Supplementary appendix from Patterns of Oncogene Coexpression at Single-Cell Resolution Influence Survival in Lymphoma
Authors: Hoppe Michal Marek , Jaynes Patrick , Shuangyi Fan , Peng Yanfen , Sridhar Shruti , Hoang Phuong Mai , Liu Clementine Xin , De Mel Sanjay , Poon Limei , Chan Esther Hian Li , Lee Joanne , Ong Choon Kiat , Tang Tiffany , Lim Soon Thye , Nagarajan Chandramouli , Grigoropoulos Nicholas F. , Tan Soo-Yong , Hue Susan Swee-Shan , Chang Sheng-Tsung , Chuang Shih-Sung , Li Shaoying , Khoury Joseph D. , Choi Hyungwon , Harris Carl , Bottos Alessia , Gay Laura J. , Runge Hendrik F.P. , Moutsopoulos Ilias , Mohorianu Irina , Hodson Daniel J. , Farinha Pedro , Mottok Anja , Scott David W. , Pitt Jason J. , Chen Jinmiao , Kumar Gayatri , Kannan Kasthuri , Chng Wee Joo , Chee Yen Lin , Ng Siok-Bian , Tripodo Claudio , Jeyasekharan Anand D. 
Published: May 2023

<p>Supplementary methods.
Supplementary Figure 1. Phenotyping of B-cells in non-malignant tissues. A, Quantitation of marker positivity across ten tonsil and two reactive lymph node samples (rLN). Analysis is spatially resolved between the GC and extra-GC zones. B, Spatial map of cellular coordinates based on cell segmentation of images in Figure 1B. Marker-positivity is indicated, and a total proportion of positive and negative cells is depicted as a pie chart. These maps were used to derive sub-population phenotypes depicted in Figure 1C. Scale bar is 100μm. C, Proliferation analysis (i.e., Ki67-positivity) among sub-populations in five tonsil samples. Median with interquartile range, whiskers denote 10th and 90th percentile.
Supplementary figure 2. Example pseudo-colored mfIHC images for MYC, BCL2, BCL6 cases in DLBCL. Images of a range of mean fluorescent intensities are shown with equal scaling for reference.
Supplementary figure 3. Global distribution of MYC, BCL2 and BCL6 sub-populations within DLBCL cohorts. Heat-maps displaying the percentage extent of individual markers and each sub-population within the DLBCL NUH, CMMC, SGH and MDA cohorts. Hierarchical k-means clustering of patients according to sub-population extent is applied. Positivity shading for single markers ranges between 0-100% positivity, whereas shading for sub-populations reflects 0-50% positivity and remains fully saturated until 100%. IPI Risk Group - International Prognostic Index Risk Group, FISH - fluorescence in situ hybridization.
Supplementary figure 4. Intra-tumor heterogeneity of sub-populations. A, Correlation of sub-population extent quantification between two biopsies of the same patient for which at least two tissue microarray (TMA) biopsies are available. Correlation is shown separately for lymph node and extranodal biopsies. Spearman rho is indicated for each correlation. Axes are in exponential and equivalent in all panels. B, Sub-population percentage extent quantification across multiple TMA cores (columns) of the same patient (rows). Pie charts are ordered according to decreasing cell numbers evaluated per core. All patients from the NUH cohort with at least five cores are evaluated. A heterogenous cluster is highlighted by the red box.
Supplementary figure 5. Spatial heterogeneity of sub-population interactions. A, Conceptual schematic of pair correlation function (PCF) plots depicting a clustered distribution (left, green) and a random distribution (right, grey). Representative counterpart spatial maps are above each plot. B, PCF analysis for sub-populations to investigate spatial clustering (top). Mean results for two independent cohorts (shading is cohort standard deviation). An example tissue microarray core is shown as physical distance reference for spatial analyses (bottom left). Absolute number of neighboring cells expected within a given radius (data from 3500 randomly selected cells across all images, mean with standard deviation) (bottom right). C, Actual spatial map of sub-populations of an example DLBCL case (top). Extent of all sub-populations within the sample is shown on the left. Simulated, hypothetical random distribution of cells for the same case (middle). PCF analysis for the shown sample and its matched simulated random distribution (bottom). Scale bars in B and C are 100µm. D, Mean deviations from expected neighbor abundance (Δ%) summarizing cell-cell interactions between sub-populations for the sample shown in (C). E, Sub-population interaction matrices from spatially distinct biopsies (cores in tissue microarray) for example DLBCL patients. Biopsies of stable, spatially homogenous, sub-population interaction profiles are grouped (top), whereas biopsies of a differing, heterogenous, interaction profile are grouped separately (bottom).
Supplementary figure 6. Global deviations from expected spatial neighbor abundance (Δ%). Hierarchical clustering (minimum variance method) of measured Δ% for all cases in the SGH and MDA cohorts. Extents of sub-populations are indicated for reference (top). For the MDA cohort, multiple biopsies (n = 1-3) from the same patient were included in the analysis to determine spatial interaction similarity across spatially distinct regions (bottom).
Supplementary figure 7. Correlation of predicted MYC, BCL2 and BCL6 sub-population percentage extent based on single oncogene positivity and observed percentage extent in DLBCL cohorts. Spearman rho, axes are equivalent in all panels.
Supplementary figure 8. Variance of M+2+6- percentage extent in the context of positivity calling across a 15% cut-off. A, M+2+6- scoring variance across multiple pathological imaging fields. All whole-tissue DLBCL sections from University of Palermo (UP), and samples from the NUH TMA with at least four fields scored per patient and a mean M+2+6- score above 5% are shown. Mean with SD. Ordinates between 50-100% are compressed for clarity. Dashed line denotes M+2+6- 15% positivity. B, Stability of M+2+6- case positivity calling across scoring increasing number of imaging fields. All cases from panel A with at least five fields scored in this study are shown. Only one case is called M+2+6- Low (<15%) at the first image scored, and subsequently called M+2+6- High (≥15%) after two or more fields scored.
Supplementary figure 9. Mapping of mRNA expression data into percentage extent data. A, Cumulative histogram of MYC, BCL2 and BCL6 protein percentage extent positivity in DLBCL cohorts (data transformed from Figure 4A) (top). B, Aggregated single oncogene cumulative distribution of MYC, BCL2 and BCL6 protein percentage extent positivity across all measured protein cohorts and its smoothed empirical cumulative distribution function (eCDF).C, Distribution of inferred single oncogene percentage extent in GEP cohorts. (see Supplementary table 6 for all values).
Supplementary figure 10. Analysis of the GOYA clinical trial. A, Correlation of MYC mRNA with quantitative IHC score. Linear regression (left) and Wilcoxon rank sum test (right). B, Analysis as in (A) for BCL2. C, Kaplan-Meier curves for PFS and OS for patients stratified across the 15% M+2+6- metric (GEP-derived). Multivariate Cox proportional hazards model is available in Supplementary table 9. PFS - progression free survival, OS - overall survival.
Supplementary figure 11. Proliferative advantage of cyclin D2 (CCND2) overexpressing B-cells. Representative FACS plots documenting to the expansion over time of the cyclin D2 positive GC B-cell population in cyclin D2 overexpressing GC B-cells (CCND2-Lyt2) and non-cyclin D2 overexpressing GC B-cells (Empty vector Lyt2). All GC B-cells co-overexpress BCL2, BCL6, MYC and GFP.
Supplementary table 3. Non-parametric correlation of sub-population percentage extent with clinicopathological features.
Supplementary table 4. Pooled univariate analysis for MYC, BCL2 and BCL6 single oncogene and sub-populations percentage extents as a continuous variable at 5% increments as predictors for overall survival (OS) in mfIHC cohorts of DLBCL (Cox proportional hazards model).
Supplementary table 5. Univariate analysis of clinicopathological features as a predictor of overall survival (OS) after first-line R-CHOP treatment in the NUH, SGH and MDA cohorts of DLBCL (Cox proportional hazards model).
Supplementary table 7. Pooled univariate analysis for sub-population metrics as a continuous variable at 5% increments as predictors for overall survival (OS) in GEP DLBCL cohorts (Cox proportional hazards model).
Supplementary table 8. Multivariate analysis of continuous M+2+6- metric at 5% increments as a predictor of overall survival (OS) in cohorts with gene-expression data (Cox proportional hazards model).
Supplementary table 9. Univariate and multivariate analysis of continuous M+2+6- metric as a continuous variable at 5% increments as predictor of progression-free survival (PFS) and overall survival (OS) in the GOYA trial cohort (Cox proportional hazards model).
Supplementary table 10. Multivariate analysis of M+2+6- metric dichotomized at 15% as a predictor of overall survival (OS) in cohorts with gene-expression data (Cox proportional hazards model).
Supplementary table 15. Clinicopathologic characteristics of DLBCL patients evaluated by multiplexed fluorescent immunohistochemistry (mfIHC) in this study.
Supplementary table 16. Manual multiplexed fluorescent immunohistochemistry (mfIHC) staining protocol performed on the NUH and CMMC cohort TMA.
Supplementary table 17. Automated multiplexed fluorescent immunohistochemistry (mfIHC) staining protocol performed on the SGH, MDA and BCA cohort TMA.</p>