Translating a gene expression signature for multiple myeloma prognosis into a robust high-throughput assay for clinical use

Plasma cell quantification and separation

Processing of bone marrow aspirate specimens submitted for MyPRS® analysis occurs largely as previously described [23]. CD138+ plasma cell isolation from red blood cell lysed bone marrow aspirates is performed by immunomagnetic bead selection with monoclonal mouse antihuman CD138 antibodies using the AutoMACS Pro automated separation system (Miltenyi-Biotec, Auburn, CA). Minimum PC purity of ≥80% homogeneity is confirmed by 2-color flow cytometry using CD38+/CD45− post-sort (after immunomagnetic bead selection) criteria (Becton Dickinson, San Jose, CA).

Determination of CD138+ cell presence is performed on the initial whole bone marrow aspirate by removing an aliquot from the gently homogenized bone marrow aspirate that was mixed with EDTA at the time of collection. This aliquot is incubated with CD138 PE and CD45 FITC antibodies (Miltenyi, CA), and then the red blood cells are lysed. The remaining cells are washed with phosphate buffered saline (PBS) and flow cytometry is performed. The pre-sort cell percentage (prior to immunomagnetic bead selection) is determined by identifying the CD138+/CD45- cells from the total population after red blood cell (RBC) lysis. This determination is performed on either the FACS Calibur system or the FACS Aria III system (Becton Dickinson, NJ). Once the presence of CD138+ cells has been confirmed, the bone marrow aspirate undergoes RBC-lysis and is washed with autoMACS Running Buffer (Miltenyi, CA).

Cell count is determined using the Nucleocounter NC-100 (Chemometec, Denmark) according to manufacturer recommendations. Immunomagnetic beads are then bound to the cells and the remaining unbound beads are removed through a second Running Buffer wash. The CD138+ cells are isolated from the remaining cells using the AutoMACS Pro Separator (Miltenyi, CA) according to manufacturer recommendations. If 80% cell homogeneity is not obtained, the specimen either undergoes a second immunomagnetic isolation and/or enriched using CD38 PE and CD45 FITC antibodies on the FACS Aria III (Becton Dickinson, NJ).

In keeping with institutional, federal, and Helsinki Declaration guidelines, all identifiable patients gave written informed consent for undergoing bone marrow sampling for gene expression profiling and the institutional review board of the University of Arkansas for Medical Sciences approved the research studies. Consent was not obtained from patients where data were analyzed anonymously and not associated with any identifiable or longitudinal information.

RNA isolation and microarray analysis

Cell lysis and total-RNA isolation from purified CD138+ plasma cells is performed using the RNeasy Micro Kit (Qiagen, Germany). RNA concentration and purity is determined using a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington) and the integrity is assessed using the Agilent Bioanalyzer 2100 system (CA). Double-stranded complementary DNA (cDNA) and amplified biotinylated antisense RNA (aRNA or cRNA) are synthesized from total RNA using the Affymetrix 3′ IVT Express Kit. The aRNA is fragmented and hybridized to whole-genome U133 Plus 2.0 GeneChip microarrays (Affymetrix, Santa Clara, CA), according to manufacturer recommendations. Hybridized GeneChips are scanned with the Affymetrix GeneChip Scanner 3000DX V2, an FDA-cleared, CE-IVD marked system. Scanned GeneChip files (CEL files) are normalized and assessed for hybridization success and sample quality by a proprietary gene expression data quality control system (ResultsPX™), previously described [2].

GEP70 risk scores are calculated using the method originally described by Shaughnessy et al. [6], with the additional modification of scaling the score to a range of 0 to 100 to assist in interpretation. This scaling is done using the equation [original GEP70 + 1.6] * 20 = scaled GEP70.

Control sample analysis

Positive and negative control specimens are analyzed alongside all clinical MyPRS® samples. Positive control-sample analysis is performed using the multiple myeloma cell line H929 which is grown as recommended (American Type Culture Collection, Chantilly, VA). To prepare the cell line for repeated control sample use, cells are consolidated and analyzed for homogeneity and for CD138+ presence. Once +80% CD138+ homogeneity is confirmed, the cells are pelleted, placed on RLT (plus 2-mercaptoethanol) buffer and frozen at −80°C. Each aliquot of H929 cells is tested over several months to generate sufficient data in order to calculate the standard deviation of its GEP70 risk score. A Levy Jennings plot is used to analyze the positive control specimen processed in parallel to each batch of clinical specimens, with results outside of the median +/−3SD range being rejected.

Negative control analysis is performed using aliquots of RLT (+2-mercaptoethanol) buffer and frozen. The negative control is inserted into a batch of samples at RNA isolation and is carried all the way through aRNA amplification. Detection of aRNA in the negative control specimen prior to microarray hybridization results is a sign of contamination and cause for rejection.

Replication of GEP70 signature between UAMS and Signal Genetics laboratories

To evaluate the difference between GEP70 risk scores generated in the original research laboratory (UAMS) and the clinical laboratory (Signal Genetics LLC, AR) a series of 99 bone marrow aspirates were analyzed. Specimen preparation, microarray hybridization and data analysis methods were performed as described above. GEP70 scores were compared by performing intra-class correlation, Passing and Bablok regression and chi-square analysis of high/low risk group classification in MedCalc 12.7.8 (MedCalc Software bvba, Belgium) [24].

Interpretation of GEP70 score using personalized gene expression heat maps

The GEP70 risk score for each MyPRS analysis performed was visualized by creation of a personalized two-dimensional heat map generated by the ResultsPX™ genomic data management platform developed by Signal Genetics. This system uses Microsoft SQL Server (Redmond, WA) databases, R [25] and Bioconductor [26] and custom scripts to display the expression profile of the prognostic GEP70 gene score within the context of the 559 multiple myeloma patients from two previously published datasets, including data from patients used to develop the algorithm [6]. The 5-year relapse status of each patient is shown at the top of each gene profile (red: relapse, blue: no relapse) along with the corresponding risk score.

No ‘batch effect’ modification is performed during this procedure as the 70 gene x 559 patient dataset was that used to originally develop the GEP70 algorithm. These data were produced by the UAMS MIRT laboratory and were used in the validation studies performed herein to ensure the 70-gene assay produces statistically equivalent results when performed in the Signal Genetics clinical laboratory.

Genomic profiling of paired aliquots of patient CD138+ cells in research and clinical laboratories shows high correlation of GEP70 scores

Ninety-nine patient bone marrow aspirate specimens were split into two aliquots and processed as described in both the UAMS research laboratory and at Signal Genetics’ CLIA-certified laboratory (Figure 1). The intraclass correlation coefficient for the set of 99 GEP70 scores generated by UAMS and Signal Genetics is 0.98 (95% CI: 0.97 to 0.99), indicating a high level of consistency. The Cusum test for linearity revealed no significant deviation from linearity (P = 0.17) [24].

In order to assess the clinical implications of the small difference in risk scores observed between the two laboratories, ROC analysis was performed using 5-year relapse-free survival as the binary outcome metric. The AUC of the UAMS risk score was 0.67 (95% CI 0.57 to 0.76) compared to the Signal Genetics AUC of 0.66 (95% CI: 0.56 to 0.75), a statistically insignificant difference (P = 0.402). This indicates that no significant difference exists in the association of the GEP70 score with multiple myeloma relapse risk based on the processing of a specimen in the research or clinical laboratory setting.

Analysis of control specimen GEP70 risk score shows high level of consistency in MyPRS® analyses over time

Along with each batch of clinical bone marrow aspirate samples analyzed, an aliquot of RNA from a multiple myeloma cell-line (H929) is analyzed and its GEP70 score is assessed for stability. Over a twelve-month period from August 2012 to August 2013, 102 control sample analyses were performed. As shown in Figure 2, the median value of the GEP70 score over time was 91.2, with a standard deviation of 2.7 and 3.0% coefficient of variance.

Next we sought to evaluate the reproducibility of the GEP70 scores across the dynamic range of the assay (i.e. low risk 0 to 45.2, high risk 45.2 to 100). Thirty specimens of MM RNA were analyzed in duplicate, approximately one month apart (Figure 3). A high degree of correlation was observed between the repeated measurements (r² = 0.99) with no statistically significant deviation from linearity detected by the Cusum test (P = 0.98).

Variation in clinical bone marrow aspirate specimen plasma cell content has negligible impact on RNA isolation and gene expression profile quality

Bone marrow aspirate specimens of varying absolute and relative CD138+ plasma cell content are submitted for GEP70 analysis from treatment centers throughout the United States. Immunomagnetic and fluorescence based isolation of CD138+ plasma cells is routinely performed using methods described on every specimen to isolate, and if necessary enrich, the target plasma cells in the specimen.

We investigated the association between the relative malignant cell content, RNA integrity and the resulting GEP70 risk scores by performing a retrospective analysis of data generated from routine bone marrow aspirate specimens submitted to Signal Genetics over a period of twelve months. Agilent Bioanalzyer RNA integrity number (RIN; range 0–10) and the GEP70 risk score (range 0–100) data were compiled from 1000 randomly selected specimens submitted to Signal Genetics for routine GEP70 analysis between August 2012 and July 2013.

Within this series of 1000 specimens, the CD138+ cell content ranged from the lower acceptance threshold of 0.25% to 96.2% (mean: 12.80%, median: 4.63%). Despite this wide range of cellularity, skewed toward the lower end of the spectrum, Figure 4a and b show that only a weak correlation exists between the RNA integrity number, GEP70 score and pre-sorted specimen percentage of CD138+ cells (r² = 0.13 and 0.010, respectively). After cell sorting, specimens with less than 80% purity are excluded from further analysis in order to ensure the genomic profile represents the cells of interest rather than other potentially contaminating material. Inspection of specimens with cell content between 80 and 100% revealed there is no significant association between the relative CD138+ plasma cell content and RNA integrity, or the GEP70 risk score, r² = 0.017 and r² = <0.001, respectively (Figure 4c-d).

These data show that the specimen preparation methods used to isolate the malignant cells from a patient’s bone marrow aspirate are robust and not impacted by natural variations in specimen quality and relative quantity of malignant plasma cells. This ensures the GEP70 prognostic risk score is an accurate and reproducible prediction of patient prognosis, with negligible impact from biological or other sources of specimen variation.

Determining the minimum amount of CD138+ plasma cell RNA required for reliable gene expression profiling

As stated, patient bone marrow aspirate specimens vary in terms of plasma cell number, viability and purity. To determine minimum number of viable CD138+ plasma cells necessary to generate a high quality GEP and reproducible GEP70 score, we performed two titration studies in which varying amounts of pooled MM aRNA were hybridized to Affymetrix microarrays in triplicate.

These data also showed no significant changes in GEP data quality metrics across the range assessed. All hybridizations successfully passed the automated series quality control metrics developed by Signal Genetics, which are comprised of chip and data assessments (with associated pass/warning/fail thresholds) that have been established by analyzing large databases GeneChip quality data generated using aRNA concentrations at or above those recommended by the manufacturer [2].

Consequently a threshold of > =3 ng/μL of total RNA and > =280 ng/μL aRNA was set for routine MyPRS testing. This amount of total RNA can be isolated from approximately 20,000 CD138+ plasma cells. After GeneChip hybridization, each profile must pass the ResultsPX GeneChip QC model before being used to calculate a patient’s GEP70 risk score, ensuring result integrity.

Personalized gene expression heat-map assists in interpreting a patient’s GEP70 score in the context of patients with known outcomes

For each MyPRS® specimen analyzed, the 70 gene expression values used to compute the patients risk score are combined with a matrix of 70-gene data from 559 patients used to originally train and validate the prognostic algorithm [9] (data available at NCBI GEO ID: GSE2658). These gene expression profiles were generated from newly diagnosed patients who were enrolled in Total Therapy 2 (Thalidomide/Dexamethasone or Dex + high dose melphalan (Mel) supported autologous stem cell transplantation (ASCT)) or 3 (TT2; TT3; bortezomib-thalidomide-dexamethasone + Mel-ASCT) at UAMS prior to the commencement of their treatment.

In order to visualize the relationship between direction of differential gene expression and risk of relapse, hierarchical clustering is used to arrange the matrix rows (genes), while the columns (patients) are ordered by increasing GEP70 risk score. The published relapse-free-survival (RFS) times for patients in this trial are used to label each patient as <5 yrs RFS (blue) or >5 yrs RFS (red), allowing the physician to interpret the gene expression patterns in context with the end point of interest.

As the example shown in Figure 5 illustrates, the GEP profile of the test patient is highlighted (yellow line), allowing the physician to visually compare the expression of the 70 prognostic genes in their patient compared to a large number of other multiple myeloma patients with known outcomes.