Reduction in Breast Cancer Death With Adjuvant Chemotherapy Among US Women According to Race, Ethnicity, and the 21-Gene Recurrence Score.

BACKGROUND: We previously showed the 21-gene breast recurrence score (RS) has lower prognostic accuracy for non-Hispanic Black (NHB) compared with non-Hispanic White (NHW) women with estrogen receptor (ER)-positive/HER2-negative breast cancer. The purpose of this study was to determine the clinical validity of the RS for predicting chemotherapy benefit as recommended in the current NCCN Guidelines for Breast Cancer among women from diverse racial/ethnic groups. METHODS: Using the SEER Oncotype database, we estimated propensity score-weighted hazard ratios (HRs) and 95% confidence intervals for breast cancer death with chemotherapy for women with ER-positive/HER2-negative, AJCC stages I-II, axillary node-negative, invasive breast cancer according to race/ethnicity. RESULTS: We included 6,033 (8.2%) Asian/Pacific Islander (API), 5,697 (7.8%) NHB, 6,688 (9.1%) Hispanic, and 54,945 (74.9%) NHW women. Breast cancer death was reduced with chemotherapy for NHB (HR, 0.48, 95% CI, 0.28-0.81), Hispanic (HR, 0.48; 95% CI, 0.25-0.94), and NHW (HR, 0.80; 95% CI, 0.65-0.99) women with an RS of 26 to 100. There was a nonsignificant reduction for API women (HR, 0.59; 95% CI, 0.28-1.24). For women with an RS of 11 to 25, there was no reduction in death for any racial/ethnic group. Among women aged ≤50 years, the reduction in breast cancer death with chemotherapy differed according to race (NHB: HR, 0.37 [95% CI, 0.20-0.67]; NHW: HR, 0.56 [95% CI, 0.44-0.74]; Pinteraction for chemotherapy * race <.0499). An exploratory subgroup analysis found that young NHB women may benefit from chemotherapy at a lower RS cutoff than other women. CONCLUSIONS: The RS was clinically validated as a predictive biomarker for NHB, Hispanic, and NHW women with ER-positive, axillary node-negative breast cancer, but it may underestimate the benefit of chemotherapy for young NHB women. If this finding is confirmed, the RS cutoff for recommending adjuvant chemotherapy for young NHB women with ER-positive, axillary node-negative breast cancer may need to be lower than for other women.

Clinical Validation of a Circulating Tumor Cell Assay Using Density Centrifugation and Automated Immunofluorescence Microscopy.

OBJECTIVES: The US Food and Drug Administration (FDA)-approved CELLSEARCH assay (Menarini Silicon Biosystems) for circulating tumor cells (CTCs) relies on expression of an epithelial cell adhesion molecule to enrich for CTCs. We sought to validate a CTC assay (RareCyte) for clinical use that instead collects a buffy coat preparation enriched for CTCs. METHODS: Normal peripheral blood specimens spiked with cultured breast and prostate cancer cells and 47 clinical samples were used to validate assay performance. Specimens were enriched for buffy coat cells and applied onto 8 glass slides. The slides were immunofluorescently stained and imaged by automated microscopy and computer-aided image analysis. RESULTS: The assay was 100% specific for detecting spiked tumor cells. For samples spiked with 25, 50, and 125 cells, the percentage coefficients of variation were 42%, 21%, and 3.7%, respectively. Linearity studies demonstrated a slope of 0.99, an intercept of 1.6, and R2 of 0.96. Recoveries at the 25-, 50-, and 125-cell levels were 92%, 111%, and 100%, respectively. Clinical samples run on both CELLSEARCH and RareCyte correlated with an R2 of 0.8 after log-transformation and demonstrated 87.5% concordance using the CELLSEARCH criteria for predicting adverse outcomes. CONCLUSIONS: The RareCyte CTC assay has comparable performance to the FDA-cleared method and is ready for further clinical validation studies.

Correction to: a Phase 2 Study of 16α-[18F]-Fluoro-17ß-Estradiol Positron Emission Tomography (FES-PET) as a Marker of Hormone Sensitivity in Metastatic Breast Cancer (MBC).

Two data points from Table 1. (continued) were published in error. The corrected data in Table 1. (continued) are shown, in italic, below.

A phase 2 study of 16α-[18F]-fluoro-17ß-estradiol positron emission tomography (FES-PET) as a marker of hormone sensitivity in metastatic breast cancer (MBC).

PURPOSE: 16α-[(18)F]-fluoro-17ß-estradiol positron emission tomography (FES-PET) quantifies estrogen receptor (ER) expression in tumors and may provide diagnostic benefit. PROCEDURES: Women with newly diagnosed metastatic breast cancer (MBC) from an ER-positive primary tumor were imaged before starting endocrine therapy. FES uptake was evaluated qualitatively and quantitatively, and associated with response and with ER expression. RESULTS: Nineteen patients underwent FES imaging. Fifteen had a biopsy of a metastasis and 15 were evaluable for response. Five patients had quantitatively low FES uptake, six had at least one site of qualitatively FES-negative disease. All patients with an ER-negative biopsy had both low uptake and at least one site of FES-negative disease. Of response-evaluable patients, 2/2 with low FES standard uptake value tumors had progressive disease within 6 months, as did 2/3 with qualitatively FES-negative tumors. CONCLUSIONS: Low/absent FES uptake correlates with lack of ER expression. FES-positron emission tomography can help identify patients with endocrine resistant disease and safely measures ER in MBC.

In situ breast cancer and microchimerism.

Microchimeric cells of fetal origin persistent in the maternal circulation post-partum are associated with protection against invasive breast cancer. Here using quantitative genomic methods, we evaluated for the presence of male fetal microchimerism in buffy coat cells from women with a prior history of breast carcinomas in situ (CIS) and in healthy controls. Fetal microchimerism was detected in 75 of 88 controls (85%) and in 57 of 89 CIS patients (64%). The odds ratio for protection against non-invasive breast disease was 0.26 (95% confidence interval 0.12-0.56; p < 0.001 adjusted for age and body mass index). Similar to women with invasive breast cancer, women with CIS who are naturally at high risk for future invasive disease were deficient for fetal microchimerism. In addition to autologous anti-tumor immune responses, the maintenance of haploidentical microchimerism may impart an allogeneic edge in immunosurveillance.

Prospective assessment of fetal-maternal cell transfer in miscarriage and pregnancy termination.

BACKGROUND: Fetal cells (microchimerism) are acquired by women during pregnancy. Fetal microchimerism persists decades later and includes cells with pluripotent capacity. Persistent microchimerism has the capacity for both beneficial and detrimental maternal health consequences. Both miscarriage and termination of pregnancy can result in fetal microchimerism. We sought to determine whether cellular fetal microchimerism is acquired during management of pregnancy loss and further explored factors that could influence fetal cell transfer, including viability of fetal tissue, surgical versus medical management and gestational age. METHODS: Pregnant women (n= 150 samples from 75 women) with singleton pregnancies undergoing a TOP (n= 63) or treatment for embryonic or fetal demise (miscarriage, n= 12) were enrolled. Mononuclear cells were isolated from blood samples drawn before, and 30 min after, treatment. Fetal cellular microchimerism concentrations were determined using quantitative PCR for a Y chromosome-specific sequence, expressed as genome equivalents of fetal DNA per 100 000 maternal cell equivalents (gEq/10(5)). Detection rate ratios were determined according to clinical characteristics. RESULTS: Cellular fetal microchimerism was found more often in post- compared with pretreatment samples, 24 versus 5% (P= 0.004) and at higher concentrations, 0-36 versus 0-0.7 gEq/10(5) (P< 0.001). Likelihood of microchimerism was higher in surgical than medical management, detection rate ratio 24.7 (P= 0.02). The detection rate ratio for TOP versus miscarriage was 16.7 for known male fetuses (P= 0.02). Microchimerism did not vary with gestational age. CONCLUSIONS: Significant fetal cell transfer occurs during miscarriage and TOP. Exploratory analyses support relationships between obstetric clinical factors and acquisition of fetal cellular microchimerism; however, our limited sample size precludes definitive analysis of these relationships, and confirmation is needed. In addition, the long-term persistence and potential consequences of fetal microchimerism on maternal health merit further investigation.

Acquisition of the rheumatoid arthritis HLA shared epitope through microchimerism.

OBJECTIVE: HLA-DRB1 alleles associated with risk of rheumatoid arthritis (RA) encode similar HLA-DRB1 sequences, called the shared epitope (SE). The most common SE sequences are QKRAA and QRRAA. Nevertheless, a substantial number of RA patients lack the SE. Bidirectional fetal-maternal trafficking results in long-term persistence of fetal cells in the mother and maternal cells in her offspring, a process known as microchimerism. This study was undertaken to discover whether RA patients who lack the SE can acquire it through microchimerism. METHODS: We studied a total of 86 female subjects who were genotypically negative for the SE, comprising 52 patients with RA and 34 healthy controls. We developed specific real-time quantitative polymerase chain reaction assays for the SE-encoded sequences QKRAA and QRRAA, and used them to test DNA extracted from peripheral blood mononuclear cells. RESULTS: Microchimerism with the SE was found significantly more often in RA patients than controls (odds ratio 4.1 [95% confidence interval 1.6-10.0], P = 0.003). Concentrations of SE microchimerism were also significantly higher among RA patients than controls (P = 0.002). In separate analyses for SE type, the prevalence of QKRAA microchimerism in RA patients versus healthy controls was 17% versus 3% (9 of 52 versus 1 of 34; P = 0.03) and the prevalence of QRRAA microchimerism was 40% versus 18% (21 of 52 versus 6 of 34; P = 0.04), respectively. Microchimerism concentrations were also higher in RA patients than healthy subjects for QKRAA (P = 0.03) and QRRAA (P = 0.03). CONCLUSION: These results indicate that RA patients who genotypically lack the SE can acquire the SE as persistent microchimerism from fetal-maternal cell exchange, suggesting that SE-encoding microchimerism could be a risk factor for RA.

Antibiotic-mediated chemoprotection enhances adaptation of E. coli PNP for herpes simplex virus-based glioma therapy.

The E. coli PNP suicide gene sensitizes solid tumors to nucleoside prodrugs, such as 6-methylpurine-2'-deoxyriboside (MeP-dR). In this study using lentiviral, MuLv, and HSV-based gene transfer, we quantified thresholds for inhibition of tumor growth and bystander killing by E. coli PNP and tested the role of intestinal flora in this process. Regressions of human glioma tumors following retroviral transduction exhibited dose dependence on both the level of PNP expression and the dose of MeP-dR administered, including strong tumor inhibition when 90-99% bystander cells comprised the tumor mass. A replication competent, non-neurovirulent herpes simplex virus (HSV) deficient in both copies of the gamma-1 34.5 gene was next engineered to express E. coli PNP under the egr-1 promoter (HSV-PNP). HSV-PNP injected intratumorally (17 million pfu/0.05 ml) in nude mice bearing 300 mg human glioma flank tumors produced a delay in tumor growth (approximately 24 days delay to one doubling). MeP-dR treatment after antibiotic therapy (to eliminate enteric flora encoding PNP enzymes) resulted in antitumor enhancement, with arrest of tumor growth (delay to doubling >50 days). Bystander killing of the magnitude described here has been difficult to accomplish with other suicide genes, such as HSV-tk or cytosine deaminase. The results establish a model for applying E. coli PNP to HSV treatment of glioma.

In vivo sensitization of ovarian tumors to chemotherapy by expression of E. coli purine nucleoside phosphorylase in a small fraction of cells.

This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing. Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells. The E. coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro. In vivo bystander killing at or near this magnitude has not been reported previously. In the present experiments, transfection of cells with the E. coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)). This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR. Much earlier killing was observed using a tat transactivated E. coli PNP vector (approximately seven-fold higher activity, 230 CU). In vivo effects on tumor growth were next examined. Human ovarian tumors transfected with E. coli PNP were excised 5 days after i.p. implantation from the peritoneal cavities of mice in order to determine both E. coli PNP enzymatic activity and the fraction of cells expressing the gene. PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization. The expression of E. coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors. Our observations lead to the conclusion that pronounced bystander killing by E. coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.

Gene therapy of cancer: activation of nucleoside prodrugs with E. coli purine nucleoside phosphorylase.

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

Components of human papillomavirus that activate transcription and support plasmid replication in human airway cells.

Human papillomaviruses (HPVs) such as types 6 and 11 can establish lifelong infections in airway epithelial cells in patients, and long-term infection can lead to pulmonary involvement and death. The mechanisms underlying this persistence depend on both the transcriptional activity of the viral enhancers and promoters and the ability of this virus to maintain its double-stranded circular DNA genome in infected tissues. We investigated the transcription and replication properties of HPV sequence elements and protein products in a human airway cell line. We showed that incorporation of the upstream regulatory region and cotransfection with expression vectors of two virus-encoded proteins, E1 and E2, conferred approximately 5,000-fold stimulation of reporter gene expression. Transient plasmid replication in transfected human airway cells and lungs of FVB/N-C57BL/6 mice was demonstrated by a modified transient replication assay. These results have important implications for viral pathogenesis in airway cells and the potential of HPV-based replicons for gene transfer into airway epithelium.

Bystander killing of melanoma cells using the human tyrosinase promoter to express the Escherichia coli purine nucleoside phosphorylase gene.

We used a gene transfer-based system to generate highly toxic purine bases in tumor cells transfected with the Escherichia coli purine nucleoside phosphorylase (PNP) gene. Because these toxic purines are membrane permeant, they mediate effective killing of neighboring cells that do not express E. coli PNP ("bystander" toxicity). In mixed cultures containing increasing percentages of cells with gene expression, 100% cancer cell growth arrest and total population killing was demonstrated when as few as 1-2% of cells expressed E. coli PNP. We used E. coli PNP to test bystander killing of human melanoma cells. A 529-bp region upstream of the human tyrosinase gene start site was shown to direct melanoma-specific expression in human cell lines. When this human tyrosinase regulatory region was used to control E. coli PNP expression, profound toxicity was observed in melanoma cells after treatment with the relatively nontoxic substrate 6-methylpurine-deoxyriboside, which is converted by E. coli PNP into the highly toxic purine base 6-methylpurine. Bystander toxicity was estimated as at least 100 cells killed for each cell expressing E. coli PNP, a level substantially higher than that of other tumor sensitization genes currently being used in clinical trails. These results suggest that the high bystander activity of the system could lead to significant antimelanoma responses in vivo.