Identification of an optimal mutant allele frequency to detect activating KRAS, NRAS, and BRAF mutations in a commercial cell-free DNA next-generation sequencing assay in colorectal and pancreatic adenocarcinomas.

Background: Evaluation for activating mutations in KRAS, NRAS, and BRAF in colorectal cancer (CRC) and in KRAS in pancreatic ductal adenocarcinoma (PDAC) is essential for clinical care. Plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) allows convenient assessment of a tumor's molecular profile, however low tumor DNA shedding limits sensitivity. We investigated mutant allele frequency (MAF) of other oncogenic dominant genes to identify a threshold for accurate detection of KRAS, NRAS, and BRAF (RAS/RAF) mutations in cfDNA. Methods: Molecular and clinical data were obtained from the Duke Molecular Registry of Tumors and the SCRUM-Japan GOZILA study. Patients with CRC or PDAC and a KRAS, NRAS, or BRAF activating single nucleotide variant (SNV) present on tissue NGS and with available cfDNA assays were included. Recursive partitioning and Wilcoxon-rank statistics methods identified potential cut-points for discriminative MAF values. Results: One hundred and thirty-five CRC and 30 PDAC cases with 198 total cfDNA assays met criteria. Greatest non-RAS/RAF dominant gene MAF of 0.34% provided maximum discrimination for predicting RAS/RAF SNV detection. Sensitivity for RAS/RAF SNVs increased with dominant gene MAF, with MAF ≥1% predicting sensitivity >98%, MAF between 0.34 and 1% predicting sensitivity of 84.0%, and MAF £0.34% predicting sensitivity of 50%. For 43 cfDNA assays that did not detect RAS/RAF SNVs, 18 assays detected 34 other oncogenic variants, of which 80.6% were not also detected on tissue. Conclusions: Non-RAS/RAF dominant oncogenic mutation MAF ≥1% on cfDNA NGS predicts high sensitivity to detect RAS/RAF oncogenic SNVs in CRC and PDAC. MAF £0.34% indicates an assay may not reliably detect RAS/RAF SNVs, despite detection on tissue testing. Most variants from assays that did not detect RAS/RAF had MAF <1% and were not detected on tissue, suggesting potential confounding. These data suggest a practical approach to determining cfDNA assay adequacy, with implications for guiding clinical decisions in CRC and PDAC.

Concordance Between Genomic Alterations Detected by Tumor and Germline Sequencing: Results from a Tertiary Care Academic Center Molecular Tumor Board.

OBJECTIVE: The majority of tumor sequencing currently performed on cancer patients does not include a matched normal control, and in cases where germline testing is performed, it is usually run independently of tumor testing. The rates of concordance between variants identified via germline and tumor testing in this context are poorly understood. We compared tumor and germline sequencing results in patients with breast, ovarian, pancreatic, and prostate cancer who were found to harbor alterations in genes associated with homologous recombination deficiency (HRD) and increased hereditary cancer risk. We then evaluated the potential for a computational somatic-germline-zygosity (SGZ) modeling algorithm to predict germline status based on tumor-only comprehensive genomic profiling (CGP) results. METHODS: A retrospective chart review was performed using an academic cancer center's databases of somatic and germline sequencing tests, and concordance between tumor and germline results was assessed. SGZ modeling from tumor-only CGP was compared to germline results to assess this method's accuracy in determining germline mutation status. RESULTS: A total of 115 patients with 146 total alterations were identified. Concordance rates between somatic and germline alterations ranged from 0% to 85.7% depending on the gene and variant classification. After correcting for differences in variant classification and filtering practices, SGZ modeling was found to have 97.2% sensitivity and 90.3% specificity for the prediction of somatic versus germline origin. CONCLUSIONS: Mutations in HRD genes identified by tumor-only sequencing are frequently germline. Providers should be aware that technical differences related to assay design, variant filtering, and variant classification can contribute to discordance between tumor-only and germline sequencing test results. In addition, SGZ modeling had high predictive power to distinguish between mutations of somatic and germline origin without the need for a matched normal control, and could potentially be considered to inform clinical decision-making.

Automated next-generation profiling of genomic alterations in human cancers.

The lack of validated, distributed comprehensive genomic profiling assays for patients with cancer inhibits access to precision oncology treatment. To address this, we describe elio tissue complete, which has been FDA-cleared for examination of 505 cancer-related genes. Independent analyses of clinically and biologically relevant sequence changes across 170 clinical tumor samples using MSK-IMPACT, FoundationOne, and PCR-based methods reveals a positive percent agreement of >97%. We observe high concordance with whole-exome sequencing for evaluation of tumor mutational burden for 307 solid tumors (Pearson r = 0.95) and comparison of the elio tissue complete microsatellite instability detection approach with an independent PCR assay for 223 samples displays a positive percent agreement of 99%. Finally, evaluation of amplifications and translocations against DNA- and RNA-based approaches exhibits >98% negative percent agreement and positive percent agreement of 86% and 82%, respectively. These methods provide an approach for pan-solid tumor comprehensive genomic profiling with high analytical performance.

Implementation of a Molecular Tumor Registry to Support the Adoption of Precision Oncology Within an Academic Medical Center: The Duke University Experience.

Comprehensive genomic profiling to inform targeted therapy selection is a central part of oncology care. However, the volume and complexity of alterations uncovered through genomic profiling make it difficult for oncologists to choose the most appropriate therapy for their patients. Here, we present a solution to this problem, The Molecular Registry of Tumors (MRT) and our Molecular Tumor Board (MTB). PATIENTS AND METHODS: MRT is an internally developed system that aggregates and normalizes genomic profiling results from multiple sources. MRT serves as the foundation for our MTB, a team that reviews genomic results for all Duke University Health System cancer patients, provides notifications for targeted therapies, matches patients to biomarker-driven trials, and monitors the molecular landscape of tumors at our institution. RESULTS: Among 215 patients reviewed by our MTB over a 6-month period, we identified 176 alterations associated with therapeutic sensitivity, 15 resistance alterations, and 51 alterations with potential germline implications. Of reviewed patients, 17% were subsequently treated with a targeted therapy. For 12 molecular therapies approved during the course of this work, we identified between two and 71 patients who could qualify for treatment based on retrospective MRT data. An analysis of 14 biomarker-driven clinical trials found that MRT successfully identified 42% of patients who ultimately enrolled. Finally, an analysis of 4,130 comprehensive genomic profiles from 3,771 patients revealed that the frequency of clinically significant therapeutic alterations varied from approximately 20% to 70% depending on the tumor type and sequencing test used. CONCLUSION: With robust informatics tools, such as MRT, and the right MTB structure, a precision cancer medicine program can be developed, which provides great benefit to providers and patients with cancer.

Next-Generation Sequencing Concordance Analysis of Comprehensive Solid Tumor Profiling between a Centralized Specialty Laboratory and the Decentralized Personal Genome Diagnostics elio Tissue Complete Kitted Solution.

Genomic tumor profiling by next-generation sequencing (NGS) allows for large-scale tumor testing to inform targeted cancer therapies and immunotherapies, and to identify patients for clinical trials. These tests are often underutilized in patients with late-stage solid tumors and are typically performed in centralized specialty laboratories, thereby limiting access to these complex tests. Personal Genome Diagnostics Inc., elio tissue complete NGS solution is a comprehensive DNA-to-report kitted assay and bioinformatics solution. Comparison of 147 unique specimens from >20 tumor types was performed using the elio tissue complete solution and Foundation Medicine's FoundationOne test, which is of similar size and gene content. The analytical performance of all genomic variant types was evaluated. In general, the overall mutational profile is highly concordant between the two assays, with agreement in sequence variants reported between panels demonstrating >95% positive percentage agreement for single-nucleotide variants and insertions/deletions in clinically actionable genes. Both copy number alterations and gene translocations showed 80% to 83% positive percentage agreement, whereas tumor mutation burden and microsatellite status showed a high level of concordance across a range of mutation loads and tumor types. The Personal Genome Diagnostics Inc., elio tissue complete assay is comparable to the FoundationOne test and will allow more laboratories to offer a diagnostic NGS assay in house, which will ultimately reduce time to result and increase the number of patients receiving molecular genomic profiling and personalized treatment.

Predictive Value of Combining Biomarkers for Clinical Outcomes in Advanced Non-Small Cell Lung Cancer Patients Receiving Immune Checkpoint Inhibitors.

INTRODUCTION: A high tumor mutational burden (TMB) (≥10 mut/Mb) has been associated with improved clinical benefit in non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICI) and is a tumor agnostic indication for pembrolizumab across tumor types. We explored whether combining TMB with programmed cell death ligand 1 (PD-L1) and pretreatment neutrophil-lymphocyte ratio (NLR) was associated with improved outcomes in ICI-treated NSCLC. METHODS: We retrospectively analyzed patients treated with ICI with Foundation One genomic testing, including TMB. Optimal cutoff for prediction of response by TMB was determined by receiver operating characteristic analysis, and area under the curve (AUC) was calculated for all 3 biomarkers and combinations. Cox model was used to assess prognostic factors of overall survival (OS) and time to progression (TTP). Survival cutoffs calculated with Kaplan-Meier survival curves were TMB ≥10 mut/Mb, PD-L1 ≥50%, NLR <5, and combined biomarkers. RESULTS: Data from 88 patients treated were analyzed. The optimal TMB cutoff was 9.24 mut/Mb (AUC, 0.62), improving to 0.74 combining all 3 biomarkers. Adjusted Cox model showed that TMB ≥10 mut/Mb was an independent factor of OS (hazard ratio [HR], 0.31; 95% confidence interval; 0.14-0.69; P = .004) and TTP (HR, 0.46; 95% CI, 0.27-0.77; P = .003). The combination of high TMB with positive PD-L1 and low NLR was significantly associated with OS (P = .038) but not TTP. CONCLUSIONS: TMB has modest predictive and prognostic power for clinical outcomes after ICI treatment. The combination of TMB, PD-L1, and NLR status improves this power.

Donor-Derived Neuroendocrine Carcinoma Transmission to Two Kidney Transplant Recipients Demonstrated by Short Tandem Repeat Analysis: A Case Report.

Cancer transmission from a donor organ to a transplant recipient is a rare but not infrequently fatal event. We report a case of lung cancer transmission from a deceased donor to 2 kidney recipients. Approximately 1 year after uneventful kidney transplantation, both recipients developed acute kidney failure. Computed tomography imaging of abdomen and pelvis for both recipients showed masses in the transplanted kidneys along with innumerable masses in the livers. Pathologic examinations for both cases demonstrated high-grade neuroendocrine carcinoma with "mirror image" histologic findings in the transplant kidneys with liver metastases. Short tandem repeat (STR) analyses were performed to determine the origin of the tumors. STRs of both tumors were nearly identical to that of the donor, proving that both tumors were from the same donor. Immunohistochemical analyses showed that both tumors were positive for thyroid transcription factor 1, supporting a lung primary. One recipient died as a direct sequela to metastatic tumor, and the other required transplant nephrectomy and chemotherapy. Awareness of this largely nonpreventable complication and prompt molecular testing if cancer transmission is suspected are important.

Early experience with universal preprocedural testing for SARS-CoV-2 in a relatively low-prevalence area.

We implemented universal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing of patients undergoing surgical procedures as a means to conserve personal protective equipment (PPE). The rate of asymptomatic coronavirus disease 2019 (COVID-19) was <0.5%, which suggests that early local public health interventions were successful. Although our protocol was resource intensive, it prevented exposures to healthcare team members.

Therapeutic outcomes in non-small cell lung cancer with BRAF mutations: a single institution, retrospective cohort study.

BACKGROUND: Data describing therapeutic outcomes in patients with non-small cell lung cancers (NSCLC) with BRAF mutations remains limited. METHODS: We conducted a retrospective cohort study of 31 patients with metastatic NSCLC treated at Duke University Hospital who had been identified by next-generation sequencing methods to bear a BRAF mutation in their tumor in order to evaluate clinical response to immunotherapy and chemotherapy. RESULTS: Sixty-five percent of patients identified in this cohort were current or former smokers. Fourteen (45.2%) of patients had a BRAF V600E mutation and 17 (54.8%) had a non-V600E mutation. Median progression-free survival (PFS) in the 23 patients who received first-line chemotherapy was 6.4 months [95% confidence interval (CI), 2.3 to 13.0]. Overall survival (OS) in patients who received first-line chemotherapy showed a median survival of 18 months (95% CI, 7.4 to 28.6). OS comparing patients who had never received immunotherapy at any point was 18.4 months (95% CI, 4.1 to NE) compared to 19.0 months (95% CI, 9.9 to 28.6) in those who had received immunotherapy. We did not find a statistically significant difference in OS in patients with BRAF V600E, BRAF amplification, or non-V600E mutations. There was also no difference in OS in patients treated with targeted BRAF inhibitors compared to those who were not treated with targeted BRAF inhibitors. CONCLUSIONS: We describe therapeutic outcomes for patients with metastatic NSCLC with BRAF mutations treated with either cytotoxic chemotherapy or immunotherapy. Although the sample size is small, the survival curves do not suggest improved clinical activity in this population when treated with immunotherapy.

Molecular testing for indeterminate thyroid nodules: Performance of the Afirma gene expression classifier and ThyroSeq panel.

BACKGROUND: The ThyroSeq mutational panel and Afirma gene expression classifier (GEC) are used to risk stratify cytologically indeterminate thyroid nodules. In the current study, the authors evaluated the performance of these tests within the context of ultrasonographic features and with the incorporation of the noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) nomenclature. METHODS: The authors reviewed nodules using ThyroSeq or Afirma GEC testing. For nodules that were surgically resected, both tests were studied within the context of ultrasound findings, comparing performance stratified by the 2015 American Thyroid Association guideline (ATA 2015) sonographic patterns and assessing the positive predictive value (PPV) of these tests both including and excluding NIFTP in the malignant category. RESULTS: A total of 304 cases were identified, 119 of which were resected. All cases that met the criteria for NIFTP on excision demonstrated either high-risk mutations on ThyroSeq or a "suspicious" result on Afirma GEC. When NIFTP cases were shifted from the malignant to nonmalignant category, the PPV of "positive" tests for both ThyroSeq and Afirma GEC decreased from 42.9% to 14.3% (an absolute decrease of 28.6%) and 30.1% to 25.3% (an absolute decrease of 4.8%), respectively. No cases of malignancy were found in the ATA 2015 "very low suspicion" group, even with a "suspicious" Afirma GEC result. CONCLUSIONS: Both the ThyroSeq and Afirma GEC tests demonstrated decreases in the PPV when NIFTP was considered nonmalignant. In the era of NIFTP, a "positive" test result for either the Afirma GEC or ThyroSeq should be interpreted in light of clinical factors and should not exclude conservative (ie, lobectomy) surgical management. ATA 2015 "very low suspicion" nodules, even with "suspicious" Afirma GEC results, were not found to demonstrate malignancy in this series. Cancer Cytopathol 2018. © 2018 American Cancer Society.

Young Investigator Challenge: Molecular testing in noninvasive follicular thyroid neoplasm with papillary-like nuclear features.

BACKGROUND: Molecular testing provides an important ancillary study for thyroid nodules with indeterminate cytology. The nomenclature shift to "noninvasive follicular thyroid neoplasm with papillary-like nuclear features" (NIFTP) will impact the performance of molecular tests. For the current study, the authors reviewed the performance of the Afirma gene-expression classifier (GEC) and the University of Pittsburgh Medical Center (UPMC) targeted mutation panel tests in thyroid nodules that were subsequently diagnosed as NIFTP on surgical resection. METHODS: In total, 302 nodules were sent for molecular testing between June 2012 and June 2016. These cases were retrospectively reviewed to identify patients who underwent subsequent surgical resection and were diagnosed with follicular variant of papillary thyroid carcinoma (FVPTC). Twenty-five nodules that were diagnosed as FVPTC met the initial inclusion criteria. These cases were reviewed using strict criteria to identify NIFTP. RESULTS: Eight cases met criteria for NIFTP, and 4 NIFTPs underwent Afirma testing. Cytology diagnoses were all Bethesda category III, with 3 diagnosed as atypia of undetermined significance (AUS) and 1 diagnosed as follicular lesion of undetermined significance (FLUS). All of these nodules were identified as "suspicious" using GEC. Four NIFTPs underwent testing at UPMC, all using ThyroSeq V2. The cytology diagnoses for these nodules also were category III, with the exception of 1 nodule that was category IV, suspicious for follicular neoplasm. All NIFTPs were positive for mutations, all of which were RAS mutations (NRAS, KRAS). One patient who had a nodule classified as NIFTP had metastatic carcinoma identified in a lymph node. Another who had a 6-cm tumor had coexisting NRAS and TERT mutations. CONCLUSIONS: The current results indicate that NIFTP is a rare tumor if defined by strict criteria, that both the GEC and UPMC methods indicate abnormalities in NIFTP, and further independent study will be needed to better characterize the molecular and clinical characteristics of NIFTP. Cancer Cytopathol 2016;124:893-900. © 2016 American Cancer Society.

Molecular testing for the BRCA1 and BRCA2 Ashkenazi Jewish founder mutations: a report on the College of American Pathologists proficiency testing surveys.

PURPOSE: The purpose of this study was to analyze laboratory performance on proficiency testing surveys offered jointly by the College of American Pathologists/American College of Medical Genetics and Genomics biannually for the three common Ashkenazi Jewish founder mutations in the BRCA1 and BRCA2 genes. METHODS: Survey responses were analyzed for accuracy of genotype determination and the associated clinical interpretation. Data on an individual laboratory's participation over time, number of samples tested, turnaround time, and test methodology were also reviewed. RESULTS: Between 2003 and 2012, 23 US laboratories and 39 international laboratories participated. There were six genotyping errors, with a corresponding analytical sensitivity of 99.0% (479/484 challenges; 95% confidence interval: 97.6-99.7%) and an analytic specificity of 99.9% (870/871; 95% confidence interval: 99.4-99.9%). Among the 1,325 clinical interpretations, 92.5% (1,226/1,325; 95% confidence interval: 91.0-93.9%) matched the intended response. Most of the 99 discrepancies-81% (80/99)-incorrectly interpreted the risk for a negative test result as having a lifetime risk of breast cancer "that is the same as that in the general population" instead of "that cannot be determined without BRCA mutation testing of the affected relative." CONCLUSION: Clinical laboratories demonstrated excellent analytical sensitivity and specificity. The clinical interpretation requires additional education, focusing on the clinical interpretation of negative test results for these three mutations.

False positives in multiplex PCR-based next-generation sequencing have unique signatures.

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.

Genes with bimodal expression are robust diagnostic targets that define distinct subtypes of epithelial ovarian cancer with different overall survival.

In some cancer types, certain genes behave as molecular switches, with on and off expression states. These genes tend to define tumor subtypes associated with different treatments and different patient survival. We hypothesized that clinically relevant molecular switch genes exist in epithelial ovarian cancer. To test this hypothesis, we applied a bimodal discovery algorithm to a publicly available ovarian cancer expression microarray data set, GSE9891 [285 tumors: 246 malignant serous (MS), 20 endometrioid (EM), and 18 low malignant potential (LMP) ovarian carcinomas]. Genes with robust bimodal expression patterns were identified across all ovarian tumor types and also within selected subtypes: 73 bimodal genes demonstrated differential expression between LMP versus MS and EM; 22 bimodal genes distinguished MS from EM; and 14 genes had significant association with survival among MS tumors. When these genes were combined into a single survival score, the median survival for patients with a favorable versus unfavorable score was 65 versus 29 months (P < 0.0001, hazard ratio = 0.4221). Two independent data sets [high-grade, advanced-stage serous (n = 53) and advanced-stage (n = 119) ovarian tumors] validated the survival score performance. We conclude that genes with bimodal expression patterns not only define clinically relevant molecular subtypes of ovarian carcinoma but also provide ideal targets for translation into the clinical laboratory.

Type III TGF-ß receptor enhances colon cancer cell migration and anchorage-independent growth.

The type III TGF-ß receptor (TßRIII or betagylcan) is a TGF-ß superfamily coreceptor with emerging roles in regulating TGF-ß superfamily signaling and cancer progression. Alterations in TGF-ß superfamily signaling are common in colon cancer; however, the role of TßRIII has not been examined. Although TßRIII expression is frequently lost at the message and protein level in human cancers and suppresses cancer progression in these contexts, here we demonstrate that, in colon cancer, TßRIII messenger RNA expression is not significantly altered and TßRIII expression is more frequently increased at the protein level, suggesting a distinct role for TßRIII in colon cancer. Increasing TßRIII expression in colon cancer model systems enhanced ligand-mediated phosphorylation of p38 and the Smad proteins, while switching TGF-ß and BMP-2 from inhibitors to stimulators of colon cancer cell proliferation, inhibiting ligand-induced p21 and p27 expression. In addition, increasing TßRIII expression increased ligand-stimulated anchorage-independent growth, a resistance to ligand- and chemotherapy-induced apoptosis, cell migration and modestly increased tumorigenicity in vivo. In a reciprocal manner, silencing endogenous TßRIII expression decreased colon cancer cell migration. These data support a model whereby TßRIII mediates TGF-ß superfamily ligand-induced colon cancer progression and support a context-dependent role for TßRIII in regulating cancer progression.

Donor cell leukemia in umbilical cord blood transplant patients: a case study and literature review highlighting the importance of molecular engraftment analysis.

Donor cell neoplasms are rare complications of treatment regimens that involve stem cell transplantation for hematological malignancies, myelodysplastic processes, or certain genetic or metabolic disorders. We report a case of donor cell leukemia in a pediatric patient with a history of acute myeloid leukemia that manifested as recurrent AML FAB type M5 fourteen months after umbilical cord blood transplantation. Although there was some immunophenotypic drift from the patient's original AML and their posttransplant presentation, the initial pathological impression was of recurrent disease. Bone marrow engraftment analysis by multiplex PCR of short tandem repeat markers performed on the patient's diagnostic specimen showed complete engraftment by donor cells, with a loss of heterozygosity in the donor alleles on chromosome 7. This led to the reinterpretation of this patient's disease as donor-derived leukemia. This interpretation was supported by a routine karyotype and fluorescence in situ hybridization analysis showing loss of chromosome 7 and a male (donor) chromosome complement in this female patient. Also noted was a loss of the patient's presenting chromosomal abnormality, t(11;19)(q23;p13). This case highlights the need for close coordination between all aspects of clinical testing for the transplant patient, including molecular engraftment studies, when distinguishing the very common complication of recurrent disease from the exceedingly rare complication of donor cell leukemia.

Intratumor heterogeneity and precision of microarray-based predictors of breast cancer biology and clinical outcome.

PURPOSE: Identifying sources of variation in expression microarray data and the effect of variance in gene expression measurements on complex predictive and diagnostic models is essential when translating microarray-based experimental approaches into clinical assays. The technical reproducibility of microarray platforms is well established. Here, we investigate the additional impact of intratumor heterogeneity, a largely unstudied component of variance, on the performance of several microarray-based assays in breast cancer. PATIENTS AND METHODS: Genome-wide expression profiling was performed on 50 core needle biopsies from 18 breast cancer patients using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Global profiles of expression were characterized using unsupervised clustering methods and variance components models. Array-based measures of estrogen receptor (ER) and progesterone receptor (PR) status were compared with immunohistochemistry. The precision of genomic predictors of ER pathway status, recurrence risk, and sensitivity to chemotherapeutics was evaluated by interclass correlation. RESULTS: Global patterns of gene expression demonstrated that intratumor variation was substantially less than the total variation observed across the patient population. Nevertheless, a fraction of genes exhibited significant intratumor heterogeneity in expression. A high degree of reproducibility was observed in single-gene predictors of ER (intraclass correlation coefficient [ICC] = 0.94) and PR expression (ICC = 0.90), and in a multigene predictor of ER pathway activation (ICC = 0.98) with high concordance with immunohistochemistry. Substantial agreement was also observed for multigene signatures of cancer recurrence (ICC = 0.71) and chemotherapeutic sensitivity (ICC = 0.72 and 0.64). CONCLUSION: Intratumor heterogeneity, although present at the level of individual gene expression, does not preclude precise microarray-based predictions of tumor behavior or clinical outcome in breast cancer patients.

SMAD4 is required for development of maximal endotoxin tolerance.

Initial exposure of monocytes/macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Molecular mechanisms responsible for endotoxin tolerance are not well defined. We and others have shown that IL-1R-associated kinase (IRAK)-M and SHIP-1 proteins, negative regulators of TLR4 signaling, increase in tolerized cells. TGF-beta1, an anti-inflammatory cytokine, is upregulated following LPS stimulation, mediating its effect through SMAD family proteins. Using a monocytic cell line, THP1, we show that LPS activates endogenous SMAD4, inducing its migration into the nucleus and increasing its expression. Secondary challenge with high dose LPS following initial low-dose LPS exposure does not increase IRAK-M or SHIP1 protein expression in small hairpin (sh)SMAD4 THP-1 cells compared with control shLUC THP1 cells. TNF-alpha concentrations in culture supernatants after second LPS challenge are higher in shSMAD4 THP-1 cells than shLUC THP1 cells, indicating failure to induce maximal tolerance in absence of SMAD4 signaling. Identical results are seen in primary murine macrophages and mouse embryonic fibroblasts, demonstrating the biological significance of our findings. TGF-beta1 treatment does not increase IRAK-M or SHIP1 protein expression in shSMAD4 THP-1 cells, whereas it does so in shLUC THP1 cells, indicating that TGF-beta1 regulates IRAK-M and SHIP1 expression through a SMAD4-dependent pathway. Knockdown of endogenous SHIP1 by shSHIP1 RNA decreases native and inducible IRAK-M protein expression and prevents development of endotoxin tolerance in THP1 cells. We conclude that in THP-1 cells and primary murine cells, SMAD4 signaling is required for maximal induction of endotoxin tolerance via modulation of SHIP1 and IRAK-M.