Maria J. Worsham

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.

High-resolution mapping of molecular events associated with immortalization, transformation, and progression to breast cancer in the MCF10 model

SummaryBackgroundA comprehensive and consistent picture of the genetic changes that underlie breast cancer initiation, development, and progression remains unresolved. The MCF10 series of cell lines represents many steps in that progression. We performed high resolution mapping of the MCF10 series of cell lines to identify specific gene targets to elucidate the molecular correlates of immortalization, development, and progression of breast cancer at the level of individual genes.DesignWe evaluated the initial untransformed outgrowths (MCF-10MS and MCF-10A) with six transformed cell lines with benign proliferations (MCF-10AT1, MCF-10AT1kcl2), carcinoma in situ (MCF-10CA1h cl13), and invasive carcinoma (MCF-10CA1h cl2, MCF-10CA1a cl1, MCF-10CA1d cl1). Losses and gains of loci at 112 unique human genome sites were interrogated using the multiplex ligation-dependent probe amplification assay (MLPA).ResultsCytogenetic alterations in the four benign progenitors that persisted in the CIS and invasive cell lines corresponded to gains and losses of genes by MLPA. MCF-10MS had only normal gene copies. The untransformed MCF-10A had cytogenetic gain of 5q13-qter with corresponding gains of the IL3, IL4 and IL12B genes at 5q31-q33; gain of distal 19q12-qter was reflected in gains in KLK3 and BAX gene loci at 19q13-q13.4. The observed genic gain of cMYC at 8q24.12 was not indicated by cytogenetics. The apparently balanced t(3;9) component of the t(3;9)(p13;p22)t(3;5)(p26;q31) resulted in complete loss of the CDKN2A and CDKN2B genes at 9p21. Additional clonal cytogenetic changes in the DCIS cell line (MCF-10A1h cl13) involving chromosomes 1, 3 and 10 persisted in the invasive progeny, with gain of corresponding genes at 1p13 (BCAR2, BCAR3, NRAS, TGFB2), at 3p12–13 (IL12A), and 3q21–27 (MME, PIK3CA, BCL6).ConclusionsOur study adopted a comprehensive exploration of genetic changes using high resolution molecular probes applied to the MCF10 family of cell lines to identify individual genes in a continuum starting from normal breast epithelial cells and progressing through immortalization, transformation and invasive malignancy. Homozygous loss of CDKN2A and CDKN2B genes and gain of MYC were initiating immortalization events. Transformation and progression to malignancy event were marked by gains of IL13, VEGF, HRAS, TRAF2, and BCAS2, IL12A, and MME, respectively.

Breast cancer incidence in a cohort of women with benign breast disease from a multiethnic, primary health care population.

Abstract:  Women with benign breast diseases (BBD), particularly those with lesions classified as proliferative, have previously been reported to be at increased risk for subsequent development of breast cancer (BC). A cohort of 4970 women with biopsy‐proven BBD, identified after histopathology review of BBD biopsies, was studied for determination of subsequent development of BC. We report on 4537 eligible women, 28% of whom are African‐American, whose BBD mass was evaluable for pathologic assessment of breast tissue. Ascertainment of subsequent progression to BC from BBD was accomplished through examination of the tumor registries of the Henry Ford Health system, the Detroit SEER registry, and the State of Michigan cancer registry. Incidence rates (IR) are reported per 100,000 person years at risk (100 k pyr). Poisson regression models were used to evaluate the association of demographic and lesion characteristics with BC incidence, using person years at the time of BBD diagnosis as the offset variable. The estimated overall BC IR for this cohort is 452 (95% confidence interval [CI] = 394–519) per 100 k pyr. Incidence for women age 50 and older is 80% greater than for younger women (p = 0.007, IRR = 1.8, 95% CI = 1.36–2.36). Neither marital status (p = 0.91, IRR = 0.97, 95% CI = 0.73–1.29) nor race (p = 0.67, IRR = 0.9, 95% CI = 0.54–1.48) is associated with differences in BC IR. Compared with women having nonproliferative lesions, the risk for BC is greater for women with atypical ductal hyperplasia of (IRR = 5.0; 95%CI = 2.26–11.0; p < 0.001) and other proliferative lesions (IR = 1.7, 95% CI = 1.02–2.95; p = 0.04). BC risk for woman with atypical lesions is significantly higher than for women with proliferative lesions without atypia (IRR = 2.58, 95% CI = 1.35–4.90; p = 0.0039). Neither race nor marital status was a factor for BC incidence from BBD in this cohort. Age retained its importance as a predictor of risk. BBD lesion histopathology in the outcome categories of either proliferative without atypia or proliferative with atypia are significant risk factors for BC, even when adjusted for the influence of demographic characteristics. The risks associated with BBD histological classifications were not different across races.

Human papillomavirus outcomes in an access-to-care laryngeal cancer cohort.

Objective. Human papillomavirus (HPV), particularly HPV16, is a causative agent for 25% of head and neck squamous cell cancer, including laryngeal squamous cell cancer (LSCC). HPV-positive (HPV+ve) patients, particularly those with oropharyngeal SCC, have improved prognosis. For LSCC patients, this remains to be established. The goal was to determine stage and survival outcomes in LSCC in the context of HPV infection. Study Design. Historical cohort study. Setting. Primary care academic health system. Subjects and Methods. In 79 patients with primary LSCC, HPV was determined using real-time quantitative polymerase chain reaction. χ2 or Fisher exact test was used to test the association of HPV+ve with 21 risk factors including race, stage, gender, age, smoking, alcohol, treatment, and health insurance. Kaplan-Meier and log-rank tests were used to study the association of HPV and LSCC survival outcome. Results. HPV16 was detected in 27% of LSCC patients. Caucasian American (CA) patients had higher HPV prevalence (33%) than did African American (AA) LSCC patients (16%; P = .058). HPV was significantly associated with gender (P = .016) and insurance type (P = .001). There were no differences in survival between HPV+ve and HPV-negative (HPV-ve) patients. There was no association with HPV and other risk factors including stage (early vs late). Conclusion. We found a high prevalence of HPV in men and a lower prevalence of HPV infection in AA compared with CA. Despite the strikingly better survival of patients with HPV+ve oropharyngeal tumors, even when adjusted for smoking, this correlation does not seem to hold true in the larynx. Larger multiethnic LSCC cohorts are needed to more clearly delineate HPV-related survival across ethnicities.

Improved survival with HPV among African Americans with oropharyngeal cancer

Purpose: A major limitation of studies reporting a lower prevalence rate of human papilloma virus (HPV) in African American patients with oropharyngeal squamous cell cancer (OPSCC) than Caucasian Americans, with corresponding worse outcomes, was adequate representation of HPV-positive African American patients. This study examined survival outcomes in HPV-positive and HPV-negative African Americans with OPSCC. Experimental Design: The study cohort of 121 patients with primary OPSCC had 42% African Americans. Variables of interest included age, race, gender, HPV status, stage, marital status, smoking, treatment, and date of diagnosis. Results: Caucasian Americans are more likely to be HPV positive (OR = 3.28; P = 0.035), as are younger age (age < 50 OR = 7.14; P = 0.023 compared with age > 65) or being married (OR = 3.44; P = 0.016). HPV positivity and being unmarried were associated with being late stage (OR = 3.10; P = 0.047 and OR = 3.23; P = 0.038, respectively). HPV-negative patients had 2.7 times the risk of death as HPV-positive patients (P = 0.004). Overall, the HPV-race groups differed (log-rank P < 0.001), with significantly worse survival for HPV-negative African Americans versus (i) HPV-positive African Americans (HR = 3.44; P = 0.0012); (ii) HPV-positive Caucasian Americans (HR = 3.11; P = < 0.049); and (iii) HPV-negative Caucasian Americans (HR = 2.21; P = 0.049). Conclusions: HPV has a substantial impact on overall survival in African American patients with OPSCC. Among African American patients with OPSCC, HPV-positive patients had better survival than HPV negative. HPV-negative African Americans also did worse than both HPV-positive Caucasian Americans and HPV-negative Caucasian Americans. This study adds to the mounting evidence of HPV as a racially linked sexual behavior life style risk factor impacting survival outcomes for both African American and Caucasian American patients with OPSCC. Clin Cancer Res; 19(9); 2486–92. ©2013 AACR.

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.

Molecular differentiation of early and late stage laryngeal squamous cell carcinoma: an exploratory analysis.

Background A current shortcoming in cancer prognostication and treatment is a lack of methods that adequately address the complexity and diversity of the disease. Prognostic marker systems based on single parameters have generally proven inadequate. Thus, multiparametric methods, which rely on many pieces of information, are ideally suited to the grouping of tumor subtypes and the identification of specific patterns of disease progression. Design This study investigated, on an exploratory basis, whether genome wide alterations of loss and gain, using a panel of 122 gene probes (112 unique genes), discriminated between early stage (stage 1 and 2) and late stage (stage 3 and 4) laryngeal squamous cell carcinomas (LSCC). The LSCC cohort comprised 29 patients, 12 early and 17 late staged. Formalin-fixed LSCC DNA was interrogated by a genome wide candidate gene panel (122 genes) using the multiplex ligation-dependent probe amplification assay. Results Statistical analysis employed the nonparametric Wilcoxon 2-sample test. Significant differences between tumor stages of early versus late were seen for the following genes: ERBB4, CASP2, RECQL4, and BCL7A. Loss of ERBB4 (P=0.045) and BCL7A (P=0.019) significantly discriminated between early and late stage LSCC. Gain of RECQL4 copy number (P=0.043) was associated with late LSCC. Gain of CASP2 (P=0.043) marked early LSCC, whereas loss was associated with late LSCC. Conclusions High-throughput genome wide approaches have the potential to yield discrete gene repertoires of early and late stage LSCC differentiation.

Multiplicity of Benign Breast Lesions Is a Risk Factor for Progression to Breast Cancer

Purpose: Benign breast disease (BBD) in women encompasses a broad spectrum of histopathologic lesions. Studies on BBD have focused on the risks for subsequent breast cancer associated with three broad categories of lesions, classified as nonproliferative, proliferative, or proliferative with atypia, without addressing the issue of the contribution of concurrent multiple BBD lesions. There is very limited information with regard to the issue of BBD lesion multiplicity and breast cancer risk. Experimental Design: We evaluated a detailed microscopic spectrum of 18 BBD lesions from fibrosis to atypical hyperplasia in a BBD cohort of 4,544 subjects, within which 4.5% (n = 201) developed breast cancer during an average follow-up period of 10.3 years. Lesions were defined as nonproliferative (8 diagnoses; risk level 1 = no risk or low risk), proliferative without atypical hyperplasia (8 diagnoses; risk level 2 = intermediate risk), and proliferative with atypical hyperplasia (2 diagnoses; risk level 3 = highest risk level). Twenty variables including age (≥50 or <50 years) at the time of BBD diagnosis and race (African American or non–African American) were assessed. A categorical variable, surrogate for lesion type and number, was represented initially by four levels: 1, nonproliferative = 1 (reference); 2, nonproliferative > 1; 3, proliferative = 1; and 4, proliferative > 1. Results: The majority of BBD subjects in our cohort (almost 70%) had more than one BBD lesion. Concurrent multiple nonproliferative or proliferative BBD lesions with or without atypia in a BBD biopsy and age are significant predictors of risk for progression of BBD to breast cancer. The presence of atypical hyperplasia in a BBD biopsy alone or in conjunction with other lesions without atypia conferred higher risks. Women with fibrosis had a reduced risk for progression to breast cancer. Race was not a significant predictor of progression to breast cancer. The effect of age, fibrosis, and multiple lesions (whether nonproliferative, proliferative, or atypia) on breast cancer progression was not influenced by race. Conclusion: BBD lesion multiplicity is frequent, and teasing out the heterogeneity of multiple concurrent BBD lesions is warranted to refine and improve risk estimates for progression of breast cancer from BBD.

Epigenetic events underlie the pathogenesis of sinonasal papillomas

Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.

Molecular classification of breast carcinoma in situ.

Pleomorphic variant of invasive lobular carcinoma (PILC) is an aggressive variant of invasive lobular carcinoma (ILC). Its in situ counterpart, pleomorphic lobular carcinoma in situ (PLCIS) is a recently described entity. Morphologically it has the typical architectural pattern of LCIS, but the neoplastic cells resemble intermediate grade DCIS. Molecular signatures that distinguish PLCIS from DCIS and LCIS would provide additional tools to aid in the histopathologic classification of PLCIS as a lesion distinct from LCIS and DCIS. CIS lesions, obtained from a study cohort of 38 breast cancer patients, were divided into 18 DCIS, 14 PLCIS and 6 LCIS. DNA from microdissected archival tissue was interrogated for loss or gain of 112 breast-cancer-specific genes using the Multiplex Ligation-dependent Probe Amplification Assay (MLPA). Classification Regression Tree (CART) analysis was employed to develop a gene-based molecular classification to distinguish or separate out PLCIS from DCIS and LCIS. Molecular classification via CART, based on gene copy number, agreed with histopathology in 34/38 CIS cases. Loss of CASP1 was predictive of LCIS (n=4) with one misclassified PLCIS. Gain of RELA predicted only the LCIS classification (n=2 cases). STK15 and TNFRSF1B were predictive only for DCIS with no misclassifications. Gain of EHF and TNFRSF1B and loss of NCOA3 were predictive of PLCIS, but not without misclassification. Molecular reclassification by CART was accomplished in 4 CIS cases: 1 PLCIS was reclassified as LCIS, 1 LCIS reclassified as PLCIS, and 2 DCIS cases as PLCIS. This study provides additional rationale for molecular modeling strategies in the evaluation of CIS lesions. This diagnostic aid may serve to minimize misclassification between PLCIS and DCIS, and PLCIS and LCIS, aiding to increase accuracy in the differential diagnosis of CIS lesions.