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Analysis_all_clustered_prop_den.Rmd
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---
title: "R Notebook- All clustered, all merged"
output:
html_document:
df_print: paged
---
```{r Load libraries, echo=F, message=F, warning=F}
library(ggpubr)
library(dplyr)
#library(tidyverse)
library(RColorBrewer)
library(qwraps2)
library(table1)
library(SingleCellExperiment)
#library(uwot)
library(tidyr)
library(scater)
library(ggridges)
library(ggsci)
library(viridis)
library(viridisLite)
library(ggplot2)
library(data.table)
library(CATALYST)
library(gridExtra)
library(Rphenograph)
library(ComplexHeatmap)
library(CATALYST)
library(scales)
library(survival)
library(broom)
library(pheatmap)
library(FlowSOM)
library(Seurat)
library(Rphenoannoy)
library(dplyr)
library(data.table)
library(ggthemes)
library(diffcyt)
library(edgeR)
library(rstatix)
library(dendextend)
library(ggdendro)
library(dendextend)
library(FactoMineR)
library(factoextra)
library(survminer)
library(corrplot)
library(rstatix)
library(graphics)
library(cowplot)
library(cluster)
library(glmnet)
library(fastDummies)
library(igraph)
library(SingleCellExperiment)
library(S4Vectors)
library(stringr)
library(DT)
library(dplyr)
library(tidyr)
library(mclust)
library(ggplot2)
library(RColorBrewer)
library(scater)
library(Rphenoannoy)
library(factoextra)
library(cluster)
library(dendextend)
library(ggthemes)
set.seed(101100)
```
```{r}
#set working directory
wd <-dirname(getwd())
#clinical.data
#data_folder <-(file.path(wd,"sce_objects","final objects with categories","FINAL"))
#all.sce <- readRDS("/mnt/lena_processed2/NSCLC_NEW/sce_objects/final objects with categories/FINAL/FINAL_All_Clustered_FILTERED_inc_tumour_vessel_immune_CAF_minusOther.rds")
#all.sce$DX.name[is.na(all.sce$DX.name)]<-"NA"
#all.sce$tcell_subtype <-all.sce$cell_subtype
#all.sce$tcell_subtype[all.sce$cell_category=="Fibroblast"] <- all.sce[, all.sce$cell_category=="Fibroblast"]$cell_type
#unique(all.sce$tcell_subtype)
data_folder <-file.path(wd,"sce_objects","merge_plus_tumour")
all.sce_pat.roi <-readRDS(file=file.path(data_folder, paste("ALL_minus-Tumour_sce_pat_roi_rem.rds",sep="")))
all.sce_pat <-readRDS(file=file.path(data_folder, paste("ALL_plus-Tumour_sce_pat_rem.rds",sep="")))
unique(all.sce_pat.roi$cell_category)
```
```{r}
#clinical.data <-as.data.frame(colData(immune.sce))
clinical.data <- read.csv(file=file.path(wd,"sce_objects","clinical_data", "clinical_data_ROI_ac_combined_CORRECT.csv"))
clinical.data = clinical.data[!duplicated(clinical.data$Patient_ID),]
clinical.data$X <-NULL
clinical.data$X.1 <-NULL
head(clinical.data)
```
```{r, eval=F}
#All Fibros together
tbl <- as.data.frame(table(all.sce[,all.sce$Patient_ID!="Control" ]$Patient_ID))
colnames(tbl) <-c("Patient ID", "Cell number overall")
#write.csv(tbl, file=file.path(plot_folder, paste("Immune cell numbers per patient overall including T-excluding undefined cells.csv")))
print(tbl)
summary(tbl$`Cell number overall`) #3-6259
tbl[tbl$`Cell number overall` <=100,] #43 patients have less than 100 F
#Lowest 10% of all patients' Cell numbers
tbl[order(tbl$`Cell number overall`),]$`Cell number overall`[1:100] #2-2114 = lowest 10%, 5-503 = lowest 5%
#Highest 10% of all patients' Cell numbers
tbl[order(-tbl$`Cell number overall`),]$`Cell number overall`[1:100] #760-2378
tbl[tbl$`Cell number overall` <=100,] #43 patients have less than 100 Fibros=10%. 77 patients have less than 15 Fibros
all_cells_pat <- tbl[tbl$`Cell number overall` <=2000,]$`Patient ID`
length(all_cells_pat)
length(unique(all.sce$Patient_ID))
```
```{r, eval=F}
#All Immune cells together
tbl <- as.data.frame(table(all.sce[,all.sce$Patient_ID!="Control"]$RoiID))
colnames(tbl) <-c("ROI ID", "Cell number overall")
#write.csv(tbl, file=file.path(plot_folder, paste("Cell numbers per ROI overall including Fibros-excluding undefined.csv")))
print(tbl)
summary(tbl$`Cell number overall`) #1-5075
tbl[tbl$`Cell number overall` <=100,] #93 images have less than 50 Fibros (10% of median)
#Lowest 10% of all patients' Cell numbers
tbl[order(tbl$`Cell number overall`),]$`Cell number overall`[1:200] #1-110
#Highest 10% of all patients' Cell numbers
tbl[order(-tbl$`Cell number overall`),]$`Cell number overall`[1:200] #1385-5075
#per image cut at 50 Fibros per image equals lowest 5% -> ensures that there's at least 50 Fibros per patient
all_cells_roi <- tbl[tbl$`Cell number overall` <=1000,]$`ROI ID`
length(all_cells_roi)
```
Remove patients and roi
```{r patient roi and pat_roi removal, echo=FALSE, message=FALSE, warning=FALSE, eval=F}
#Patient removal
all.sce_pat <- all.sce[,all.sce$Patient_ID!="Control"&
!all.sce$Patient_ID%in%all_cells_pat]
length(unique(all.sce_pat$Patient_ID)) #1025
#Roi removal
all.sce_roi <- all.sce[,all.sce$Patient_ID!="Control"&
!all.sce$RoiID%in%all_cells_roi]
length(unique(all.sce_roi$Patient_ID)) #1039
#Patient & Roi removal
all.sce_pat.roi <- all.sce[,all.sce$Patient_ID!="Control"&
!all.sce$Patient_ID%in%all_cells_pat&
!all.sce$RoiID%in%all_cells_roi]
length(unique(all.sce_pat.roi$Patient_ID)) #1025
```
```{r Save SCE patient and roi removed, echo=FALSE,warning=FALSE, message=FALSE, eval=FALSE}
data_folder <-file.path(wd,"sce_objects","merge_plus_tumour")
unique(all.sce$cell_category)
all.sce[, all.sce$cell_category!="Other"]
saveRDS(all.sce_pat.roi,file=file.path(data_folder, paste("ALL_plus-Tumour_sce_pat_roi_rem.rds",sep="")))
saveRDS(all.sce_roi,file=file.path(data_folder, paste("ALL_plusTumour_sce_roi_rem.rds",sep="")))
saveRDS(all.sce_pat,file=file.path(data_folder, paste("ALL_plus-Tumour_sce_pat_rem.rds",sep="")))
all.sce_pat.roi <-readRDS(file=file.path(data_folder, paste("ALL_minus-Tumour_sce_pat_roi_rem.rds",sep="")))
all.sce_roi <-readRDS(file=file.path(data_folder, paste("ALL_plusTumour_sce_roi_rem.rds",sep="")))
all.sce_pat <-readRDS(file=file.path(data_folder, paste("ALL_plus-Tumour_sce_pat_rem.rds",sep="")))
```
##Analysis
#Proportions Fibro CATEGORY
## **Analysis**
In the following, only tumours that were identified as adenocarcinomas or squamous cell carcinomas by pathologists will be used.
## **Fibro Category**
## **Proportions**
Optimal number of patient metaclusters Fibro category proportions
```{r optimal number of clusters for non Fibro category type proportion clusters, message=FALSE, warning=FALSE, echo=FALSE, out.width="50%", fig.width=6, fig.height=4, eval=F}
#Calculate Proportions
neoadj_pat <- clinical.data %>% filter(NeoAdj == "NeoAdjuvantTherapy") %>% select(Patient_ID)
tdat <-as.data.frame(table( all.sce_pat[, all.sce_pat$Patient_ID!="Control"&
all.sce_pat$DX.name=="Adenocarcinoma"|
all.sce_pat$DX.name=="Squamous cell carcinoma"]$Patient_ID,
all.sce_pat[, all.sce_pat$Patient_ID!="Control"&
all.sce_pat$DX.name=="Adenocarcinoma"|
all.sce_pat$DX.name=="Squamous cell carcinoma"]$tcell_subtype))
colnames(tdat)<-c("Patient_ID","Phenotype","n")
tdat <-tdat %>%
dplyr::group_by(Patient_ID) %>%
dplyr::mutate(freq = n / sum(n))
tdat[is.na(tdat)] <- 0
tdat$Phenotype <-droplevels(tdat$Phenotype)
tdat$Patient_ID <-droplevels(tdat$Patient_ID)
tdat_wide <- pivot_wider(tdat,id_cols=c("Patient_ID"),names_from = "Phenotype", values_from ="freq",names_prefix = "")
tdat_wide <-tdat_wide[!tdat_wide$Patient_ID%in% neoadj_pat$Patient_ID,]
tdat_m <-as.matrix(tdat_wide)
rownames(tdat_m)<-tdat_wide$Patient_ID
#tdat_m <-tdat_m[,1:2]
hc <- hclust(dist(tdat_m[,-1], method="euclidian"),"ward.D2")
#devtools::install_github("kassambara/factoextra")
sbg <-cutree(hc, k=2)
fviz_cluster(list(data = tdat_wide[,-1], cluster = sbg))
fviz_nbclust(tdat_wide[,-1], FUN = hcut, method = "wss", k.max=15)
fviz_nbclust(tdat_wide[,-1], FUN = hcut, method = "gap_stat", k.max=15)
fviz_nbclust(tdat_wide[,-1], FUN = hcut, method = "silhouette", k.max=15)
#install.packages("cluster")
gap_stat <- clusGap(tdat_wide[,-1], FUN = hcut, nstart = 25, K.max = 10, B = 100)
fviz_gap_stat(gap_stat)
# Compute distance matrix
#res.dist <- dist(tdat_wide[,-1], method = "euclidean")
```
## Patients are ordered by hierarchical clustering (euclidian, wards.D2)
```{r Fibro category proportions ordered by hierarchical clustering, message=FALSE, warning=FALSE, echo=FALSE}
tdat <-as.data.frame(table( all.sce_pat[, all.sce_pat$Patient_ID!="Control"&
all.sce_pat$DX.name=="Adenocarcinoma"|
all.sce_pat$DX.name=="Squamous cell carcinoma"]$Patient_ID,
all.sce_pat[, all.sce_pat$Patient_ID!="Control"&
all.sce_pat$DX.name=="Adenocarcinoma"|
all.sce_pat$DX.name=="Squamous cell carcinoma"]$tcell_subtype))
colnames(tdat)<-c("Patient_ID","Phenotype","n")
tdat <-tdat %>%
dplyr::group_by(Patient_ID) %>%
dplyr::mutate(freq = n / sum(n))
tdat[is.na(tdat)] <- 0
tdat$Phenotype <-droplevels(tdat$Phenotype)
tdat$Patient_ID <-droplevels(tdat$Patient_ID)
tdat_wide <- pivot_wider(tdat,id_cols=c("Patient_ID"),names_from = "Phenotype", values_from ="freq",names_prefix = "")
tdat_m <-as.matrix(tdat_wide)
rownames(tdat_m)<-tdat_wide$Patient_ID
hc <- hclust(dist(tdat_m[,-1], method="euclidian"),"ward.D2")
tdat_wide$Patient_ID <- factor(tdat_wide$Patient_ID, levels = hc$labels[hc$order])
tdat$Patient_ID <- factor(tdat$Patient_ID, levels = hc$labels[hc$order])
tdat_wide
```
Patient based metaclusters split by metacluster
```{r Barplot Fibro category proportions, message=FALSE, warning=FALSE, echo=FALSE, fig.width=12, fig.height=6}
#Barplot proportions ordered by
p <-ggplot(tdat,aes(x=freq, y=Patient_ID, fill=Phenotype))+geom_bar(stat="identity")+scale_fill_igv()
#plot(p)
#ggsave(plot=p, file=file.path(plot_folder, paste("Barplot_Tcell-Category_over_FILTEREDpatients_ordered-hc.pdf")), width=6, height=6)
#dendrogram coloured by metacluster
ggdend <-hc%>% as.dendrogram() %>% dendextend::set("branches_k_color", k =15) %>% as.ggdend()
p1 <-ggplot(ggdend, labels = F)+scale_x_continuous(expand = c(0, 0))+
theme_void()+
coord_flip()+
scale_y_reverse()
#n
p2<-ggplot(tdat,aes(x=n, y=Patient_ID, fill=Phenotype))+geom_bar(stat="identity")+scale_fill_igv()+theme(
axis.text.y=element_blank(),
axis.ticks.y=element_blank(),
axis.title.y=element_blank())
plt <-p1+
p+theme(legend.position = "none", axis.text.y=element_blank(),
axis.ticks.y=element_blank(),
axis.title.y=element_blank())+
p2
plot(plt)
#ggsave(plot=plt, file=file.path(plot_folder, paste("Barplot_n_hc_cell_type_proportions.pdf")), width=6, height=6)
```
```{r merge hc metacluster into t_dat Fibro category proportions, message=FALSE, warning=FALSE, echo=FALSE, fig.width=12, fig.height=6}
clinical.data = clinical.data[!duplicated(clinical.data$Patient_ID),]
#cut dendrogram, add metacluster info to tdat
ct <-as.data.frame(cutree(hc, k= 15))
colnames(ct)<-"metacluster"
ct$metacluster <-as.factor(ct$metacluster)
ct$Patient_ID <- rownames(ct)
tdat_wide_ct <-left_join(tdat_wide, ct, by="Patient_ID")
tdat_wide_ct <- left_join(tdat_wide_ct, clinical.data, by="Patient_ID")
table(tdat_wide_ct$metacluster)
#split barlot by metacluster
tdat_ct <-left_join(tdat, ct, by="Patient_ID")
p <-ggplot(tdat_ct,aes(y=freq,x=Patient_ID, fill=Phenotype))+geom_bar(stat="identity")+scale_fill_igv()+
theme(legend.position="bottom")+
facet_grid(~ metacluster, scales = "free", space = "free")
#ggsave(plot=p, file=file.path(plot_folder, paste("Barplot_split_metacluster_tcell-category_proportions.pdf")))
plot(p)
```
```{r, fig.width=12, fig.height=6}
m <- match(order.histo,clin$SaNr)
fq_df <- cbind(data.table("sample" = order.histo),clin[m,sel.clin])
fq_df$sample <- factor(fq_df$sample,levels = order.histo)
tdat_pat <- unique(tdat$Patient_ID)
df <- clinical.data %>% select(Grade, DX.name, Patient_ID, T.new, N, M.new,Relapse) %>%
reshape2::melt(id.vars =c("Patient_ID"), variable.name = "clinical_features", value.name = "value")
length(unique(df$Patient_ID))
df <-df[df$Patient_ID %in% tdat_pat,]
df$Patient_ID <- factor(df$Patient_ID, levels = hc$labels[hc$order])
p<- ggplot(df, aes(x=Patient_ID, y=clinical_features, fill= value))+
geom_tile(color="white")+
# scale_fill_manual(values = color_clusters_3)+
theme_bw()+ theme(
panel.grid = element_blank(),
legend.key.size = unit(4, "mm"),
axis.text = element_text(color = "black"),
axis.text.x = element_text(angle = 45, hjust = 1),
axis.title.x = element_blank(),
axis.ticks.x = element_blank())
p
df <- clinical.data %>% select(Grade, DX.name, Patient_ID, T.new, N, M.new,Relapse)
df <-df[df$Patient_ID %in% tdat_pat,]
df$Patient_ID <- factor(df$Patient_ID, levels = hc$labels[hc$order])
df$Relapse <- factor(df$Relapse)
df$Grade <- factor(df$Grade)
df$T.new <- factor(df$T.new)
df$N <- factor(df$N)
df$M.new <- factor(df$M.new)
Heatmap(df$DX.name, name = "Type", width = unit(5, "mm"))+
Heatmap(df$Grade, name = "Grade", width = unit(5, "mm"))+
Heatmap(df$T.new, name = "T", width = unit(5, "mm"))+
Heatmap(df$N, name = "N", width = unit(5, "mm"))+
Heatmap(df$M.new, name = "M", width = unit(5, "mm"))+
Heatmap(df$Relapse, name = "Relapse", width = unit(5, "mm"))
col<- palette("Tableau 10")
```
```{r cell proportions over tumour size, fig.width=12, fig.height=12}
c.param <- c("Size")
for(i in c.param){
print(i)
for(k in unique(colnames(tdat_wide_ct[,2:30]))){
print(k)
print(cor.test(tdat_wide_ct[[k]],tdat_wide_ct[[i]], method="pearson", exact=F))
}
}
tdat_c <- left_join(tdat, clinical.data, by="Patient_ID")
tdat_c %>% ggplot(aes(x=Size, y=freq, color=Phenotype))+facet_wrap(~Phenotype, scales="free_y")+geom_point()+geom_smooth(method=lm,color="darkred")
```
## **Survival analysis** - Fibro category
Fibro categories (CD4 and CD8) are grouped into "high" and "low" using the proportion's median as a cut-off.
- high > median
- low < median
Patients who received neoadjuvant treatment are excluded from survival analysis.
```{r survival data Fibro category proportions high-low, message=FALSE, warning=FALSE, echo=FALSE}
neoadj_pat <- clinical.data %>% filter(NeoAdj == "NeoAdjuvantTherapy") %>% select(Patient_ID)
surv_dat <-as.data.table(tdat_wide_ct)
surv_dat<- surv_dat[is.na(surv_dat$OS) !=T,]
# Censoring for OS (1 = event (death due to disease), 0 = censored)
surv_dat[,censoringOS := 0]
surv_dat[Ev.O == 1,censoringOS := 1]
# Censoring for disease free survival (1 = event (recurrence), 0 = censored)
surv_dat[ ,censoringDFS := 0]
surv_dat[ OS > DFS, censoringDFS := 1]
surv_dat$censoringDFS <-as.numeric(as.character(surv_dat$censoringDFS))
surv_dat <-surv_dat[surv_dat$DX.name=="Adenocarcinoma"|surv_dat$DX.name=="Squamous cell carcinoma",]
surv_dat <-surv_dat[!surv_dat$Patient_ID%in% neoadj_pat,]
#high low by median
surv_dat$Collagen_CAF_G <- ifelse(surv_dat$Collagen_CAF >summary(surv_dat$Collagen_CAF)[3],"Collagen_CAF high","Collagen_CAF low")
surv_dat$hypoxic_CAF_G <- ifelse(surv_dat$hypoxic_CAF >summary(surv_dat$hypoxic_CAF)[3],"hypoxic_CAF high","hypoxic_CAF low")
surv_dat$mCAF_G <- ifelse(surv_dat$mCAF >summary(surv_dat$mCAF)[3],"mCAF high","mCAF low")
surv_dat$SMA_CAF_G <- ifelse(surv_dat$SMA_CAF >summary(surv_dat$SMA_CAF)[3],"SMA_CAF high","SMA_CAF low")
surv_dat$tpCAF_G <- ifelse(surv_dat$tpCAF >summary(surv_dat$tpCAF)[3],"tpCAF high","tpCAF low")
surv_dat$iCAF_G <- ifelse(surv_dat$iCAF >summary(surv_dat$iCAF)[3],"iCAF high","iCAF low")
surv_dat$vCAF_G <- ifelse(surv_dat$vCAF >summary(surv_dat$vCAF)[3],"vCAF high","vCAF low")
surv_dat$dCAF_G <- ifelse(surv_dat$dCAF >summary(surv_dat$dCAF)[3],"dCAF high","dCAF low")
surv_dat$hypoxic_tpCAF_G <- ifelse(surv_dat$hypoxic_tpCAF >summary(surv_dat$hypoxic_tpCAF)[3],"hypoxic_tpCAF high","hypoxic_tpCAF low")
surv_dat$IDO_CAF_G <- ifelse(surv_dat$IDO_CAF >summary(surv_dat$IDO_CAF)[3],"IDO_CAF high","IDO_CAF low")
surv_dat$PDPN_CAF_G <- ifelse(surv_dat$PDPN_CAF >summary(surv_dat$PDPN_CAF)[3],"PDPN_CAF high","PDPN_CAF low")
surv_dat$Neutrophil_G <- ifelse(surv_dat$Neutrophil >summary(surv_dat$Neutrophil)[3],"Neutrophil high","Neutrophil low")
surv_dat$Bcell_G <- ifelse(surv_dat$Bcell >summary(surv_dat$Bcell)[3],"B cell high","B cell low")
surv_dat$Myeloid_G <- ifelse(surv_dat$Myeloid >summary(surv_dat$Myeloid)[3],"Myeloid high","Myeloid low")
surv_dat$CD4_G <- ifelse(surv_dat$CD4 >summary(surv_dat$CD4)[3],"CD4 high","CD4 low")
surv_dat$CD4_Treg_G <- ifelse(surv_dat$CD4_Treg >summary(surv_dat$CD4_Treg)[3],"CD4 Treg high","CD4 Treg low")
surv_dat$CD4_IDO_G <- ifelse(surv_dat$IDO_CD4 >summary(surv_dat$IDO_CD4)[3],"CD4 IDO high","CD4 IDO low")
surv_dat$CD4_PD1_G <- ifelse(surv_dat$PD1_CD4 >summary(surv_dat$PD1_CD4)[3],"CD4 PD1 high","CD4 PD1 low")
surv_dat$CD4_TCF_G <- ifelse(surv_dat$`TCF1/7_CD4` >summary(surv_dat$`TCF1/7_CD4`)[3],"CD4 TCF1/7 high","CD4 TCF1/7 low")
surv_dat$CD4_ki67_G <- ifelse(surv_dat$ki67_CD4 >summary(surv_dat$ki67_CD4)[3],"dividing CD4 high","dividing CD4 low")
surv_dat$CD8_IDO_G <- ifelse(surv_dat$IDO_CD8 >summary(surv_dat$IDO_CD8)[3],"CD8 IDO high","CD8 IDO low")
surv_dat$CD8_TCF_G <- ifelse(surv_dat$`TCF1/7_CD8` >summary(surv_dat$`TCF1/7_CD8`)[3],"CD8 TCF1/7 high","CD8 TCF1/7 low")
surv_dat$CD8_G <- ifelse(surv_dat$CD8 >summary(surv_dat$CD8)[3],"CD8 high","CD8 low")
surv_dat$CD8_ki67_G <- ifelse(surv_dat$ki67_CD8 >summary(surv_dat$ki67_CD8)[3],"dividing CD8 high","dividing CD8 low")
surv_dat$normal_G <- ifelse(surv_dat$normal >summary(surv_dat$normal)[3],"normal high","normal low")
surv_dat$hypoxic_G <- ifelse(surv_dat$hypoxic >summary(surv_dat$hypoxic)[3],"hypoxic high","hypoxic low")
surv <-c("PDPN_CAF_G","IDO_CAF_G","hypoxic_tpCAF_G","dCAF_G","vCAF_G","iCAF_G","tpCAF_G","SMA_CAF_G","mCAF_G","hypoxic_CAF_G","Collagen_CAF_G","Bcell_G","Neutrophil_G","Myeloid_G", "metacluster","CD8_G","CD4_G","CD4_Treg_G","CD4_IDO_G","CD4_PD1_G","CD4_TCF_G","CD4_ki67_G", "CD8_IDO_G","CD8_TCF_G","CD8_ki67_G","normal_G","hypoxic_G")
```
#CoxPH for Fibro category proportions corrected for Stage, Grade and M
```{r Coxph Fibro category proportions, echo=F, message=FALSE, warning=FALSE}
surv_dat
surv_dat<-surv_dat[!surv_dat$OS == 0, ]
time <- surv_dat$OS
status <- surv_dat$censoringOS
surv_dat$metacluster <-as.factor(surv_dat$metacluster)
surv_dat$Grade <-as.numeric(surv_dat$Grade)
rownames(surv_dat) <- surv_dat$Patient_ID
res.cox <- coxph(Surv(time, status) ~ metacluster, data = surv_dat)
res.cox
res.cox <- coxph(Surv(time, status) ~ PDPN_CAF + IDO_CAF+hypoxic_tpCAF+dCAF+vCAF+iCAF+tpCAF+mCAF+hypoxic_CAF+SMA_CAF+Collagen_CAF+Neutrophil+Bcell+ CD4 + CD8+`TCF1/7_CD4`+ki67_CD8+`TCF1/7_CD8`+PD1_CD4+IDO_CD8 +ki67_CD4+IDO_CD4+CD4_Treg + Myeloid + normal+hypoxic+ Stage+Grade+M.new, data = surv_dat)
#res.cox <- coxph(Surv(time, status) ~ `CD4+`+tpCAF+iCAF+mCAF+dCAF+vCAF+`SMA+ CAF`+`IDO+ CAF`+`dividing tpCAF`+`hypoxic CAF`+Grade, data = surv_dat)
summary(res.cox)
td_res = tidy(res.cox, exponentiate = TRUE)
td_res = td_res %>% arrange(estimate) %>% mutate(term = factor(term,levels = term))
td_res<-td_res %>%
rowwise() %>%
dplyr::mutate(conf.high = estimate + std.error)
td_res<-td_res %>%
rowwise() %>%
dplyr::mutate(conf.low = estimate - std.error)
td_res<-td_res[!td_res$std.error == 0, ]
td_res <-td_res[td_res$term !="Grade"&
td_res$term !="Stage"&
td_res$term !="M.new",]
p <-ggplot(td_res, aes(x = term, y = estimate,color =p.value < 0.05)) +
geom_point(size = 4) +
geom_errorbar(aes(ymax = conf.high, ymin = conf.low))+
scale_colour_viridis_d("p value < 0.05")+
coord_flip()+
#xlim(0,2)+
geom_hline(yintercept = 1,color = 'black')+
theme_bw()+
scale_colour_tableau()
#
plot(p)
p <-ggplot(td_res, aes(x = term, y = estimate,color =p.value < 0.05)) +
geom_point(size = 4) +
#geom_errorbar(aes(ymax = conf.high, ymin = conf.low))+
scale_colour_viridis_d("p value < 0.05")+
coord_flip()+
#xlim(0,2)+
geom_hline(yintercept = 1,color = 'black')+
theme_bw()+
scale_colour_tableau()+
scale_y_continuous(trans = "log10")
#
plot(p)
```
## Not split by tumour type (AC and SQC together)
```{r survival Fibro category proportions not split high low, message=FALSE, warning=FALSE, echo=FALSE,out.width="50%", fig.height=4, fig.width=6}
#not split by Tumour Type (AC/SQC)
for (i in surv){
#for (k in unique(surv_dat$DX.name)){
#dat <-surv_dat[surv_dat$DX.name==k,]
dat <-surv_dat
k="AC & SCC together"
#OS
pw<-pairwise_survdiff(Surv(OS, censoringOS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
# abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(OS, censoringOS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = F,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,5000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("OS over",i,"per",k))
# facet_grid(.~surv_dat[[i]])
print(p)
#print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
facet_wrap(~strata)+theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#km_trt_fit
#print(p)
#disease free survival
pw<-pairwise_survdiff(Surv(DFS, censoringDFS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
# abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(DFS, censoringDFS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = T,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,5000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("DFS over",i,"per",k))#+
# facet_grid(.~surv_dat[[i]])
print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
#facet_wrap(~strata)+
theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#print(p)
#km_trt_fit
}
```
## Split by tumour type (AC and SQC individually)
```{r survival Fibro category proportions split high low, message=FALSE, warning=FALSE, echo=FALSE,out.width="50%", fig.height=4, fig.width=6}
#OS survival
#surv <-c("PDPN_CAF_G","IDO_CAF_G","hypoxic_tpCAF_G","dCAF_G","vCAF_G","iCAF_G","tpCAF_G","SMA_CAF_G","mCAF_G","hypoxic_CAF_G","Collagen_CAF_G", "metacluster")
#split by Tumour Type (AC/SQC)
for (i in surv){
for (k in unique(surv_dat$DX.name)){
dat <-surv_dat[surv_dat$DX.name==k,]
dat$Collagen_CAF_G <- ifelse(dat$Collagen_CAF >summary(dat$Collagen_CAF)[3],"Collagen_CAF high","Collagen_CAF low")
dat$hypoxic_CAF_G <- ifelse(dat$hypoxic_CAF >summary(dat$hypoxic_CAF)[3],"hypoxic_CAF high","hypoxic_CAF low")
dat$mCAF_G <- ifelse(dat$mCAF >summary(dat$mCAF)[3],"mCAF high","mCAF low")
dat$SMA_CAF_G <- ifelse(dat$SMA_CAF >summary(dat$SMA_CAF)[3],"SMA_CAF high","SMA_CAF low")
dat$tpCAF_G <- ifelse(dat$tpCAF >summary(dat$tpCAF)[3],"tpCAF high","tpCAF low")
dat$iCAF_G <- ifelse(dat$iCAF >summary(dat$iCAF)[3],"iCAF high","iCAF low")
dat$vCAF_G <- ifelse(dat$vCAF >summary(dat$vCAF)[3],"vCAF high","vCAF low")
dat$dCAF_G <- ifelse(dat$dCAF >summary(dat$dCAF)[3],"dCAF high","dCAF low")
dat$hypoxic_tpCAF_G <- ifelse(dat$hypoxic_tpCAF >summary(dat$hypoxic_tpCAF)[3],"hypoxic_tpCAF high","hypoxic_tpCAF low")
dat$IDO_CAF_G <- ifelse(dat$IDO_CAF >summary(dat$IDO_CAF)[3],"IDO_CAF high","IDO_CAF low")
dat$PDPN_CAF_G <- ifelse(dat$PDPN_CAF >summary(dat$PDPN_CAF)[3],"PDPN_CAF high","PDPN_CAF low")
dat$Neutrophil_G <- ifelse(dat$Neutrophil >summary(dat$Neutrophil)[3],"Neutrophil high","Neutrophil low")
dat$Bcell_G <- ifelse(dat$Bcell >summary(dat$Bcell)[3],"B cell high","B cell low")
dat$Myeloid_G <- ifelse(dat$Myeloid >summary(dat$Myeloid)[3],"Myeloid high","Myeloid low")
dat$CD4_G <- ifelse(dat$CD4 >summary(dat$CD4)[3],"CD4 high","CD4 low")
dat$CD4_Treg_G <- ifelse(dat$CD4_Treg >summary(dat$CD4_Treg)[3],"CD4 Treg high","CD4 Treg low")
dat$CD4_IDO_G <- ifelse(dat$IDO_CD4 >summary(dat$IDO_CD4)[3],"CD4 IDO high","CD4 IDO low")
dat$CD4_PD1_G <- ifelse(dat$PD1_CD4 >summary(dat$PD1_CD4)[3],"CD4 PD1 high","CD4 PD1 low")
dat$CD4_TCF_G <- ifelse(dat$`TCF1/7_CD4` >summary(dat$`TCF1/7_CD4`)[3],"CD4 TCF1/7 high","CD4 TCF1/7 low")
dat$CD4_ki67_G <- ifelse(dat$ki67_CD4 >summary(dat$ki67_CD4)[3],"dividing CD4 high","dividing CD4 low")
dat$CD8_IDO_G <- ifelse(dat$IDO_CD8 >summary(dat$IDO_CD8)[3],"CD8 IDO high","CD8 IDO low")
dat$CD8_TCF_G <- ifelse(dat$`TCF1/7_CD8` >summary(dat$`TCF1/7_CD8`)[3],"CD8 TCF1/7 high","CD8 TCF1/7 low")
dat$CD8_G <- ifelse(dat$CD8 >summary(dat$CD8)[3],"CD8 high","CD8 low")
dat$CD8_ki67_G <- ifelse(dat$ki67_CD8 >summary(dat$ki67_CD8)[3],"dividing CD8 high","dividing CD8 low")
dat$hypoxic_G <- ifelse(dat$hypoxic >summary(dat$hypoxic)[3],"hypoxic high","hypoxic low")
dat$normal_G <- ifelse(dat$normal >summary(dat$normal)[3],"normal high","normal low")
#OS
pw<-pairwise_survdiff(Surv(OS, censoringOS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
#abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(OS, censoringOS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = F,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,2000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("OS over",i,"per",k))
# facet_grid(.~surv_dat[[i]])
print(p)
#print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
#facet_wrap(~strata)+
theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#km_trt_fit
#print(p)
#disease free survival
pw<-pairwise_survdiff(Surv(DFS, censoringDFS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
# abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(DFS, censoringDFS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = T,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,2000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("DFS over",i,"per",k))#+
# facet_grid(.~surv_dat[[i]])
print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
#facet_wrap(~strata)+
theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#print(p)
#km_trt_fit
}
}
```
## Lasso-regressed cox-ph model
Including clinical parameters, patient metacluster and Fibro categories (CD4 vs CD8) classified as high / low as well as the proportion (continuous).
```{r coxph Fibro category proportions, message=FALSE, warning=FALSE, echo=FALSE,out.width="50%", fig.width=10, fig.height=6}
surv_dat$metacluster <-as.numeric(surv_dat$metacluster)
fd_test <- fastDummies::dummy_cols(surv_dat, select_columns = c(paste(surv),"Typ","Grade","T.new","N","M.new","Stage","Smok","Gender", "metacluster")) #
fd_test<-fd_test[!fd_test$OS == 0, ]
time <- fd_test$OS
status <- fd_test$censoringOS
rownames(fd_test) <- fd_test$Patient_ID
fd_test <-fd_test %>%select(-c(paste(surv),"metacluster",colnames(clinical.data),contains("censor"))) #))
fd_test[is.na(fd_test)] <- 0
fd_test_matrix <-data.matrix(fd_test)
#determine lambda
cv.fit <- cv.glmnet(fd_test_matrix, Surv(time, status), family = "cox", maxit = 1000)
fit <- glmnet(fd_test_matrix, Surv(time, status), family = "cox", maxit = 1000)
#plot(cv.fit)
#cv.fit$lambda.min
#loop to determine optimal lambda for lasso regression
MSEs <- NULL
for (i in 1:100){
cv <- cv.glmnet(fd_test_matrix, Surv(time, status), family = "cox", maxit = 1000)
MSEs <- cbind(MSEs, cv$cvm)
}
rownames(MSEs) <- cv$lambda
lambda.min <- as.numeric(names(which.min(rowMeans(MSEs))))
#coef(fit, s = lambda.min)
Coefficients <- coef(fit, s = lambda.min)
Active.Index <- which(Coefficients != 0)
Active.Coefficients <- Coefficients[Active.Index]
#Coefficients
active_coefficients <- Coefficients[,1] != 0
x <- fd_test_matrix[,active_coefficients]
x <- data.frame(x)
vct <-colnames(x)
coxdf<-coxph(Surv(time,status)~.,data=x)
td_res = tidy(coxdf, exponentiate = TRUE)
td_res = td_res %>% arrange(estimate) %>% mutate(term = factor(term,levels = term))
td_res<-td_res %>%
rowwise() %>%
dplyr::mutate(conf.high = estimate + std.error)
td_res<-td_res %>%
rowwise() %>%
dplyr::mutate(conf.low = estimate - std.error)
td_res<-td_res[!td_res$std.error == 0, ]
na_td_res <- td_res[-grep("NA",td_res$term),]
p <-ggplot(na_td_res, aes(x = term, y = sqrt(estimate),color =p.value < 0.05)) +
geom_point(size = 4) +
#geom_errorbar(aes(ymax = conf.high, ymin = conf.low))+
scale_colour_viridis_d("p value < 0.05")+
coord_flip()+
geom_hline(yintercept = 1,color = 'black')+
theme_bw()+
scale_colour_tableau()+
scale_y_continuous(trans = "log10")
p
#ggsave(file=file.path(plot_folder, paste("CoxPH_Tcell-Category_Proportions_high-low_Lasso.pdf")), width=10, height=10, plot=p)
ggforest(coxdf, data = (fd_test %>% select(-contains("NA"))))
#pdf(file=file.path(plot_folder,paste("COX_selectedFactors_glmnet.pdf")))
#ggforest(coxdf, data = fd_test)
#dev.off()
```
## Cell groups categorised into "high", "mid", "low".
- high > 3rd quantile
- mid >1st quantile and < 3rd quantile
- low < 1st quantile
```{r survival Fibro category proportions classified as high mid low, message=FALSE, warning=FALSE, echo=FALSE}
surv_dat$mCAF_G[surv_dat$mCAF >summary(surv_dat$mCAF)[5]] <-"mCAF high"
surv_dat$mCAF_G[surv_dat$mCAF <=summary(surv_dat$mCAF)[2]] <-"mCAF low"
surv_dat$mCAF_G[surv_dat$mCAF >summary(surv_dat$mCAF)[2]& surv_dat$mCAF<=summary(surv_dat$mCAF)[5]] <-"mCAF medium"
surv_dat$vCAF_G[surv_dat$vCAF >summary(surv_dat$vCAF)[5]] <-"vCAF high"
surv_dat$vCAF_G[surv_dat$vCAF <=summary(surv_dat$vCAF)[2]] <-"vCAF low"
surv_dat$vCAF_G[surv_dat$vCAF >summary(surv_dat$vCAF)[2]& surv_dat$vCAF<=summary(surv_dat$vCAF)[5]] <-"vCAF medium"
surv_dat$tpCAF_G[surv_dat$tpCAF >summary(surv_dat$tpCAF)[5]] <-"tpCAF high"
surv_dat$tpCAF_G[surv_dat$tpCAF <=summary(surv_dat$tpCAF)[2]] <-"tpCAF low"
surv_dat$tpCAF_G[surv_dat$tpCAF >summary(surv_dat$tpCAF)[2]& surv_dat$tpCAF<=summary(surv_dat$tpCAF)[5]] <-"tpCAF medium"
surv_dat$iCAF_G[surv_dat$iCAF >summary(surv_dat$iCAF)[5]] <-"iCAF high"
surv_dat$iCAF_G[surv_dat$iCAF <=summary(surv_dat$iCAF)[2]] <-"iCAF low"
surv_dat$iCAF_G[surv_dat$iCAF >summary(surv_dat$iCAF)[2]& surv_dat$iCAF<=summary(surv_dat$iCAF)[5]] <-"iCAF medium"
surv_dat$dCAF_G[surv_dat$dCAF >summary(surv_dat$dCAF)[5]] <-"dCAF high"
surv_dat$dCAF_G[surv_dat$dCAF <=summary(surv_dat$dCAF)[2]] <-"dCAF low"
surv_dat$dCAF_G[surv_dat$dCAF >summary(surv_dat$dCAF)[2]& surv_dat$dCAF<=summary(surv_dat$dCAF)[5]] <-"dCAF medium"
surv_dat$Collagen_CAF_G[surv_dat$Collagen_CAF >summary(surv_dat$Collagen_CAF)[5]] <-"Collagen_CAF high"
surv_dat$Collagen_CAF_G[surv_dat$Collagen_CAF <=summary(surv_dat$Collagen_CAF)[2]] <-"Collagen_CAF low"
surv_dat$Collagen_CAF_G[surv_dat$Collagen_CAF >summary(surv_dat$Collagen_CAF)[2]& surv_dat$Collagen_CAF<=summary(surv_dat$Collagen_CAF)[5]] <-"Collagen_CAF medium"
surv_dat$hypoxic_CAF_G[surv_dat$hypoxic_CAF >summary(surv_dat$hypoxic_CAF)[5]] <-"hypoxic_CAF high"
surv_dat$hypoxic_CAF_G[surv_dat$hypoxic_CAF <=summary(surv_dat$hypoxic_CAF)[2]] <-"hypoxic_CAF low"
surv_dat$hypoxic_CAF_G[surv_dat$hypoxic_CAF >summary(surv_dat$hypoxic_CAF)[2]& surv_dat$hypoxic_CAF<=summary(surv_dat$hypoxic_CAF)[5]] <-"hypoxic_CAF medium"
surv_dat$SMA_CAF_G[surv_dat$SMA_CAF >summary(surv_dat$SMA_CAF)[5]] <-"SMA_CAF high"
surv_dat$SMA_CAF_G[surv_dat$SMA_CAF <=summary(surv_dat$SMA_CAF)[2]] <-"SMA_CAF low"
surv_dat$SMA_CAF_G[surv_dat$SMA_CAF >summary(surv_dat$SMA_CAF)[2]& surv_dat$SMA_CAF<=summary(surv_dat$SMA_CAF)[5]] <-"SMA_CAF medium"
surv_dat$hypoxic_tpCAF_G[surv_dat$hypoxic_tpCAF >summary(surv_dat$hypoxic_tpCAF)[5]] <-"hypoxic_tpCAF high"
surv_dat$hypoxic_tpCAF_G[surv_dat$hypoxic_tpCAF <=summary(surv_dat$hypoxic_tpCAF)[2]] <-"hypoxic_tpCAF low"
surv_dat$hypoxic_tpCAF_G[surv_dat$hypoxic_tpCAF >summary(surv_dat$hypoxic_tpCAF)[2]& surv_dat$hypoxic_tpCAF<=summary(surv_dat$hypoxic_tpCAF)[5]] <-"hypoxic_tpCAF medium"
surv_dat$IDO_CAF_G[surv_dat$IDO_CAF >summary(surv_dat$IDO_CAF)[5]] <-"IDO_CAF high"
surv_dat$IDO_CAF_G[surv_dat$IDO_CAF <=summary(surv_dat$IDO_CAF)[2]] <-"IDO_CAF low"
surv_dat$IDO_CAF_G[surv_dat$IDO_CAF >summary(surv_dat$IDO_CAF)[2]& surv_dat$IDO_CAF<=summary(surv_dat$IDO_CAF)[5]] <-"IDO_CAF medium"
surv_dat$PDPN_CAF_G[surv_dat$PDPN_CAF >summary(surv_dat$PDPN_CAF)[5]] <-"PDPN_CAF high"
surv_dat$PDPN_CAF_G[surv_dat$PDPN_CAF <=summary(surv_dat$PDPN_CAF)[2]] <-"PDPN_CAF low"
surv_dat$PDPN_CAF_G[surv_dat$PDPN_CAF >summary(surv_dat$PDPN_CAF)[2]& surv_dat$PDPN_CAF<=summary(surv_dat$PDPN_CAF)[5]] <-"PDPN_CAF medium"
surv_dat$normal_G[surv_dat$normal >summary(surv_dat$normal)[5]] <-"normal high"
surv_dat$normal_G[surv_dat$normal <=summary(surv_dat$normal)[2]] <-"normal low"
surv_dat$normal_G[surv_dat$normal >summary(surv_dat$normal)[2]& surv_dat$normal<=summary(surv_dat$normal)[5]] <-"normal medium"
surv_dat$hypoxic_G[surv_dat$hypoxic >summary(surv_dat$hypoxic)[5]] <-"hypoxic high"
surv_dat$hypoxic_G[surv_dat$hypoxic <=summary(surv_dat$hypoxic)[2]] <-"hypoxic low"
surv_dat$hypoxic_G[surv_dat$hypoxic >summary(surv_dat$hypoxic)[2]& surv_dat$hypoxic<=summary(surv_dat$hypoxic)[5]] <-"hypoxic medium"
surv_dat$Bcell_G[surv_dat$Bcell >summary(surv_dat$Bcell)[5]] <-"Bcell high"
surv_dat$Bcell_G[surv_dat$Bcell <=summary(surv_dat$Bcell)[2]] <-"Bcell low"
surv_dat$Bcell_G[surv_dat$Bcell >summary(surv_dat$Bcell)[2]& surv_dat$Bcell<=summary(surv_dat$Bcell)[5]] <-"Bcell medium"
surv_dat$Myeloid_G[surv_dat$Myeloid >summary(surv_dat$Myeloid)[5]] <-"Myeloid high"
surv_dat$Myeloid_G[surv_dat$Myeloid <=summary(surv_dat$Myeloid)[2]] <-"Myeloid low"
surv_dat$Myeloid_G[surv_dat$Myeloid >summary(surv_dat$Myeloid)[2]& surv_dat$Myeloid<=summary(surv_dat$Myeloid)[5]] <-"Myeloid medium"
surv_dat$Neutrophil_G[surv_dat$Neutrophil >summary(surv_dat$Neutrophil)[5]] <-"Neutrophil high"
surv_dat$Neutrophil_G[surv_dat$Neutrophil <=summary(surv_dat$Neutrophil)[2]] <-"Neutrophil low"
surv_dat$Neutrophil_G[surv_dat$Neutrophil >summary(surv_dat$Neutrophil)[2]& surv_dat$Neutrophil<=summary(surv_dat$Neutrophil)[5]] <-"Neutrophil medium"
surv_dat$CD4_G[surv_dat$CD4 >summary(surv_dat$CD4)[5]] <-"CD4 high"
surv_dat$CD4_G[surv_dat$CD4 <=summary(surv_dat$CD4)[2]] <-"CD4 low"
surv_dat$CD4_G[surv_dat$CD4 >summary(surv_dat$CD4)[2]& surv_dat$CD4<summary(surv_dat$CD4)[5]] <-"CD4 medium"
surv_dat$CD4_Treg_G[surv_dat$CD4_Treg >summary(surv_dat$CD4_Treg)[5]] <-"CD4 Treg high"
surv_dat$CD4_Treg_G[surv_dat$CD4_Treg <=summary(surv_dat$CD4_Treg)[2]] <-"CD4 Treg low"
surv_dat$CD4_Treg_G[surv_dat$CD4_Treg >summary(surv_dat$CD4_Treg)[2]& surv_dat$CD4_Treg<summary(surv_dat$CD4_Treg)[5]] <-"CD4 T reg medium"
surv_dat$CD4_IDO_G[surv_dat$IDO_CD4 >summary(surv_dat$IDO_CD4)[5]] <-"IDO_CD4 high"
surv_dat$CD4_IDO_G[surv_dat$IDO_CD4 <=summary(surv_dat$IDO_CD4)[2]] <-"IDO_CD4 low"
surv_dat$CD4_IDO_G[surv_dat$IDO_CD4 >summary(surv_dat$IDO_CD4)[2]& surv_dat$IDO_CD4<summary(surv_dat$IDO_CD4)[5]] <-"IDO_CD4 medium"
surv_dat$CD4_PD1_G[surv_dat$PD1_CD4 >summary(surv_dat$PD1_CD4)[5]] <-"PD1_CD4 high"
surv_dat$CD4_PD1_G[surv_dat$PD1_CD4 <=summary(surv_dat$PD1_CD4)[2]] <-"PD1_CD4 low"
surv_dat$CD4_PD1_G[surv_dat$PD1_CD4 >summary(surv_dat$PD1_CD4)[2]& surv_dat$PD1_CD4<summary(surv_dat$PD1_CD4)[5]] <-"PD1_CD4 medium"
surv_dat$CD4_TCF_G[surv_dat$`TCF1/7_CD4` >summary(surv_dat$`TCF1/7_CD4`)[5]] <-"CD4 TCF1/7 high"
surv_dat$CD4_TCF_G[surv_dat$`TCF1/7_CD4` <=summary(surv_dat$`TCF1/7_CD4`)[2]] <-"CD4 TCF1/7 low"
surv_dat$CD4_TCF_G[surv_dat$`TCF1/7_CD4` >summary(surv_dat$`TCF1/7_CD4`)[2]& surv_dat$`TCF1/7_CD4`<summary(surv_dat$`TCF1/7_CD4`)[5]] <-"CD4 TCF1/7 medium"
surv_dat$CD4_ki67_G[surv_dat$ki67_CD4 >summary(surv_dat$ki67_CD4)[5]] <-"CD4 dividing high"
surv_dat$CD4_ki67_G[surv_dat$ki67_CD4 <=summary(surv_dat$ki67_CD4)[2]] <-"CD4 dividing low"
surv_dat$CD4_ki67_G[surv_dat$ki67_CD4 >summary(surv_dat$ki67_CD4)[2]& surv_dat$ki67_CD4<summary(surv_dat$ki67_CD4)[5]] <-"CD4 dividing medium"
surv_dat$CD8_G[surv_dat$CD8 >summary(surv_dat$CD8)[5]] <-"CD8 high"
surv_dat$CD8_G[surv_dat$CD8 <=summary(surv_dat$CD8)[2]] <-"CD8 low"
surv_dat$CD8_G[surv_dat$CD8 >summary(surv_dat$CD8)[2]& surv_dat$CD8<summary(surv_dat$CD8)[5]] <-"CD8 medium"
surv_dat$CD8_IDO_G[surv_dat$IDO_CD8 >summary(surv_dat$IDO_CD8)[5]] <-"CD8 IDO high"
surv_dat$CD8_IDO_G[surv_dat$IDO_CD8 <=summary(surv_dat$IDO_CD8)[2]] <-"CD8 IDO low"
surv_dat$CD8_IDO_G[surv_dat$IDO_CD8 >summary(surv_dat$IDO_CD8)[2]& surv_dat$IDO_CD8<summary(surv_dat$IDO_CD8)[5]] <-"CD8 IDO medium"
surv_dat$CD8_TCF_G[surv_dat$`TCF1/7_CD8` >summary(surv_dat$`TCF1/7_CD8`)[5]] <-"CD8 TCF1/7 high"
surv_dat$CD8_TCF_G[surv_dat$`TCF1/7_CD8` <=summary(surv_dat$`TCF1/7_CD8`)[2]] <-"CD8 TCF1/7 low"
surv_dat$CD8_TCF_G[surv_dat$`TCF1/7_CD8` >summary(surv_dat$`TCF1/7_CD8`)[2]& surv_dat$`TCF1/7_CD8`<summary(surv_dat$`TCF1/7_CD8`)[5]] <-"CD8 TCF1/7 medium"
surv_dat$CD8_ki67_G[surv_dat$ki67_CD8 >summary(surv_dat$ki67_CD8)[5]] <-"CD8 dividing high"
surv_dat$CD8_ki67_G[surv_dat$ki67_CD8 <=summary(surv_dat$ki67_CD8)[2]] <-"CD8 dividing low"
surv_dat$CD8_ki67_G[surv_dat$ki67_CD8 >summary(surv_dat$ki67_CD8)[2]& surv_dat$ki67_CD8<summary(surv_dat$ki67_CD8)[5]] <-"CD8 dividing medium"
#surv <-c("PDPN_CAF_G","IDO_CAF_G","hypoxic_tpCAF_G","dCAF_G","vCAF_G","iCAF_G","tpCAF_G","SMA_CAF_G","mCAF_G","hypoxic_CAF_G","Collagen_CAF_G", "metacluster")
```
## Survival Fibro category proportions high mid low not split
```{r survival Fibro category proportions not split high mid low, message=FALSE, warning=FALSE, echo=FALSE,out.width="50%", fig.height=4, fig.width=6}
#not split by Tumour Type (AC/SQC)
for (i in surv){
#for (k in unique(surv_dat$DX.name)){
#dat <-surv_dat[surv_dat$DX.name==k,]
dat <-surv_dat
k="AC & SCC together"
#OS
pw<-pairwise_survdiff(Surv(OS, censoringOS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
# abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(OS, censoringOS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = F,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,5000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("OS over",i,"per",k))
# facet_grid(.~surv_dat[[i]])
print(p)
#print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
#facet_wrap(~strata)+
theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#km_trt_fit
#print(p)
#disease free survival
pw<-pairwise_survdiff(Surv(DFS, censoringDFS) ~ metacluster,data = surv_dat)
#print(pw)
#print(symnum(pw$p.value, cutpoints = c(0, 0.0001, 0.001, 0.01, 0.05, 0.1, 1),
# symbols = c("****", "***", "**", "*", "+", "."),
# abbr.colnames = FALSE, na = " "))
km_trt_fit <- survfit(Surv(DFS, censoringDFS) ~ dat[[i]], data=dat)
km_trt_fit
p <-ggsurvplot(km_trt_fit,
data=dat,
conf.int = T,
pval = T,
#conf.int.style = "step",
surv.median.line = "hv",
xlim=c(0,5000))+ # Specify median survival,
#legend.title = paste(i),
#legend.labs=c("Grade 1","Grade 2","Grade 3"),
#legend.labs=c(paste(i,sort(unique(surv_dat[[i]][!is.na(surv_dat[[i]])])))))+
labs(title=paste("DFS over",i,"per",k))#+
# facet_grid(.~surv_dat[[i]])
print(p)
p <-p$plot+
theme_bw()+
theme(panel.grid.major = element_blank(), panel.grid.minor = element_blank())+
#facet_wrap(~strata)+
theme(axis.text.x = element_text(angle=45, hjust=1), axis.ticks.x = element_blank())
#print(p)
#km_trt_fit
}
```
## Survival Fibro category proportions high mid low split by tumour type
```{r survival Fibro category proportions split high mid low, message=FALSE, warning=FALSE, echo=FALSE,out.width="50%", fig.height=4, fig.width=6}
#split by Tumour Type (AC/SQC)
for (i in surv){
for (k in unique(surv_dat$DX.name)){
dat <-surv_dat[surv_dat$DX.name==k,]
dat$mCAF_G[dat$mCAF >summary(dat$mCAF)[5]] <-"mCAF high"
dat$mCAF_G[dat$mCAF <=summary(dat$mCAF)[2]] <-"mCAF low"
dat$mCAF_G[dat$mCAF >summary(dat$mCAF)[2]& dat$mCAF<=summary(dat$mCAF)[5]] <-"mCAF medium"
dat$vCAF_G[dat$vCAF >summary(dat$vCAF)[5]] <-"vCAF high"
dat$vCAF_G[dat$vCAF <=summary(dat$vCAF)[2]] <-"vCAF low"
dat$vCAF_G[dat$vCAF >summary(dat$vCAF)[2]& dat$vCAF<=summary(dat$vCAF)[5]] <-"vCAF medium"
dat$tpCAF_G[dat$tpCAF >summary(dat$tpCAF)[5]] <-"tpCAF high"
dat$tpCAF_G[dat$tpCAF <=summary(dat$tpCAF)[2]] <-"tpCAF low"
dat$tpCAF_G[dat$tpCAF >summary(dat$tpCAF)[2]& dat$tpCAF<=summary(dat$tpCAF)[5]] <-"tpCAF medium"
dat$iCAF_G[dat$iCAF >summary(dat$iCAF)[5]] <-"iCAF high"
dat$iCAF_G[dat$iCAF <=summary(dat$iCAF)[2]] <-"iCAF low"
dat$iCAF_G[dat$iCAF >summary(dat$iCAF)[2]& dat$iCAF<=summary(dat$iCAF)[5]] <-"iCAF medium"
dat$dCAF_G[dat$dCAF >summary(dat$dCAF)[5]] <-"dCAF high"
dat$dCAF_G[dat$dCAF <=summary(dat$dCAF)[2]] <-"dCAF low"
dat$dCAF_G[dat$dCAF >summary(dat$dCAF)[2]& dat$dCAF<=summary(dat$dCAF)[5]] <-"dCAF medium"
dat$Collagen_CAF_G[dat$Collagen_CAF >summary(dat$Collagen_CAF)[5]] <-"Collagen_CAF high"
dat$Collagen_CAF_G[dat$Collagen_CAF <=summary(dat$Collagen_CAF)[2]] <-"Collagen_CAF low"
dat$Collagen_CAF_G[dat$Collagen_CAF >summary(dat$Collagen_CAF)[2]& dat$Collagen_CAF<=summary(dat$Collagen_CAF)[5]] <-"Collagen_CAF medium"
dat$hypoxic_CAF_G[dat$hypoxic_CAF >summary(dat$hypoxic_CAF)[5]] <-"hypoxic_CAF high"
dat$hypoxic_CAF_G[dat$hypoxic_CAF <=summary(dat$hypoxic_CAF)[2]] <-"hypoxic_CAF low"
dat$hypoxic_CAF_G[dat$hypoxic_CAF >summary(dat$hypoxic_CAF)[2]& dat$hypoxic_CAF<=summary(dat$hypoxic_CAF)[5]] <-"hypoxic_CAF medium"
dat$SMA_CAF_G[dat$SMA_CAF >summary(dat$SMA_CAF)[5]] <-"SMA_CAF high"
dat$SMA_CAF_G[dat$SMA_CAF <=summary(dat$SMA_CAF)[2]] <-"SMA_CAF low"
dat$SMA_CAF_G[dat$SMA_CAF >summary(dat$SMA_CAF)[2]& dat$SMA_CAF<=summary(dat$SMA_CAF)[5]] <-"SMA_CAF medium"
dat$hypoxic_tpCAF_G[dat$hypoxic_tpCAF >summary(dat$hypoxic_tpCAF)[5]] <-"hypoxic_tpCAF high"
dat$hypoxic_tpCAF_G[dat$hypoxic_tpCAF <=summary(dat$hypoxic_tpCAF)[2]] <-"hypoxic_tpCAF low"
dat$hypoxic_tpCAF_G[dat$hypoxic_tpCAF >summary(dat$hypoxic_tpCAF)[2]& dat$hypoxic_tpCAF<=summary(dat$hypoxic_tpCAF)[5]] <-"hypoxic_tpCAF medium"
dat$IDO_CAF_G[dat$IDO_CAF >summary(dat$IDO_CAF)[5]] <-"IDO_CAF high"
dat$IDO_CAF_G[dat$IDO_CAF <=summary(dat$IDO_CAF)[2]] <-"IDO_CAF low"
dat$IDO_CAF_G[dat$IDO_CAF >summary(dat$IDO_CAF)[2]& dat$IDO_CAF<=summary(dat$IDO_CAF)[5]] <-"IDO_CAF medium"
dat$PDPN_CAF_G[dat$PDPN_CAF >summary(dat$PDPN_CAF)[5]] <-"PDPN_CAF high"
dat$PDPN_CAF_G[dat$PDPN_CAF <=summary(dat$PDPN_CAF)[2]] <-"PDPN_CAF low"
dat$PDPN_CAF_G[dat$PDPN_CAF >summary(dat$PDPN_CAF)[2]& dat$PDPN_CAF<=summary(dat$PDPN_CAF)[5]] <-"PDPN_CAF medium"
dat$normal_G[dat$normal >summary(dat$normal)[5]] <-"normal high"
dat$normal_G[dat$normal <=summary(dat$normal)[2]] <-"normal low"
dat$normal_G[dat$normal >summary(dat$normal)[2]& dat$normal<=summary(dat$normal)[5]] <-"normal medium"