated with fluorescence in situ hybridisation (FISH) on the original tumour nuclei. METHODS: Specific karyotypic aberrations identified in cultures of two squamous cell carcinomas were targets for FISH analysis on tumour sections. Chromosome painting mixtures were selected based on in vitro karyotypic data. FISH was performed on cultured interphase and metaphase cells, and on histological sections from the original tumours. RESULTS: The 9cen and 17cen probes yielded FISH signals consistent with the aneusomies predicted for the respective chromosomes from the culture karyotypes. Whole chromosome 9 paint confirmed the prior existence in the tumours of i(9p) and i(9q), although only the latter hybridised with the 9cen probe. FISH data also supported in vivo representation of the diploid and tetraploid tumour subclones observed in cultures. In tumour HFH-SCC-8a, FISH results were generally concordant between cultured interphase and metaphase cells and the histological sections, and improved the interpretation of marker chromosomes identified in culture. CONCLUSION: The karyotypes obtained in these cases after prolonged passage in culture were consistent with the genetic alterations in the original tumours.

Enhanced assessment of allogeneic bone marrow transplant engraftment using automated fluorescent-based typing.

Traditional qualitative gel electrophoresis approaches lack accurate' and quantitative assessment of mixed chimerism in BMT patients. The likelihood of informative markers is greatly increased using simultaneous amplification of 10 highly polymorphic loci with fluorescent-labeled primers in an automated DNA sequencer. This allows for more precise interpretation of mixed chimerism with a detection level approximating 1%. To evaluate this approach to quantitative assessment of chimeric populations we mixed varying proportions of samples from two unrelated donors, by either mixing aliquots of DNA isolated from whole blood, or by first counting the white blood cells and mixing varying proportions of cells together prior to DNA isolation. The allelic-peak area ratios were identical to allelic-peak height ratios and corresponded to the proportion of mixed DNA, regardless of the method used to create the mixture. Formulas to provide routine, consistent and quantitative interpretation of mixed chimerism are presented. We analyzed 14 allograft recipients and one autologous BMT patient with transfusion-induced GVHD. In all cases, at least four out of nine markers were informative. Inter-laboratory concordance of results was also obtained with an eight marker panel using an automated Alf-Express. In conclusion, the automated DNA fluorescent-labeled primer approach using an eight to 10 marker panel is quantitative and informative in assessing chimerism.

A new BRCA1 mutation in a Filipino woman with a family history of breast and ovarian cancer.

Mutation of the BRCA1 gene in well-defined breast cancer families has been associated with an 87% lifetime risk for breast cancer and a 44% risk for ovarian cancer. Recent data indicate that the risk associated with these mutations is considerably lower, although still far greater than the risk for disease in the rest of the population. Approximately 81% of the mutations that have been identified have been frameshift (71%) or nonsense (10%) mutations, and either may result in a truncated protein. The protein truncation test (PTT) is often used to screen patients at high risk, because sequencing of this large (100 kb) gene with its 22 coding exons is an arduous task. The PTT was used to analyze genomic DNA and RNA from the peripheral blood of a 31-year-old Filipino woman with a poorly differentiated, stage 2A breast carcinoma and a family history of breast-ovarian cancer. PTT identified the wild-type protein fragment and an additional truncated protein fragment in the patient's sample. Subsequent direct sequencing of the appropriate coding region revealed a point mutation in exon 11 at nucleotide 2178, resulting in a C > T transition that caused a termination (stop codon) in amino acid 687. To our knowledge, this is the first report of mutation of the BRCA1 gene in a Filipino family, and this in-frame stop-codon mutation has not been reported previously.

Frequent loss of heterozygosity on chromosome arm 18q in squamous cell carcinomas. Identification of 2 regions of loss--18q11.1-q12.3 and 18q21.1-q23.

OBJECTIVES: To determine the frequency and regions of loss on chromosome arm 18q in uncultured head and neck squamous cell carcinomas. DESIGN: Polymerase chain reaction amplification of DNA extracted from 18 tumor specimens (1 patient had 2 tumors) and blood samples from 17 patients with head and neck squamous cell carcinoma was performed using primers flanking 16 microsatellite repeat polymorphisms spanning most of chromosome 18q. DNA was extracted only from specimens with greater than 70% tumor nuclei. SETTING: Research university. PATIENTS: Seventeen individuals with newly diagnosed head and neck cancer. MAIN OUTCOME MEASURE: Loss of heterozygosity (LOH). RESULTS: There was LOH at more than 1 locus in 52% (9/ 17) of the tumors; 3 tumors had LOH at all informative markers. Four had loss at only 1 locus, raising the total with loss to 12 (75%) of 16. Loss of 18q11.1-q12.3 in 4 tumors without distal loss defines a proximal region of loss. Loss of heterozygosity affecting 18q21.1 in 1 tumor, without proximal loss and LOH for 18q21.1, 18q22, or 18q23 in 9 (52%) of 17 tumors defines a distal region of loss. CONCLUSIONS: Loss of heterozygosity on chromosome arm 18q is not an artifact of in vitro culture. The finding of 18q LOH in 50% to 70% tumors makes 18q an important region for study. Regions 18q11.1-q12.3 and 18q21.1-q23 are common regions of loss, indicating that there may be more than one 18q tumor suppressor gene involved in the genesis and progression of head and neck squamous cell carcinomas.

Identifying genetic changes associated with tumor progression in squamous cell carcinoma.

Tumor behavior is the result of specific genetic changes that alter gene expression. From our cytogenetic studies chromosome 18 loss emerged as a common genetic change in squamous carcinoma cell lines. In this report we summarize data that link loss of 18 to tumor progression and reduced survival, indicating that one or more tumor suppressor gene(s) are located on this chromosome. Tumors grown in vitro were karyotyped either as short-term or permanent cultures. Loss of chromosome 18 was measured by karyotype, decreased frequency of heterozygosity at the DCC locus, and loss of heterozygosity (LOH) for microsatellite repeat polymorphisms (MSRP). Loss of any part of chromosome 18 was observed in approximately 63% of cultured tumors. Primary and secondary tumors from the same individuals sometimes differed in loss of 18 indicating that this genetic change is associated with tumor progression. Heterozygosity for DCC was present in only 3/19 cultured SCC (16%), compared with 68% (11/16) of blood samples from unrelated donors, which is consistent with LOH in roughly one half of the cases. Of 4 informative cases with normal and tumor tissue, LOH was observed in 2. Microsatellite analysis also shows loss of 18q in 55% of fresh tumors. Analysis of tumor tissue and cell lines from the same patient gave identical results. There was an excess of deaths from cancer in the group with 18 loss (20/25) when compared with the group without (5/15). Loss of chromosome 18 appears to be a marker of tumor progression in SCC. It is likely that mutation affecting DCC or another gene on 18 affects tumor growth or spread, leading to more rapid progression and reduced survival.

Two regions of homozygosity on chromosome 3p in squamous cell carcinoma of the head and neck: comparison with cytogenetic analysis.

BACKGROUND: Loss effecting the short arm of chromosome 3 occurs in nearly 60% of squamous cell carcinomas of the head and neck (SCCHN). Karyotype analysis indicated that these losses occur in two regions, 3p13-p14 and 3p21-p24. To test these findings, we examined tumor DNA from 38 SCCHN cell lines for heterozygosity and homozygosity at 6 polymorphic loci spanning this region. METHODS: The polymerase chain reaction (PCR) was used to amplify polymorphic restriction sites on 3p, the amplified products were digested with the appropriate restriction enzyme, electrophoresed on agarose gels, and assessed for the presence of one or both alleles. The 38 SCCHN cell lines were established from 31 patients and included 16 that had been karyotyped. In 6 cases two or three tumor cell lines established from separate tumors in the same patients were studied. RESULTS: The cell lines exhibited a very low frequency of heterozygosity for the regions 3p12-3p21 (D3S3, D3S30 and D3S2) and distal 3p21-3p24 (D3F15S2 and THRB), when compared with that observed in the normal population. In contrast, D3S32, located within 3p21, was heterozygous in 38% of the tumors which is close to the frequency seen in the normal population (50%). In most cases the PCR results were consistent with the cytogenetic predictions. However, in 4 cell lines 3p loss was predicted from the karyotype, but heterozygosity for D3S32 was present. CONCLUSIONS: These experiments support cytogenetic data that indicate two regions of 3p loss in SCCHN tumors. The 3p regions that show a high frequency of homozygosity may contain tumor suppressor genes involved in the development and/or progression of squamous cancer. The region surrounding D3S32 may contain an essential gene that is conserved in two copies even when much of 3p is lost.

Loss of 18q and homozygosity for the DCC locus: possible markers for clinically aggressive squamous cell carcinoma.

Karyotyping and polymorphisms within the DCC (deleted in colon cancer) locus (18q21) were used to analyze loss of chromosome 18 in squamous cell carcinomas (SCC). Tumors from 26 patients (including 7 for whom matched tumor and normal DNA samples were available) were examined for heterozygosity within DCC. Of the seven normal-tumor combinations, four were informative. Two of these had loss of heterozygosity (LOH) at DCC. For 19 SCC tumor cultures normal tissue was not available. These were scored only as homozygous or heterozygous. The majority were homozygous. Only 3/19 (15%) were heterozygous. In contrast, in a panel of normal blood samples the majority, 11/16 (69%), were heterozygous. Allelic zygosity was concordant with the chromosome 18 content in the 16 tumors that were also karyotyped. Tumors from 40 patients, 37 that were karyotyped and three that were informative at the DCC locus, were assessed for loss of chromosome 18 and patient survival. Loss of part or all of chromosome 18 occurred in tumors from 25. Twenty-seven of the 40 patients have died and 13 are alive. There was strong association between loss of 18 and overall survival. Of those who are alive only 5/13 (38%) had loss of 18, whereas among those who have died 20/27 (74%) had loss of 18. By chi 2 analysis the association of loss of 18 and death from cancer was significant (p > 0.01). The high frequency of chromosome 18 loss in SCC suggests that this region contains one or more tumor suppressor genes important in the clinical behavior of SCC. DCC is one candidate, but other regions of loss not including the DCC locus indicate that chromosome 18 probably contains more than one tumor suppressor locus. Prospective studies of chromosome 18 loss as a single prognostic indicator are strongly indicated in this tumor type since loss in early stage tumors might indicate a need for more aggressive therapy than would be given on the basis of staging alone.

Common clonal origin of synchronous primary head and neck squamous cell carcinomas: analysis by tumor karyotypes and fluorescence in situ hybridization.

Two synchronously arising primary squamous cell carcinomas (SCC) originating from separate sites in the anterior floor of mouth (FOM) and the pyriform sinus (PS) were evaluated by karyotype and fluorescence in situ hybridization (FISH) to determine whether they were of common or independent ancestry. The primary tumors were designated Henry Ford Hospital (HFH)-SCC-8a (FOM) and HFH-SCC-9a (PS), and the respective recurrent tumors after chemotherapy and radiation were designated -8b and -9b. HFH-SCC-8a and -8b were cultured and had closely related hypotetraploid karyotypes of monoclonal origin. Karyotypes could not be obtained from the second primary tumor HFH-SCC-9a or its recurrence -9b. However, we used karyotypes from HFH-SCC-8a and -8b as a guide to select FISH probes for the histological evaluation of genetic markers in tumor sections. Fluorescence in situ hybridization on metaphase chromosomes from the cell cultures was useful in modifying the tumor karyotypes. Fluorescence in situ hybridization identified a chromosome Y rearrangement that was not obvious from the HFH-SCC-8a and -8b karyotypes, and this Y rearrangement served as a unique clonal marker. Using two probes for the Y chromosome we showed that all four tumors shared the same Y rearrangement with loss of Yq (DYZ1) and retention of Ycen (DYZ3). Furthermore, FISH showed that all four tumors had the same aneuploidy patterns for chromosomes X, Y, 7, 9, 15, 16, and 17. From karyotypic and FISH analysis disomy for X and 9 centromere regions and the rearranged Y were all predicted and observed in the tumor tissue sections. Tetrasomy and trisomy for 7, 15, 16, and 17 were predicted from the karyotypes and this also was observed using FISH in all four tumors. These FISH aneuploidy patterns and the presence of a clonal Y marker in all four tumor samples indicate that the synchronous primaries and their recurrences were of monoclonal origin.