Preferences for return of incidental findings from genome sequencing among women diagnosed with breast cancer at a young age.

While experts have made recommendations, information is needed regarding what genome sequencing results patients would want returned. We investigated what results women diagnosed with breast cancer at a young age would want returned and why. We conducted 60 semi-structured, in-person individual interviews with women diagnosed with breast cancer at age 40 or younger. We examined interest in six types of incidental findings and reasons for interest or disinterest in each type. Two coders independently coded interview transcripts; analysis was conducted using NVivo 10. Most participants were at least somewhat interested in all six result types, but strongest interest was in actionable results (i.e. variants affecting risk of a preventable or treatable disease and treatment response). Reasons for interest varied between different result types. Some participants were not interested or ambivalent about results not seen as currently actionable. Participants wanted to be able to choose what results are returned. Participants distinguished between types of individual genome sequencing results, with different reasons for wanting different types of information. The findings suggest that a focus on actionable results can be a common ground for all stakeholders in developing a policy for returning individual genome sequencing results.

How will GINA influence participation in pharmacogenomics research and clinical testing?

After a 13-year battle in Congress--longer than it took to map the human genome--the Genetic Information Nondiscrimination Act (GINA) was passed into law on 21 May 2008. Before its passing, Francis Collins, then director of the National Human Genome Research Institute, testified before the 110th Congress that the success of personalized medicine hinged on the passing of the legislation. How will GINA, which takes effect in 2009, influence participation in pharmacogenomic research and clinical testing?

Reproductive factors in relation to breast cancer characterized by p53 protein expression (United States).

OBJECTIVE: To evaluate the potential etiologic heterogeneity of breast cancer by examining whether associations with reproductive and other personal characteristics differed by p53 protein expression status. METHODS: Data from the Carolina Breast Cancer Study, a population-based, case-control study of 861 cases and 790 controls, were utilized. Immunohistochemical staining for the p53 protein was performed on 638 archived tumor specimens; 46% of cases were classified as p53+. Two separate unconditional logistic regression models were used to calculate odds ratios (OR) and 95% confidence intervals (CI) for p53+ and p53- breast cancer relative to controls for reproductive and other personal characteristics. Analyses were performed separately for younger (< or = 45 years) and older (>45 years) women. RESULTS: Risk factor profiles largely overlapped for p53+ and p53- breast cancer, with the exception of oral contraceptive (OC) use among younger women and a family history of breast cancer. Prolonged OC use was more strongly associated with p53+ breast cancer [OR 3.1 (95% CI: 1.2-8.1) than p53- breast cancer (OR 1.3 (95% CI: 0.6-3.2)] among younger women only. A first-degree family history of breast cancer was associated with p53+ breast cancer among younger women [OR 1.5 (95% CI: 1.0-2.2)] and older women [OR 1.4 (95% CI: 0.9-2.3)], but not p53- breast cancer in either age-group. CONCLUSIONS: These results provide little evidence of breast cancer heterogeneity as classified by p53 expression status. However, although not statistically significant, OC use among younger women and family history of breast cancer may operate through a pathway involving p53 alterations to increase risk of breast cancer.

Environmental factors in relation to breast cancer characterized by p53 protein expression.

Findings from studies of cigarette smoking and low-dose ionizing radiation exposure and breast cancer are unclear. Laboratory studies indicate that both exposures can cause DNA damage, potentially increasing cancer risk if such mutations occur in growth control genes, such as p53. We examined the potential etiologic heterogeneity of breast cancer by evaluating whether associations between cigarette smoking and low-dose ionizing radiation and breast cancer differed by p53 protein expression status. Data were obtained from the Carolina Breast Cancer Study, a population-based, case-control study conducted among African-American and white women ages 20-74 years. Questionnaire data were available from 861 women with incident, primary invasive breast cancer and 790 community-based controls. p53 immunostaining was performed on tissue from 683 women with breast cancer; 46% were classified as p53+. Two separate unconditional logistic regression models were used to calculate odds ratios (ORs) for p53+ and p53- breast cancer, as compared with controls, in relation to smoking and low-dose ionizing radiation exposure. Smoking was not differentially associated with p53+ or p53- breast cancer, even when duration, dose, and passive smoking status were considered. Exposure to individual sources of radiation did not differ for p53+ and p53- breast cancers. However, ORs for combined exposure to chest X-rays and occupational radiation were higher for p53+ [OR, 2.2; 95% confidence interval (CI), 1.0-5.3] than p53- breast cancer (OR, 1.2; 95% CI, 0.5-3.4). Combined exposure to radiation from other medical sources as well as occupational exposure was also higher for p53+ (OR, 3.7; 95% CI, 0.8-16.8) than for p53- breast cancer (OR, 1.7; 95% CI, 0.3-10.5). Although preliminary, our results suggest that exposure to multiple sources of low-dose ionizing radiation may contribute to the development of p53+ breast cancer.

Prediction of node-negative breast cancer outcome by histologic grading and S-phase analysis by flow cytometry: an Eastern Cooperative Oncology Group Study (2192).

Histologic evaluation and reporting of invasive breast cancer has effectively used Nottingham combined histologic grade (NCHG). This approach to predict outcome in invasive breast cancer has not been tested in multicenter cooperative trials. Histologic slides from selected breast cancer cases entered on node-negative Eastern Cooperative Oncology Group trials were assigned grades. Two pathologists evaluated cases for NCHG defined from differentiation, mitotic index, and nuclear grade. The study population consisted of separate samples from low- and high-risk strata, where low risk was estrogen receptor positive with a tumor size of less than 3 cm and high risk was estrogen receptor negative or tumor size greater than or equal to 3 cm. The rate of agreement was generally good, with 80% of cases classified the same for mitotic count and 76% of the cases classified the same for combined grade. There were no cases disagreeing from the lowest to the highest of the three categories. The median follow-up is 11.6 years, but for analysis of survival, this was truncated at 5 years. Mitotic index and combined grade as assessed by both pathologists showed significant associations with survival. High combined histologic grade was predictive for response to cyclophosphamide/methotrexate/5-fluorouracil (CMF) with survival differences at 5 years of 30% in the treated high-grade patients over the untreated patients. Overall, it is clear that pathologists can have close agreement in assignment of combined histologic grades, with highly significant prediction in univariate and borderline significance in multivariate analysis in prognostication of time to recurrence as well as survival. Thus, stratification used in these trials was highly prognostic as hoped, leaving a role for histologic grading in these relatively large tumors, more powerful than S-phase analysis in this series. In the subgroups of high-risk patients randomized between CMF and observation, there was a suggestion that the high-combined-grade group was predictive of treatment efficacy. We conclude that a combined histologic grade with defined criteria may be reliably assigned by practiced pathologists using readily available criteria, and that the measure may be of use in prognostication and prediction of therapeutic responsiveness when done in a technically ideal fashion.

Handling, storage, and preparation of human tissues.

Human tissue for flow cytometry must be prepared as an adequate single-cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single-cell suspensions of whole cells from fine-needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin-embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris.

Prognostic value of histologic grade and proliferative activity in axillary node-positive breast cancer: results from the Eastern Cooperative Oncology Group Companion Study, EST 4189.

PURPOSE: The identification of a subset of patients with axillary lymph node-positive breast cancer with an improved prognosis would be clinically useful. We report the prognostic importance of histologic grading and proliferative activity in a cohort of patients with axillary lymph node-positive breast cancer and compare these parameters with other established prognostic factors. PATIENTS AND METHODS: This Eastern Cooperative Oncology Group laboratory companion study (E4189) centered on 560 axillary lymph node-positive patients registered onto one of six eligible clinical protocols. Flow cytometric (ploidy and S-phase fraction [SPF]) and histopathologic analyses (Nottingham Combined Histologic Grade and mitotic index) were performed on paraffin-embedded tissue from 368 patients. RESULTS: Disease recurred in 208 patients; in 161 (77%), within the first 5 years. Mitotic index and grade were associated with both ploidy and SPF (P

HER2 testing: laboratory, technical and clinical considerations.

HER2 measurement holds great promise in predicting response to a variety of systemic therapies an exciting step forward in the management of breast cancer patients. The enthusiasm surrounding the clinical importance of HER2, however, is tempered by the uncertainty regarding the clinical usefulness and accuracy of the different methodologies currently available to assess HER2 status. In this paper the authors address laboratory and technical issues associated with methods which measure HER2 overexpression or amplification. We also discuss how these issues can influence the clinical utility and routine application of this marker. While a tumor marker may be considered clinically relevant, it must be proven to be clinically useful. Optimally, this should occur in the setting of a randomized clinical trial, using methods that are reliable, reproducible, and biologically accurate. For HER2 testing to be a useful, routine clinical marker several critical issues need to addressed. These include the determination and validation of the clinical utility of each method to predict response to the different systemic therapies currently associated with HER2. In addition, there is need to set standards for assay performance and interpretation of assay results, based on criteria that are clinically validated.

Policy guidelines for the utilization of formalin-fixed, paraffin-embedded tissue sections: the UNC SPORE experience. University of North Carolina Specialized Program of Research Excellence.

Paraffin blocks represent a valuable resource that has allowed investigators to apply today's technology to address scientific questions in a shorter period of time and in more diverse populations than would have been possible with fresh or frozen tissue. However, in addition to being an exhaustible resource, there is concern regarding the appropriate use of these tissues, both with respect to medical or legal considerations and quality control and quality assurance practices. We describe policy guidelines to address these concerns, including: safeguards to address medical/legal and patient confidentiality issues, quality control and quality assurance for tissue sectioning, processing and storage, database management for sample tracking, and scientific review for utilization of specimens. These policies and procedures have been developed and implemented by the University of North Carolina (UNC) Specialized Program of Research Excellence (SPORE) in the Breast Cancer Immunohistochemistry (IHC) Core laboratory, in collaboration with our study pathologists, participants, and research investigators. It is our hope that the information and experience described here may stimulate discussion that can ultimately lead to a uniform policy for handling formalin-fixed paraffin-embedded tissues in research.

DNA content measurement can be obtained using archival material for DNA flow cytometry. A comparison with cytogenetic analysis in 56 pediatric solid tumors.

BACKGROUND: Although flow cytometry (FCM) has become a widely used technique for the measurement of DNA content in solid tumors, the correlation of ploidy analysis by FCM with cytogenetic analysis (CGA) is not well described. The sensitivities of G-banded CGA and FCM were compared to determine the accuracy of the DNA index value (DI) as a measurement of chromosome number. METHODS: Tumor specimens from 56 pediatric cases were analyzed for DNA content by both FCM and CGA. Nuclei for FCM were prepared from archival tissue in 53 specimens using a modification of the Hedley technique and from fresh tissue in 3 specimens. Metaphase chromosomes for CGA were prepared from standard solid tumor harvests and Giemsa-trypsin banding procedures. Ploidy status for this study was defined as (1) diploid--DI between 0.97 and 1.03 by FCM or chromosome number +/- 2 from normal by CGA (44-48); and (2) aneuploid--DI < 0.97 or > 1.03 by FCM or total chromosomes < 44 or > 48 by CGA. RESULTS: Forty-nine of the 56 pediatric specimens were evaluable by both techniques. Concordance was observed in 34 cases (69%) between the two techniques in assigning similar ploidy status to a tumor (22 diploid and 12 aneuploid). It also was observed that among the aneuploid concordant cases, the actual DI obtained from archival material could predict total chromosome number with 95% accuracy. The 15 discordant cases showed a distinct aneuploid population by FCM, but were diploid by CGA. CONCLUSIONS: A correlation of 69% was obtained between both techniques to assign a similar ploidy status (diploid versus aneuploid) in 56 pediatric solid tumors. These results support the combined use of CGA and FCM to obtain the most complete analysis of DNA content and chromosome abnormalities in pediatric solid tumors. FCM on formalin-fixed, paraffin-embedded tissue can be used to measure total DNA content.

DNA flow cytometry measurements as surrogate endpoints in chemoprevention trials: clinical, biological, and quality control considerations.

DNA flow cytometric evaluation of S-phase fraction (SPF) is a strong and consistent predictor of relapse-free survival in the node-negative breast cancer patient. As such, it can be implicated as a marker of tumor aggressiveness and has been shown to be an independent predictor of outcome in a multivariate setting. Measurement of ploidy status is less well-defined as a marker of prognosis, but may be an important marker of response to therapy. Estimation of DNA ploidy and proliferative capacity by flow cytometry can be obtained from virtually any type of specimen, including fine needle aspirates, fresh or frozen material, as well as formalin-fixed, paraffin-embedded material, as long as there is a sufficient number of tumor nuclei for assay. Therefore, the assay has clinical relevance in predicting relapse, as well as providing flexibility for sample preparation. In addition, flow cytometric measurements are biologically relevant markers. In general, DNA index is a good estimate of total chromosome number. SPF, using sophisticated modeling algorithms, shows good correlation with thymidine labeling index and/or bromodeoxyuridine incorporation, two standard assays used to measure DNA synthesis in fresh tissue. Recently, preliminary data in locally advanced breast cancer have indicated that ploidy and/or S-phase may also be useful in predicting cellular response to chemotherapy. Although there is good justification for measuring these parameters, appropriate quality control and quality assurance measures must be incorporated into all aspects of the assay--from sample handling and preparation to interpretation of cell cycle and histogram data.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic significance of S-phase fraction in good-risk, node-negative breast cancer patients.

PURPOSE: Formalin-fixed, paraffin-embedded tissues from axillary node-negative breast cancer patients were analyzed by flow cytometry to determine the prognostic significance of DNA ploidy and S-phase fraction (SPF). PATIENTS AND METHODS: All patients were registered on a good-risk control arm of an intergroup clinical trial. They had small- to intermediate-sized (less than 3 cm), estrogen receptor (ER)-positive tumors and received no adjuvant therapy after modified radical mastectomy or total mastectomy with low axillary-node sampling. The median follow-up was 4.8 years. RESULTS: Assessable ploidy results were obtained from 92% of the 298 specimens studied (51% diploid, 49% aneuploid), and SPFs were assessable for 83% of the tumors. SPFs for diploid tumors ranged from 0.7% to 11.9% (median, 3.6%), compared with a range of 1.2% to 26.7% (median, 7.6%) for aneuploid tumors (P less than .0001). No significant differences in disease-free or overall survival were observed between patients with diploid and aneuploid tumors. Using different SPF cutoffs by ploidy status (4.4% for diploid, 7.0% for aneuploid), patients with low SPFs had significantly longer disease-free survival rates than patients with high SPFs (P = .0008). The actuarial 5-year relapse rates were 15% and 32% for patients with low (n = 142) and high SPFs (n = 105), respectively. Similar relationships between SPF and clinical outcome were observed for patients with diploid tumors (P = .053) and for patients with aneuploid tumors (P = .0012). CONCLUSION: S-phase fraction provides additional prognostic information for predicting disease-free survival for axillary node-negative breast cancer patients with small, ER-positive tumors.

Prognostic potential of DNA flow cytometry measurements in node-negative breast cancer patients: preliminary analysis of an intergroup study (INT 0076).

An ancillary study (INT 0076) to the Intergroup clinical trial of node-negative breast cancer patients (INT 0011) was performed to retrospectively evaluate DNA flow cytometry measurements of ploidy (DNA content) and proliferative capacity (S-phase fraction) for their ability to predict time to recurrence. Of the 915 patients eligible for the clinical trial, 788 were registered for the ancillary flow cytometry study (INT 0076). Four hundred and three of these patients [estrogen receptor (ER)-positive, tumor size less than 3 cm] had been registered to the observation arm of the clinical trial and 385 (ER-negative and/or tumor size greater than or equal to 3 cm) had been randomly assigned to adjuvant chemotherapy (cyclophosphamide, methotrexate, fluorouracil, and prednisone for six cycles) or to observation. Paraffin blocks from 95% (748 of 788) of these patients were obtained, 712 of which had sufficient cancer tissue to be evaluable for the flow cytometric assay. DNA ploidy status (DNA diploid vs DNA aneuploid) was evaluable for 565 (79%) specimens, 64% of which were aneuploid. Proliferative capacity was estimated by the percentage of cells having an S-phase DNA content, using a trapezoidal modeling algorithm(s) as previously described. The median S-phase value for the entire group (both registered and randomly assigned patients) was 6.97%, which defined the cutoff for interpretation of high or low S-phase values. With a median follow-up time of 4.55 years, S-phase fraction, but not ploidy status, is a significant predictor for time to recurrence in both the randomly assigned and the untreated population (observed registered group and observed randomly assigned group).(ABSTRACT TRUNCATED AT 250 WORDS)

Are DNA flow cytometry measurements providing useful information in the management of the node-negative breast cancer patient?

The appropriate management of the breast cancer patient with early stage disease is a controversial, frustrating issue. If laboratory tests could accurately predict tumor behavior, however, the clinician and patient would be greatly aided in their treatment decisions. Although imperfect, there are several new and significant factors that can be used to predict patient prognosis; the most promising and well studied of these factors are DNA flow cytometry measurements. There are at least two estimates of tumor aggressiveness that we can obtain from DNA flow cytometry: one is an estimate of the tumor DNA content or ploidy and the other is an estimate of the tumor proliferative capacity. These measurements have their greatest clinical impact in the node negative patient predicting for relapse-free survival and overall survival. Estimates of proliferative capacity are independent predictors of patient prognosis. Estimates of DNA content are at times controversial and yet still are helpful in distinguishing prognostic subgroups of proliferative activity and may have additional clinical relevance. This discussion will summarize the data obtained from DNA flow cytometry measurements supporting their use as clinically important markers of prognosis in the node-negative patient.

Atypical congenital mesoblastic nephroma. Report of a case with karyotypic and flow cytometric analysis.

Atypical congenital mesoblastic nephroma is a rare infantile renal tumor that may behave aggressively in older infants. A case of atypical congenital mesoblastic nephroma occurring in an 8-month-old hispanic male was analyzed by routine histopathologic, cytogenetic, and retrospective flow cytometric analysis for DNA ploidy. Light microscopy revealed marked hypercellularity. The karyotype was abnormal, with the following configuration: 45,XY,-1,-3,-9,-9,-15,-17,-21,+del(1)(q21q25),+der(3), t(3;9;15)(q23;p22;q11),+der(9),t(3;9;15) (q23;p22;q11),+der(9),t(9;?) (p?22;?),+r21, + mar. Retrospective DNA ploidy analysis revealed a DNA index of 1.0. The significance of karyotypic changes occurring in mesenchymal renal tumors of this type is currently unknown. Cytogenetic analysis might be of prognostic value in these potentially aggressive tumors.

Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry.

More accurate prediction of the prognosis in women with node-negative breast cancer may improve physicians' ability to identify the patients most likely to benefit from systematic adjuvant therapy. With this in mind, we performed DNA flow-cytometric measurements of ploidy and the fraction of cells in the synthesis phase of the cell cycle (S-phase fraction) on 395 specimens of node-negative breast cancer from our bank of frozen tumors, using the aliquots of pulverized frozen tissue from steroid-receptor assays. The median duration of follow-up in patients still alive at the time of analysis was 59 months. Thirty-two percent of the 345 specimens that could be evaluated were diploid, and 68 percent were aneuploid. The probability of disease-free survival at five years was 88 +/- 3 percent in patients with diploid tumors and 74 +/- 3 percent in those with aneuploid tumors (P = 0.02). The S-phase fraction was not a significant additional predictor of disease-free survival in patients with aneuploid tumors. However, the probability of disease-free survival in patients with diploid tumors and low S-phase fractions was 90 +/- 3 percent at five years, as compared with 70 +/- 13 percent in those with diploid tumors and high S-phase fractions (P = 0.007). Similar differences in overall survival were noted. We conclude that DNA flow-cytometric measurements of ploidy and S-phase fraction can be performed on frozen specimens of tumors and are potentially important predictors of disease-free and overall survival in patients with node-negative breast cancer.