Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity.

BACKGROUND: HER2 gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods. METHODS: In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms. RESULTS: Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability. CONCLUSIONS: These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two probe FISH interphase analysis is inadequate and results interpreted using the HER2/CEP17 ratio should be reported "with caution" when the presence of centromeric amplification or monosomy is suspected by FISH signal gains or losses. The presence of these pericentromeric copy number changes may result in artificial skewing of the HER2/CEP17 ratio towards false negative or false positive results in breast cancer with chromosome 17 complexity. Full genomic analysis should be considered in all cases with complex chromosome 17 aneusomy as these cases are likely to have genome-wide instability, amplifications, and a poor prognosis.

Memory B cells and pneumococcal antibody after splenectomy.

Splenectomized patients are susceptible to bloodstream infections with encapsulated bacteria, potentially due to loss of blood filtering but also defective production of anticarbohydrate Ab. Recent studies propose that a lack of Ab is related to reduced numbers of IgM(+) CD27(+) memory B cells found after splenectomy. To test this, we analyzed CD27(+) memory B cell subsets, IgG, and IgM pneumococcal Ab responses in 26 vaccinated splenectomized subjects in comparison to memory B cell subsets and Ab responses in healthy controls. As shown previously, the splenectomized autoimmune subjects had fewer total, isotype switched, and IgM(+) CD27(+) memory B cells as compared with controls, but there was no difference in memory B cells subsets between controls and splenectomized subjects with spherocytosis. There was no difference between the geometric mean IgG Ab response between normal controls and splenectomized subjects (p = 0.51; p = 0.81). Control subjects produced more IgM Ab than splenectomized autoimmune subjects (p = 0.01) but the same levels as subjects with spherocytosis (p = 0.15.) There was no correlation between memory B cell subsets and IgG or IgM Ab responses for controls or splenectomized subjects. These data suggest that splenectomy alone may not be the sole reason for loss of memory B cells and reduced IgM antipneumococcal Ab. Because subjects with autoimmunity had splenectomy at a significantly older age than participants with spherocytosis, these data suggest that an age-related loss of extra splenic sites necessary for the maintenance or function of memory B cells may lead to impaired immunity in these subjects.

Comprehensive Determination of Prostate Tumor ETS Gene Status in Clinical Samples Using the CLIA Decipher Assay.

ETS family gene fusions are common in prostate cancer and molecularly define a tumor subset. ERG is the most commonly rearranged, leading to its overexpression, followed by ETV1, ETV4, and ETV5, and these alterations are generally mutually exclusive. We validated the Decipher prostate cancer assay to detect ETS alterations in a Clinical Laboratory Improvement Amendments-accredited laboratory. Benchmarking against ERG immunohistochemistry and ETV1/4/5 RNA in situ hybridization, we examined the accuracy, precision, and reproducibility of gene expression ETS models using formalin-fixed, paraffin-embedded samples. The m-ERG model achieved an area under curve of 95%, with 93% sensitivity and 98% specificity to predict ERG immunohistochemistry status. The m-ETV1, -ETV4, and -ETV5 models achieved areas under curve of 98%, 88%, and 99%, respectively. The models had 100% robustness for ETS status, and scores were highly correlated across sample replicates. Models predicted 41.5% of a prospective radical prostatectomy cohort (n = 4036) to be ERG+, 6.3% ETV1+, 1% ETV4+, and 0.4% ETV5+. Of prostate tumor biopsy samples (n = 509), 41.2% were ERG+, 8.6% ETV1+, 0.4% ETV4+, and none ETV5+. Higher Decipher risk status tumors were more likely to be ETS+ (ERG or ETV1/4/5) in the radical prostatectomy and the biopsy cohorts (P < 0.05). These results support the utility of microarray-based ETS status prediction models for molecular classification of prostate tumors.

Array-based karyotyping in plasma cell neoplasia after plasma cell enrichment increases detection of genomic aberrations.

The discovery of genomic abnormalities present in monoclonal plasma cells has diagnostic, prognostic, and disease-monitoring implications in plasma cell neoplasms (PCNs). However, technical and disease-related limitations hamper the detection of these abnormalities using cytogenetic analysis or fluorescence in situ hybridization (FISH). In this study, 28 bone marrow specimens with known PCNs were examined for the presence of genomic abnormalities using microarray analysis after plasma cell enrichment. Cytogenetic analysis was performed on 15 of 28 samples, revealing disease-related genomic aberrations in only 3 (20%) of 15 cases. FISH analysis was performed on enriched plasma cells and detected aberrations in 84.6% of specimens while array comparative genomic hybridization (aCGH) detected abnormalities in 89.3% of cases. Furthermore, aCGH revealed additional abnormalities in 24 cases compared with FISH alone. We conclude that aCGH after plasma cell enrichment, in combination with FISH, is a valuable approach for routine clinical use in achieving a more complete genetic characterization of patients with PCN.

Association of anti-mullerian hormone levels with obesity in late reproductive-age women.

OBJECTIVE: To describe anti-mullerian hormone (AMH) levels in healthy late reproductive age women and test the hypothesis that AMH levels are lower in obese compared to non-obese women. DESIGN: Cross-sectional study of AMH levels. Longitudinal analysis of a subgroup with 10 AMH measures over 8 years to support the cross-sectional results. SETTING: A population-based cohort of healthy late reproductive-age women. PARTICIPANTS: Selected from the cohort to provide comparisons of body mass index (BMI), menopausal status, age and race (n = 122). INTERVENTIONS: AMH levels were determined from blood samples collected in the parent study. MAIN OUTCOME MEASURE: Serum levels of AMH. RESULTS: AMH levels were 65% lower in obese women compared to non-obese women (0.016 ng/mL and 0.046 ng/mL, respectively). The geometric mean ratio was 0.35; 95% CI 0.13, 0.92, P=.034. AMH levels were significantly lower in the menopausal transition compared to premenopausal women and were significantly lower in all age groups > or =40 years compared to the 35-39 year-old women. BMI remained significantly associated with AMH levels in multivariable models that included adjustments for menopausal status, age, race and cycle day. In the longitudinal analysis of a subgroup, obese women had significantly lower mean AMH levels over the 8-year interval compared to the non-obese women (0.459 ng/mL; CI 0.28, 0.75 and 0.566 ng/mL; CI 0.34, 0.94, respectively; P=.016), corroborating the cross-sectional study results. CONCLUSIONS: Obese women have lower AMH levels compared to non-obese women in the late reproductive years. The findings offer further evidence of the complex relationships between obesity and reproductive hormone levels in women.

Pilot study investigating the age-related decline in ovarian function of regularly menstruating normal women.

OBJECTIVE: To use a pilot study to investigate markers of the age-related decline in ovarian function of regularly menstruating normal women. DESIGN: Prospective. SETTING: Tertiary research center. PATIENT(S): Healthy volunteers (n = 42) aged 18 to 50 years who had regular ovulatory menstrual cycles and a prior pregnancy. INTERVENTION(S): A single 300-IU dose of human recombinant FSH on day 3 of the menstrual cycle. MAIN OUTCOME MEASURE(S): Antral follicle count by transvaginal ultrasound and basal and FSH-stimulated serum markers. RESULT(S): Age correlated most strongly with FSH-stimulated inhibin B (r = -0.660), followed by antral follicle count (r = -0.578), basal FSH (r = 0.509), basal Müllerian inhibiting substance (MIS; r = -0.468), and basal inhibin B (r = -0.358). Total antral follicle count correlated most strongly with basal MIS level (r = 0.642). CONCLUSION(S): Of the parameters tested, FSH-stimulated serum inhibin B level had the strongest correlation with age. Basal serum MIS level had the strongest correlation with total antral follicle count. We confirm a previous report that in normal women, the antral follicle count as determined by transvaginal ultrasound examination correlates better with age than do basal FSH and basal inhibin B levels.