Controlling technical variation amongst 6693 patient microarrays of the randomized MINDACT trial.

Gene expression data obtained in large studies hold great promises for discovering disease signatures or subtypes through data analysis. It is also prone to technical variation, whose removal is essential to avoid spurious discoveries. Because this variation is not always known and can be confounded with biological signals, its removal is a challenging task. Here we provide a step-wise procedure and comprehensive analysis of the MINDACT microarray dataset. The MINDACT trial enrolled 6693 breast cancer patients and prospectively validated the gene expression signature MammaPrint for outcome prediction. The study also yielded a full-transcriptome microarray for each tumor. We show for the first time in such a large dataset how technical variation can be removed while retaining expected biological signals. Because of its unprecedented size, we hope the resulting adjusted dataset will be an invaluable tool to discover or test gene expression signatures and to advance our understanding of breast cancer.

Investigating the concordance in molecular subtypes of primary colorectal tumors and their matched synchronous liver metastasis.

To date, no systematic analyses are available assessing concordance of molecular classifications between primary tumors (PT) and matched liver metastases (LM) of metastatic colorectal cancer (mCRC). We investigated concordance between PT and LM for four clinically relevant CRC gene signatures. Twenty-seven fresh and 55 formalin-fixed paraffin-embedded pairs of PT and synchronous LM of untreated mCRC patients were retrospectively collected and classified according to the MSI-like, BRAF-like, TGFB activated-like and the Consensus Molecular Subtypes (CMS) classification. We investigated classification concordance between PT and LM and association of TGFBa-like and CMS classification with overall survival. Fifty-one successfully profiled matched pairs were used for analyses. PT and matched LM were highly concordant in terms of BRAF-like and MSI-like signatures, (90.2% and 98% concordance, respectively). In contrast, 40% to 70% of PT that were classified as mesenchymal-like, based on the CMS and the TGFBa-like signature, respectively, lost this phenotype in their matched LM (60.8% and 76.5% concordance, respectively). This molecular switch was independent of the microenvironment composition. In addition, the significant change in subtypes was observed also by using methods developed to detect cancer cell-intrinsic subtypes. More importantly, the molecular switch did not influence the survival. PT classified as mesenchymal had worse survival as compared to nonmesenchymal PT (CMS4 vs CMS2, hazard ratio [HR] = 5.2, 95% CI = 1.5-18.5, P = .0048; TGFBa-like vs TGFBi-like, HR = 2.5, 95% CI = 1.1-5.6, P = .028). The same was not true for LM. Our study highlights that the origin of the tissue may have major consequences for precision medicine in mCRC.

Performance Characteristics of the BluePrint® Breast Cancer Diagnostic Test.

The analytical performance of a multi-gene diagnostic signature depends on many parameters, including precision, repeatability, reproducibility and intra-tumor heterogeneity. Here we study the analytical performance of the BluePrint 80-gene breast cancer molecular subtyping test through determination of these performance characteristics. BluePrint measures the expression of 80 genes that assess functional pathways which determine the intrinsic breast cancer molecular subtypes (i.e. Luminal-type, HER2-type, Basal-type). Knowing a tumor's dominant functional pathway can help allocate effective treatment to appropriate patients. Here we show that BluePrint is a highly precise and highly reproducible test with correlations above 98% based on the generated index and subtype concordance above 99%. Therefore, BluePrint can be used as a robust and reliable tool to identify breast cancer molecular subtypes.

Decentralization of Next-Generation RNA Sequencing-Based MammaPrint® and BluePrint® Kit at University Hospitals Leuven and Curie Institute Paris.

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.

MammaPrint and BluePrint Molecular Diagnostics Using Targeted RNA Next-Generation Sequencing Technology.

Next-generation DNA sequencing is rapidly becoming an indispensable tool for genome-directed cancer diagnostics, but next-generation RNA sequencing (RNA-seq) is currently not standardly used in clinical diagnostics for expression assessment. However, multigene RNA diagnostic assays are used increasingly in the routine diagnosis of early-stage breast cancer. Two of the most widely used tests are currently available only as a central laboratory service, which limits their clinical use. We evaluated the use of RNA-seq as a decentralized method to perform such tests. The MammaPrint and BluePrint RNA-seq tests were found to be equivalent to the clinically validated microarray tests. The RNA-seq tests were highly reproducible when performed in different locations and were stable over time. The MammaPrint RNA-seq test was clinically validated. Our data demonstrate that RNA-seq can be used as a decentralized platform, yielding results substantially equivalent to results derived from the predicate diagnostic device.

A breast cancer gene signature for indolent disease.

PURPOSE: Early-stage hormone-receptor positive breast cancer is treated with endocrine therapy and the recommended duration of these treatments has increased over time. While endocrine therapy is considered less of a burden to patients compared to chemotherapy, long-term adherence may be low due to potential adverse side effects as well as compliance fatigue. It is of high clinical utility to identify subgroups of breast cancer patients who may have excellent long-term survival without or with limited duration of endocrine therapy to aid in personalizing endocrine treatment. METHODS: We describe a new ultralow risk threshold for the 70-gene signature (MammaPrint) that identifies a group of breast cancer patients with excellent 20 year, long-term survival prognosis. Tumors of these patients are referred to as "indolent breast cancer." We used patient series on which we previously established and assessed the 70-gene signature high-low risk threshold. RESULTS: In an independent validation cohort, we show that patients with indolent breast cancer had 100% breast cancer-specific survival at 15 years of follow-up. CONCLUSIONS: Our data indicate that patients with indolent disease may be candidates for limited treatment with adjuvant endocrine therapy based on their very low risk of distant recurrences or death of breast cancer.

A Computational Workflow Translates a 58-Gene Signature to a Formalin-Fixed, Paraffin-Embedded Sample-Based Companion Diagnostic for Personalized Treatment of the BRAF-Mutation-Like Subtype of Colorectal Cancers.

Colorectal cancer patients with the BRAF(p.V600E) mutation have poor prognosis in metastatic setting. Personalized treatment options and companion diagnostics are needed to better treat these patients. Previously, we developed a 58-gene signature to characterize the distinct gene expression pattern of BRAF-mutation-like subtype (accuracy 91.1%). Further experiments repurposed drug Vinorelbine as specifically lethal to this BRAF-mutation-like subtype. The aim of this study is to translate this 58-gene signature from a research setting to a robust companion diagnostic that can use formalin-fixed, paraffin-embedded (FFPE) samples to select patients with the BRAF-mutation-like subtype. BRAF mutation and gene expression data of 302 FFPE samples were measured (mutants = 57, wild-type = 245). The performance of the 58-gene signature in FFPE samples showed a high sensitivity of 89.5%. In the identified BRAF-mutation-like subtype group, 50% of tumours were known BRAF mutants, and 50% were BRAF wild-type. The stability of the 58-gene signature in FFPE samples was evaluated by two control samples over 40 independent experiments. The standard deviations (SD) were within the predefined criteria (control 1: SD = 0.091, SD/Range = 3.0%; control 2: SD = 0.169, SD/Range = 5.5%). The fresh frozen version and translated FFPE version of this 58-gene signature were compared using 170 paired fresh frozen and FFPE samples and the result showed high consistency (agreement = 99.3%). In conclusion, we translated this 58-gene signature to a robust companion diagnostic that can use FFPE samples.

Prognostic Value of MammaPrint® in Invasive Lobular Breast Cancer.

BACKGROUND: MammaPrint® is a microarray-based gene expression test cleared by the US Food and Drug Administration to assess recurrence risk in early-stage breast cancer, aimed to guide physicians in making neoadjuvant and adjuvant treatment decisions. The increase in the incidence of invasive lobular carcinomas (ILCs) over the past decades and the modest representation of ILC in the MammaPrint development data set calls for a stratified survival analysis dedicated to this specific subgroup. STUDY AIM: The current study aimed to validate the prognostic value of the MammaPrint test for breast cancer patients with early-stage ILCs. MATERIALS AND METHODS: Univariate and multivariate survival associations for overall survival (OS), distant metastasis-free interval (DMFI), and distant metastasis-free survival (DMFS) were studied in a study population of 217 early-stage ILC breast cancer patients from five different clinical studies. RESULTS AND DISCUSSION: A significant association between MammaPrint High Risk and poor clinical outcome was shown for OS, DMFI, and DMFS. A subanalysis was performed on the lymph node-negative study population. In the lymph node-negative study population, we report an up to 11 times higher change in the diagnosis of an event in the MammaPrint High Risk group. For DMFI, the reported hazard ratio is 11.1 (95% confidence interval = 2.3-53.0). CONCLUSION: Study results validate MammaPrint as an independent factor for breast cancer patients with early-stage invasive lobular breast cancer. Hazard ratios up to 11 in multivariate analyses emphasize the independent value of MammaPrint, specifically in lymph node-negative ILC breast cancers.

Equivalence of MammaPrint array types in clinical trials and diagnostics.

MammaPrint is an FDA-cleared microarray-based test that uses expression levels of the 70 MammaPrint genes to assess distant recurrence risk in early-stage breast cancer. The prospective RASTER study proved that MammaPrint Low Risk patients can safely forgo chemotherapy, which is further subject of the prospective randomized MINDACT trial. While MammaPrint diagnostic results are obtained from mini-arrays, clinical trials may be performed on whole-genome arrays. Here we demonstrate the equivalence and reproducibility of the MammaPrint test. MammaPrint indices were collected for breast cancer samples: (i) on both customized certified array types (n = 1,897 sample pairs), (ii) with matched fresh and FFPE tissues (n = 552 sample pairs), iii) for control samples replicated over a period of 10 years (n = 11,333), and iv) repeated measurements (n = 280). The array type indicated a near perfect Pearson correlation of 0.99 (95 % CI: 0.989-0.991). Paired fresh and FFPE samples showed an excellent Pearson correlation of 0.93 (95 % CI 0.92-0.94), in spite of the variability introduced by intratumoral tissue heterogeneity. Control samples showed high consistency over 10 year's time (overall reproducibility of 97.4 %). Precision and repeatability are overall 98.2 and 98.3 %, respectively. Results confirm that the combination of the near perfect correlation between array types, excellent equivalence between tissue types, and a very high stability, precision, and repeatability demonstrate that results from clinical trials (such as MINDACT and I-SPY 2) are equivalent to current MammaPrint FFPE and fresh diagnostics, and can be used interchangeably.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

MammaPrint molecular diagnostics on formalin-fixed, paraffin-embedded tissue.

MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, has been available for fresh tissue. Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Here, we describe method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrint-fresh, was performed on an independent series of matched tissue from five hospitals (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue.

Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition.

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

Independent validation of a prognostic genomic signature (ColoPrint) for patients with stage II colon cancer.

OBJECTIVES: The aim of this study was to independently validate a genomic signature developed both to assess recurrence risk in stage II patients and to assist in treatment decisions. BACKGROUND: Adjuvant therapy is recommended for high-risk patients with stage II colon cancer, but better tools to assess the patients' prognosis accurately are still required. METHODS: Previously, an 18-gene signature had been developed and validated on an independent cohort, using full genome microarrays. In this study, the gene signature was translated and validated as a robust diagnostic test (ColoPrint), using customized 8-pack arrays. In addition, clinical validation of the diagnostic ColoPrint assay was performed on 135 patients who underwent curative resection (R0) for colon cancer stage II in Munich. Fresh-frozen tissue, microsatellite instability status, clinical parameters, and follow-up data for all patients were available. The diagnostic ColoPrint readout was determined blindly from the clinical data. RESULTS: ColoPrint identified most stage II patients (73.3%) as at low risk. The 5-year distant-metastasis free survival was 94.9% for low-risk patients and 80.6% for high-risk patients. In multivariable analysis, ColoPrint was the only significant parameter to predict the development of distant metastasis with a hazard ratio of 4.28 (95% confidence interval, 1.36-13.50; P = 0.013). Clinical risk parameters from the American Society of Clinical Oncology (ASCO) recommendation did not add power to the ColoPrint classification. Technical validation of ColoPrint confirmed stability and reproducibility of the diagnostic platform. CONCLUSIONS: ColoPrint is able to predict the development of distant metastasis of patients with stage II colon cancer and facilitates the identification of patients who may be safely managed without chemotherapy.

A combined oncogenic pathway signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction.

OBJECTIVE: To develop gene expression profiles that characterise KRAS-, BRAF- or PIK3CA-activated- tumours, and to explore whether these profiles might be helpful in predicting the response to the epidermal growth factor receptor (EGFR) pathway inhibitors better than mutation status alone. DESIGN: Fresh frozen tumour samples from 381 colorectal cancer (CRC) patients were collected and mutations in KRAS, BRAF and PIK3CA were assessed. Using microarray data, three individual oncogenic and a combined model were developed and validated in an independent set of 80 CRC patients, and in a dataset from metastatic CRC patients treated with cetuximab. RESULTS: 175 tumours (45.9%) harboured oncogenic mutations in KRAS (30.2%), BRAF (11.0%) and PIK3CA (11.5%). Activating mutation signatures for KRAS (75 genes), for BRAF (58 genes,) and for PIK3CA (49 genes) were developed. The development of a combined oncogenic pathway signature-classified tumours as 'activated oncogenic', or as 'wildtype-like' with a sensitivity of 90.3% and a specificity of 61.7%. The identified signature revealed other mechanisms that can activate ERK/MAPK pathway in KRAS, BRAF and PIK3CA wildtype patients. The combined signature is associated with response to cetuximab treatment in patients with metastatic CRC (HR 2.51, p<0.0009). CONCLUSION: A combined oncogenic pathway signature allows the identification of patients with an active EGFR-signalling pathway that could benefit from downstream pathway inhibition.

Assaying endogenous phosphatidylinositol-4-phosphate 5-kinase (PIP5K) activities.

Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proliferation, migration, integrin activation, and transcription. PtdIns(4,5)P2 is at the heart of phosphoinositide signaling; its levels are controlled by enzymes that synthesize it and those that degrade it. Phosphatidylinositol-4-phosphate 5-kinases (PIP5 K) phosphorylate PtdIns4P on the 5-position and constitute the major pathway for the generation of PtdIns(4,5)P2. We will discuss how to suppress the expression of human PIP5 Kbeta using RNAi and how to measure the activity and levels of the endogenous enzyme. We describe a method to immunoprecipitate the endogenous PIP5 Kbeta and to assay its activity. Western blotting with another panel of antibodies is then used to determine the levels of endogenous PIP5 Kbeta in the immunoprecipitates.

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis.

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).

Microarray analysis of bleomycin-exposed lymphoblastoid cells for identifying cancer susceptibility genes.

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers.

Involvement of cell cycle control in bleomycin-induced mutagen sensitivity.

Bleomycin-induced chromosomal instability, generally referred to as mutagen sensitivity, is associated with an increased risk for the development of environmentally related cancer including head and neck squamous cell carcinoma and lung cancer. On average, the cultured lymphocytes of patients with these types of cancer show an increased number of chromatid breaks per cell after bleomycin exposure in the late S or G2 phase of the cell cycle as compared to lymphocytes from control persons. The aim of the present study was to investigate whether cell cycle regulation is involved in mutagen sensitivity. We determined cell cycle arrest after bleomycin-induced DNA damage in 21 lymphoblastoid cell lines that varied in mutagen sensitivity score. An ataxia telangiectasia (AT) cell line was included for comparison. Using a cut-off point of 0.70 breaks per cell, eight cell lines were classified as insensitive and 13 cell lines showed the hypersensitive phenotype. Compared to insensitive cell lines, bleomycin-treated hypersensitive cells remained at a relatively high level of DNA synthesis, as measured by thymidine incorporation, and showed a decreased accumulation of cells in G2 and M phase, as measured by flow cytometry. AT cells showed an extremely high mutagen sensitivity score, a high level of DNA synthesis, and a strong G2 block. In conclusion, mutagen sensitivity is associated with "damage-resistant growth," which is indicative of impaired cell cycle arrest. By which specific pathway(s) this checkpoint defect is explained has yet to be elucidated; however, it is probably distinct from the checkpoint defect in AT cells. Environ. Mol. Mutagen. 40:79-84, 2002.