Figures and data in Identifying prostate cancer and its clinical risk in asymptomatic men using machine learning of high dimensional peripheral blood flow cytometric natural killer cell subset phenotyping data

Figures
Tables
Additional files

9 figures, 12 tables and 2 additional files

Figures

Figure 1

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NK cell phenotypic features in men with benign prostate disease and patients with prostate cancer.

Boxplots represent the flow cytometry values of each feature for patients with benign disease and with prostate cancer.

Figure 2

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Mean and standard deviation values of flow cytometry features.

Figure 3

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Figure 4

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Figure 5

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Boxplots illustrating the performance of the proposed model using various feature sets.

(a) Average AUC values, (b) Average Optimal ROC points (TPRs), (c) Average Optimal ROC points (FPRs), (d) Average Accuracy values. Each box plot contains 30 points, where each point is the average performance evaluation value (i.e. AUC, ORP TPR, ORP FPR, Accuracy) from one 10-fold run using the various feature sets.

Figure 6

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Flow charts illustrating the process to detect the presence and risk of prostate cancer and patient outcomes.

Model 1: Distinguishes between men with benign prostate disease and prostate cancer; Model 2: predicts risk (in terms of clinical significance) in men identified as having prostate cancer in Stage 1. Note that Model 1 can detect prostate cancer in men with PSA < 20 ng ml^-1.

Figure 7

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Each box plot contains 30 points, where each point is the average performance evaluation value (i.e. AUC, FPR, TPR, Accuracy (ACC)) from one 10-fold run during (a) k-fold validation results, and (b) independent testing results (i.e. using 10 patient records).

Figure 8

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Representative gating strategy for analyzing the expression of activating and inhibitory receptors on peripheral blood natural killer (NK) cells.

Using density plots, the NK cell phenotypic profiles were determined by first gating on ‘live cells’ in the forward scatter (FSc) linear vs side scatter (SSc) linear density plot and then gating on single cells (determined by FSc Linear vs FS time of flight). The expression of activating and inhibitory receptors was determined by gating on $C D 3^{-} C D 19^{-} C D 56^{+}$ cells using fluorescence minus one (FMO) controls. The expression of each NK cell receptor was measured using the ‘Logical’ setting.

Figure 9

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Proposed Ensemble Subspace kNN model.

Ensembles combine predictions from different models to generate a final prediction. Because Ensemble approaches combine baseline predictions, they perform at least as well as the best baseline model.

Tables

Table 1

Descriptive statistics of the dataset.

		Min.		Max.		Mean		Std.		IQR		Range		Diff.
		Beni.	Canc.	Beni.	Canc.	Beni.	Canc.	Beni.	Canc.	Beni.	Canc.	Beni.	Canc.
	PSA	4.70	4.70	19.00	19.00	8.26	8.34	3.31	3.28	3.30	4.08	14.30	14.30	−0.08
$C D 56^{d i m}$ %
1	$C D 16^{+}$	83.85	73.04	96.61	96.98	90.98	90.64	3.35	5.46	4.13	5.02	12.76	23.94	0.34
2	$C D 16^{h i g h}$	24.38	49.66	87.46	89.33	72.88	73.32	11.74	10.22	15.00	10.45	63.08	39.67	−0.44
3	$C D 16^{l o w}$	5.17	6.57	64.22	44.00	17.74	16.84	10.40	7.45	8.76	7.66	59.05	37.43	0.90
4	$C D 16^{-}$	1.41	1.25	11.11	18.06	4.83	4.89	2.45	3.48	2.58	2.68	9.70	16.81	−0.06
5	$C D 56^{d i m} t o t a l$	91.29	87.24	98.70	98.70	95.81	95.53	2.02	2.58	2.96	3.02	7.41	11.46	0.28
$C D 56^{b r i g h t}$ %
6	$C D 16^{+}$	0.46	0.65	5.10	5.88	1.91	1.83	1.06	1.04	1.64	0.92	4.64	5.23	0.08
7	$C D 16^{h i g h}$	0.09	0.12	1.97	1.15	0.60	0.47	0.44	0.25	0.50	0.40	1.88	1.03	0.13
8	$C D 16^{l o w}$	0.34	0.40	3.11	4.95	1.27	1.35	0.72	0.86	0.97	0.63	2.77	4.55	−0.07
9	$C D 16^{-}$	0.61	0.58	5.78	9.09	2.28	2.64	1.14	1.82	1.42	1.75	5.17	8.51	−0.36
10	$C D 56^{b r i g h t} t o t a l$	1.30	1.30	8.71	12.76	4.19	4.47	2.02	2.58	2.95	3.01	7.41	11.46	−0.28
$C D 8$ %
11	$C D 56^{+} C D 8^{+}$	21.88	9.20	86.70	80.47	46.43	40.71	15.64	14.66	24.03	20.05	64.82	71.27	5.72
12	$C D 56^{+} C D 8^{-}$	13.30	19.53	78.12	90.80	53.57	59.29	15.64	14.66	24.03	20.05	64.82	71.27	−5.72
13	$C D 56^{d i m} C D 8^{+}$	19.63	8.60	82.38	77.47	45.18	39.11	15.31	14.10	24.72	19.36	62.75	68.87	6.07
14	$C D 56^{b r i g h t} C D 8^{+}$	0.37	0.25	4.75	6.64	1.41	1.70	1.07	1.41	0.70	1.60	4.38	6.39	−0.29
NKp30 %
15	$C D 56^{+} N K p 30^{+}$	40.69	56.80	96.74	98.43	79.78	88.56	16.42	10.41	21.80	10.44	56.05	41.63	−8.78
16	$C D 56^{+} N K p 30^{-}$	3.26	1.57	58.34	44.59	20.05	11.43	16.22	10.46	20.54	10.49	55.08	43.02	8.61
NKp46 %
17	$C D 56^{+} N K p 46^{+}$	38.11	45.37	86.52	95.82	62.65	69.82	13.49	11.58	23.90	12.71	48.41	50.45	−7.18
18	$C D 56^{+} N K p 46^{-}$	14.02	4.32	62.97	55.68	38.40	30.87	13.58	11.64	24.89	13.44	48.95	51.36	7.53
DNAM-1 %
19	$C D 56^{+} D N A M - 1^{+}$	63.69	88.56	99.18	99.60	95.35	96.46	6.81	2.59	3.37	3.49	35.49	11.04	−1.11
20	$C D 56^{+} D N A M - 1^{-}$	0.86	0.42	37.29	11.66	4.74	3.59	6.96	2.61	3.45	3.54	36.43	11.24	1.14
NKG2D %
21	$C D 56^{+} N K G 2 D^{+}$	85.17	80.79	98.77	98.96	93.49	94.07	4.45	4.87	6.81	3.83	13.60	18.17	−0.58
22	$C D 56^{+} N K G 2 D^{-}$	1.22	1.03	14.76	19.12	6.44	5.84	4.36	4.76	6.80	3.96	13.54	18.09	0.60
	PSA	4.70	4.70	19.00	19.00	8.26	8.34	3.31	3.28	3.30	4.08	14.30	14.30	−0.08
NKp44 %
23	$C D 56^{+} N K p 44^{+}$	0.43	0.28	3.71	6.77	1.16	1.34	0.82	1.20	0.78	1.25	3.28	6.49	−0.18
24	$C D 56^{+} N K p 44^{-}$	96.10	93.70	99.53	99.70	98.82	98.64	0.83	1.13	0.80	1.25	3.43	6.00	0.18
CD85j %
25	$C D 56^{+} C D 85 j^{+}$	19.53	14.21	84.73	91.59	53.37	55.10	19.04	18.34	30.49	20.23	65.20	77.38	−1.74
26	$C D 56^{+} C D 85 j^{-}$	14.93	8.50	81.54	86.08	46.94	45.24	19.21	18.43	30.28	21.48	66.61	77.58	1.69
LAIR-1 %
27	$C D 56^{+} L A I R - 1^{+}$	94.97	21.43	99.90	99.89	99.07	97.47	1.07	12.19	0.49	0.47	4.93	78.46	1.60
28	$C D 56^{+} L A I R - 1^{-}$	0.02	0.05	5.24	78.20	0.76	2.40	1.02	12.15	0.42	0.43	5.22	78.15	−1.65
NKG2A %
29	$C D 56^{+} N K G 2 A^{+}$	20.43	19.01	77.57	73.01	46.14	44.24	17.41	13.73	30.82	17.47	57.14	54.00	1.90
30	$C D 56^{+} N K G 2 A^{-}$	22.62	27.11	79.40	80.85	54.01	55.99	17.39	13.67	30.48	17.90	56.78	53.74	−1.98
2B4 %
31	$C D 56^{+} 2 B 4^{+}$	98.41	97.06	99.99	99.96	99.53	99.50	0.39	0.59	0.32	0.33	1.58	2.90	0.02
32	$C D 56^{+} 2 B 4^{-}$	0.01	0.05	1.59	2.95	0.48	0.50	0.39	0.59	0.31	0.34	1.58	2.90	−0.02

Min. is the minimum value, Max. is maximum value, Mean is the mean or average value, and Std. is Standard Deviation. Range is the difference between the minimum and maximum values. The Interquartile range (IQR) is a measure of data variability and was derived by computing the distance between the Upper Quartile (i.e. top) and Lower Quartile (i.e. bottom) of the boxes illustrated in Figure 1. Difference is computed as diff = mean(Benign)-mean(Cancer).

Table 2

Tests of normality results.

Tests of normality
		NK cell values	Kolmogorov-Smirnova			Shapiro-Wilk
			Statistic	df	Sig.	Statistic	df	Sig.
1	$C D 56^{d i m}$	$C D 16^{+}$	0.15	71.00	0.00	0.85	71.00	0.00
2	$C D 56^{d i m}$	$C D 16^{h i g h}$	0.11	71.00	0.03	0.89	71.00	0.00
3	$C D 56^{d i m}$	$C D 16^{l o w}$	0.17	71.00	0.00	0.79	71.00	0.00
4	$C D 56^{d i m}$	$C D 16^{-}$	0.19	71.00	0.00	0.82	71.00	0.00
5	$C D 56^{d i m}$	$C D 56^{d i m} t o t a l %$	0.15	71.00	0.00	0.91	71.00	0.00
6	$C D 56^{b r i g h t}$	$C D 16^{+}$	0.13	71.00	0.00	0.88	71.00	0.00
7	$C D 56^{b r i g h t}$	$C D 16^{h i g h}$	0.15	71.00	0.00	0.87	71.00	0.00
8	$C D 56^{b r i g h t}$	$C D 16^{l o w}$	0.14	71.00	0.00	0.85	71.00	0.00
9	$C D 56^{b r i g h t}$	$C D 16^{-}$	0.16	71.00	0.00	0.86	71.00	0.00
10	$C D 56^{b r i g h t}$	$C D 56^{b r i g h t} t o t a l$	0.15	71.00	0.00	0.91	71.00	0.00
11	$C D 8$	$C D 56^{+} C D 8^{+}$	0.10	71.00	0.06	0.98	71.00	0.17
12	$C D 8$	$C D 56^{+} C D 8^{-}$	0.10	71.00	0.06	0.98	71.00	0.17
13	$C D 8$	$C D 56^{d i m} C D 8^{+}$	0.09	71.00	0.20*	0.98	71.00	0.24
14	$C D 8$	$C D 56^{b r i g h t} C D 8^{+}$	0.19	71.00	0.00	0.82	71.00	0.00
15	$N K p 30$	$C D 56^{+} N K p 30^{+}$	0.21	71.00	0.00	0.81	71.00	0.00
16	$N K p 30$	$C D 56^{+} N K p 30^{-}$	0.21	71.00	0.00	0.81	71.00	0.00
17	$N K p 46$	$C D 56^{+} N K p 46^{+}$	0.08	71.00	0.20*	0.98	71.00	0.52
18	$N K p 46$	$C D 56^{+} N K p 46^{-}$	0.07	71.00	0.20*	0.99	71.00	0.57
19	$D N A M - 1$	$C D 56^{+} D N A M - 1^{+}$	0.23	71.00	0.00	0.56	71.00	0.00
20	$D N A M - 1$	$C D 56^{+} D N A M - 1^{-}$	0.23	71.00	0.00	0.55	71.00	0.00
21	$N K G 2 D$	$C D 56^{+} N K G 2 D^{+}$	0.19	71.00	0.00	0.84	71.00	0.00
22	$N K G 2 D$	$C D 56^{+} N K G 2 D^{-}$	0.18	71.00	0.00	0.85	71.00	0.00
23	$N K p 44$	$C D 56^{+} N K p 44^{+}$	0.18	71.00	0.00	0.76	71.00	0.00
24	$N K p 44$	$C D 56^{+} N K p 44^{-}$	0.17	71.00	0.00	0.78	71.00	0.00
25	$C D 85 j$	$C D 56^{+} C D 85 j^{+}$	0.11	71.00	0.05	0.96	71.00	0.02
26	$C D 85 j$	$C D 56^{+} C D 85 j^{-}$	0.10	71.00	0.07	0.96	71.00	0.02
27	$L A I R - 1$	$C D 56^{+} L A I R - 1^{+}$	0.43	71.00	0.00	0.14	71.00	0.00
28	$L A I R - 1$	$C D 56^{+} L A I R - 1^{-}$	0.43	71.00	0.00	0.14	71.00	0.00
29	$N K G 2 A$	$C D 56^{+} N K G 2 A^{+}$	0.09	71.00	0.20*	0.97	71.00	0.11
30	$N K G 2 A$	$C D 56^{+} N K G 2 A^{-}$	0.08	71.00	0.20*	0.97	71.00	0.10
31	$2 B 4$	$C D 56^{+} 2 B 4^{+}$	0.23	71.00	0.00	0.75	71.00	0.00
32	$2 B 4$	$C D 56^{+} 2 B 4^{-}$	0.23	71.00	0.00	0.75	71.00	0.00

*. This is a lower bound of the true significance.

Those values in bold are of those features whose data is normally distributed.
If the $p > 0.05$ , we can accept the null hypothesis, that there is no statistically significant difference between the data and the normal distribution, hence we can presume that the data of those features are normally distributed.

If the $p < 0.05$ , we can reject the null hypothesis because there is a statistically significant difference between the data and the normal distribution, hence we can presume that the data of those features are not normally distributed.

Table 3

Results of the Kruskal-Wallis test.

			Chi-Sq.( $χ^{2}$ )	Asy. sig. p value
	PSA		0	0.949
	NK cells
1	$C D 56^{d i m}$	$C D 16^{+}$	0.001	0.981
2	$C D 56^{d i m}$	$C D 16^{h i g h}$	0.069	0.793
3	$C D 56^{d i m}$	$C D 16^{l o w}$	0.555	0.456
4	$C D 56^{d i m}$	$C D 16^{-}$	0.033	0.857
5	$C D 56^{d i m}$	$C D 56^{d i m} t o t a l %$	0.063	0.802
6	$C D 56^{b r i g h t}$	$C D 16^{+}$	0.836	0.361
7	$C D 56^{b r i g h t}$	$C D 16^{h i g h}$	0.201	0.654
8	$C D 56^{b r i g h t}$	$C D 16^{l o w}$	0.106	0.744
9	$C D 56^{b r i g h t}$	$C D 16^{-}$	0.030	0.861
10	$C D 56^{b r i g h t}$	$C D 56^{b r i g h t} t o t a l$	2.415	0.120
11	$C D 8$	$C D 56^{+} C D 8^{+}$	2.415	0.120
12	$C D 8$	$C D 56^{+} C D 8^{-}$	2.849	0.091
13	$C D 8$	$C D 56^{d i m} C D 8^{+}$	0.417	0.518
14	$C D 8$	$C D 56^{b r i g h t} C D 8^{+}$	7.230	0.007
15	$N K p 30$	$C D 56^{+} N K p 30^{+}$	7.106	0.008
16	$N K p 30$	$C D 56^{+} N K p 30^{-}$	4.638	0.031
17	$N K p 46$	$C D 56^{+} N K p 46^{+}$	5.179	0.023
18	$N K p 46$	$C D 56^{+} N K p 46^{-}$	0.001	0.981
19	$D N A M - 1$	$C D 56^{+} D N A M - 1^{+}$	0.001	0.972
20	$D N A M - 1$	$C D 56^{+} D N A M - 1^{-}$	0.293	0.588
21	$N K G 2 D$	$C D 56^{+} N K G 2 D^{+}$	0.325	0.568
22	$N K G 2 D$	$C D 56^{+} N K G 2 D^{-}$	0.033	0.857
23	$N K p 44$	$C D 56^{+} N K p 44^{+}$	0.072	0.789
24	$N K p 44$	$C D 56^{+} N K p 44^{-}$	0.049	0.825
25	$C D 85 j$	$C D 56^{+} C D 85 j^{+}$	0.072	0.789
26	$C D 85 j$	$C D 56^{+} C D 85 j^{-}$	2.135	0.144
27	$L A I R - 1$	$C D 56^{+} L A I R - 1^{+}$	1.343	0.247
28	$L A I R - 1$	$C D 56^{+} L A I R - 1^{-}$	0.060	0.807
29	$N K G 2 A$	$C D 56^{+} N K G 2 A^{+}$	0.072	0.789
30	$N K G 2 A$	$C D 56^{+} N K G 2 A^{-}$	0.879	0.348
31	$2 B 4$	$C D 56^{+} 2 B 4^{+}$	0.890	0.346
32	$2 B 4$	$C D 56^{+} 2 B 4^{-}$	0.890	0.346

Table 4

Results of the Genetic Algorithm when searching for the best subset of features.

$λ$	No. different comb	Comb. with highest freq.	Freq. of comb.	Relative freq. (%)
2	3	17,28	16	53.3
3	2	17,27,29	23	76.7
4	1	2,20,27,28	30	100.0
5	2	3,20,27,28,32	29	96.7
6	2	3,7,20,27,28,32	26	86.7
7	3	3,7,20,23,27,28,32	24	80.0
8	4	3,7,20,22,23,27,28,32	19	63.3
9	3	3,7,19,20,22,23,27,28,32	24	80.0
10	3	2,3,7,19,20,22,23,27,28,32	21	70.0

Table 5

Naming of the models includes the feature selection method (GA) combined with the proposed Ensemble Subspace kNN classifier.

Validation results are presented at k = 10 fold cross validation.

	Results of 10-fold cross validation over 30 runs
		AUC	Orp fpr	Orp tpr	ACC	Mean std.	Rank
GA	Mean	0.776	0.296	0.833	0.781		4
	Std.	0.024	0.065	0.026	0.023	0.035
STAT	Mean	0.769	0.303	0.828	0.774		5
	Std.	0.022	0.057	0.023	0.021	0.031
GA+STAT	Mean	0.818	0.201	0.836	0.821		1
	Std.	0.021	0.027	0.021	0.020	0.022
PSA+GA+STAT	Mean	0.812	0.208	0.832	0.815		2
	Std.	0.020	0.031	0.018	0.019	0.022
PSA	Mean	0.698	0.217	0.609	0.692		6
	Std.	0.022	0.025	0.043	0.020	0.028
All features	Mean	0.812	0.213	0.836	0.815		3
	Std.	0.022	0.035	0.021	0.021	0.025

Table 6

Comparing the performance of the proposed Ensemble Subspace kNN model against conventional machine learning models when using the GA+STAT feature set.

Results of 10-fold cross validation over 30 runs.

Proposed ensemble subspace kNN (EkNN) model
(No. of learners (NL): 30; Subspace Dimension (SD): 16)
Parameters		AUC	ORP FPR	ORP TPR	ACC
NL: 30, SD:16	Mean	0.818	0.201	0.836	0.821
	Std.	0.021	0.027	0.021	0.020
Simple kNN model (Distance: Euclidean)
		AUC	ORP FPR	ORP TPR	ACC	Acc. Diff.
k						(EkNN vs. kNN)
2	Mean	0.768	0.241	0.730	0.751	+0.070
	Std.	0.119	0.160	0.393	0.128	−0.108
5	Mean	0.778	0.300	0.833	0.783	+0.038
	Std.	0.107	0.265	0.103	0.103	−0.083
10	Mean	0.753	0.371	0.845	0.758	+0.063
	Std.	0.137	0.350	0.120	0.131	−0.111
Support Vector Machine models
		AUC	ORP FPR	ORP TPR	ACC	Acc. Diff.
Kernel						(EkNN vs. SVM)
Linear	Mean	0.782	0.342	0.860	0.784	+0.037
	Std.	0.126	0.352	0.110	0.120	−0.100
Gaussian	Mean.	0.808	0.353	0.876	0.799	+0.022
	Std.	0.112	0.416	0.107	0.111	−0.091
Naive Bayes model
		AUC	ORP FPR	ORP TPR	ACC	Acc. Diff.
Predictor distributions						(EkNN vs. Naïve Bayes)
Normal	Mean.	0.695	0.132	0.455	0.662	+0.159
	Std.	0.169	0.163	0.493	0.181	−0.161

Table 7

Ad hoc test results.

	Ad hoc test
	Group 1	Group 2	Ll 95%	Diff. betw.means	Ul 95%	P
1	GA	STAT	−12.658	1.317	15.292	1.000
2	GA	GA+STAT	−22.208	−8.233	5.742	0.525
3	GA	PSA	−4.992	8.983	22.958	0.344
4	GA	PSA+GA+STAT	−20.792	−6.817	7.158	1.000
5	STAT	GA+STAT	−23.525	−9.550	4.425	0.245
6	STAT	PSA	−6.308	7.667	21.642	0.710
7	STAT	PSA+GA+STAT	−22.108	−8.133	5.842	0.555
8	GA+STAT	PSA	3.242	17.217	31.192	0.001
9	GA+STAT	PSA+GA+STAT	−12.558	1.417	15.392	1.000
10	PSA	PSA+GA+STAT	−29.775	−15.800	−1.825	0.002

The first two columns show the groups that are compared. The third and fifth columns show the lower and upper limits for 95% confidence intervals for the true mean difference. The fourth column shows the difference between the estimated group means. The sixth column contains the p-value for testing a hypothesis that the corresponding mean difference is equal to zero.

Table 8

Results of the best prediction models created during the 30 runs.

Validation results are presented at k = 10 fold cross validation.

	Best prediction model results
	AUC	Orp fpr	Orp tpr	Accuracy	Rank
GA	0.818	0.192	0.829	0.820	3
GA+STAT	0.853	0.157	0.862	0.855	1
PSA	0.734	0.218	0.685	0.730	5
PSA+GA+STAT	0.844	0.175	0.864	0.848	2
STAT	0.811	0.227	0.85	0.817	4

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Biological Sample	Hyclone fetal bovine serum (FBS)	GE Healthcare Life Sciences	SV30180.03
Antibody	Monoclonal mouse IgG1 kappa anti human DNAM-1 (CD226) (clone 11A8); FITC	BioLegend	338304	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human NKG2D (CD314) (clone 1D11); PE	eBioscience	12-5878-42	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human CD56 (clone N901); ECD (PE-Texas Red)	Beckman Coulter	A82943	2.5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human CD16 (clone 3G8); PerCP-Cy5.5	BioLegend	302028	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human NKp46 (CD335) (clone 9E2); PE-Cy7	BioLegend	331916	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human NKp30 (CD337) (clone P30-15); Alexa Fluor 647	BioLegend	325212	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human CD3 (clone UCHT1); Alexa Fluor 700	BioLegend	300424	2 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human CD19 (clone HIB19); Alexa Fluor 700	BioLegend	302226	1 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human CD8 (clone SK1); APC-Cy7	BioLegend	344714	2.5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG2b anti human CD85j (ILT2) (clone GHI/75); FITC	Miltenyi Biotec	130-098-437	10 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human LAIR-1 (CD305) (clone DX26); PE	BD Biosciences	550811	20 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG2b anti human NKG2A (CD159a) (clone Z199); PE-Cy7(PC7)	Beckman Coulter	B10246	20 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human NKp44 (CD336) (clone P44-8); Alexa Fluor 647	BioLegend	325112	5 μl per tube / 10⁶cells
Antibody	Monoclonal mouse IgG1 kappa anti human 2B4 (CD244.2) (clone C1.7); FITC	BioLegend	329506	5 μl per tube / 10⁶cells
Chemical Compound	LIVE/DEAD Fixable Violet Dead Stain	Thermo Fisher Scientific	L34955	1 μl in 1 μl
Chemical Compound	Novagen Benzonase Nuclease	Merck Millipore	70664
Chemical Compound	CTL Wash Solution	Cellular Technology Limited	CTLW-010
Chemical Compound	Trypan Blue viability stain	Santa Cruz	sc-216028
Chemical Compound	Dimethyl sulfoxide (DMSO)	Santa Cruz	sc-202581
Chemical Compound	Calbiochem bovine serum albumin (BSA)	Merck Millipore	2905-OP
Chemical Compound	Sigma-Aldrich sodium azide	Merck Millipore	S8032
Chemical Compound	Sigma-Aldrich lithium heparin	Merck Millipore	H0878
Chemical Compound	Ficoll-Paque	GE Healthcare Life Sciences	17-1440-03
Chemical Compound	Isoton II isotonic buffered saline solution	Beckman Coulter	844 80 11
Chemical Compound	RPMI medium	Lonza	12-167Q
Chemical Compound	Phosphate Buffered Saline (PBS)	Lonza	17-517Q
Other	Leucosep tubes	Greiner Bio-One International	227290
Software	Kaluza v1.3	Beckman Coulter

Table 9

Patient clinical features.

Patient group	Gleason score	Number of patients	Age range (years)	PSA range (ng/ml)
Benign	Benign	9	64-71	5.3–15
Benign	HGPIN	9	54–70	5.1–12
Benign	Atypia	10	50–76	4.7–19
Benign	ASAP	2	59–60	5.3–7.8
Cancer	Gleason 6	16	55–80	4.7–11
Cancer	Gleason 7	23	53–77	4.7–19
Cancer	Gleason 9	2	65–75	6.3–18

Table 10

Dataset used for differentiating between patients with L/I and H cancer.

Patient group	Count	%
L/I	38	70.37
H	16	29.63

Table 11

Antibody panels for measuring the phenotype of Natural Killer cells.

Antibody	Fluorochrome	Clone no.	Supplier
Panel 1
DNAM-1 (CD226)	FITC	11A8	BioLegend
NKG2D (CD314)	PE	1D11	eBioscience
CD56	ECD (PE-Texas Red)	N901	Beckman Coulter
CD16	PerCP-Cy5.5	3G8	BioLegend
NKp46 (CD335)	PE-Cy7	9E2	BioLegend
NKp30 (CD337)	Alexa Fluor 647	P30-15	BioLegend
CD3	Alexa Fluor 700	UCHT1	BioLegend
CD19	Alexa Fluor 700	HIB19	BioLegend
CD8	APC-Cy7	SK1	BioLegend
Live/Dead	Dye (violet)		Thermo Fisher Scientific
Panel 2
CD85j (ILT2)	FITC	GHI/75	Miltenyi Biotec
LAIR-1 (CD305)	PE	DX26	BD Biosciences
CD56	ECD (PE-Texas Red)	N901	Beckman Coulter
CD16	PerCP-Cy5.5	3G8	BioLegend
NKG2A (CD159a)	PC7 (PE-Cy7)	Z199	Beckman Coulter
NKp44 (CD336)	Alexa Fluor 647	P44-8	BioLegend
CD3	Alexa Fluor 700	UCHT1	BioLegend
CD19	Alexa Fluor 700	HIB19	BioLegend
CD8	APC-Cy7	SK1	BioLegend
LIVE/DEAD	Dye (violet)		Thermo Fisher Scientific
Panel 3
2B4 (CD244.2)	FITC	C1.7	BioLegend
CD56	ECD (PE-Texas Red)	N901	Beckman Coulter
CD16	PerCp-Cy5.5	3G8	BioLegend
CD3	Alexa Fluor 700	UCHT1	BioLegend
CD19	Alexa Fluor 700	HIB19	BioLegend
CD8	APC-Cy7	SK1	BioLegend
LIVE/DEAD	Dye (violet)		Thermo Fisher Scientific

Additional files

Source data 1 Prostate Cancer Dataset.: https://cdn.elifesciences.org/articles/50936/elife-50936-data1-v1.xlsx
Download elife-50936-data1-v1.xlsx
Transparent reporting form: https://cdn.elifesciences.org/articles/50936/elife-50936-transrepform-v1.docx
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Simon P Hood
Georgina Cosma
Gemma A Foulds
Catherine Johnson
Stephen Reeder
Stéphanie E McArdle
Masood A Khan
A Graham Pockley

(2020)

Identifying prostate cancer and its clinical risk in asymptomatic men using machine learning of high dimensional peripheral blood flow cytometric natural killer cell subset phenotyping data

eLife 9:e50936.

https://doi.org/10.7554/eLife.50936

Figures

NK cell phenotypic features in men with benign prostate disease and patients with prostate cancer.

Mean and standard deviation values of flow cytometry features.

Correlations between features.

PSA values by group.

Boxplots illustrating the performance of the proposed model using various feature sets.

Flow charts illustrating the process to detect the presence and risk of prostate cancer and patient outcomes.

Each box plot contains 30 points, where each point is the average performance evaluation value (i.e. AUC, FPR, TPR, Accuracy (ACC)) from one 10-fold run during (a) k-fold validation results, and (b) independent testing results (i.e. using 10 patient records).

Representative gating strategy for analyzing the expression of activating and inhibitory receptors on peripheral blood natural killer (NK) cells.

Proposed Ensemble Subspace kNN model.

Tables

Descriptive statistics of the dataset.

Tests of normality results.

Results of the Kruskal-Wallis test.

Results of the Genetic Algorithm when searching for the best subset of features.

Naming of the models includes the feature selection method (GA) combined with the proposed Ensemble Subspace kNN classifier.

Comparing the performance of the proposed Ensemble Subspace kNN model against conventional machine learning models when using the GA+STAT feature set.

Ad hoc test results.

Results of the best prediction models created during the 30 runs.

Patient clinical features.

Dataset used for differentiating between patients with L/I and H cancer.

Antibody panels for measuring the phenotype of Natural Killer cells.

Additional files

Source data 1

Transparent reporting form

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Downloads (link to download the article as PDF)

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NK cell phenotypic features in men with benign prostate disease and patients with prostate cancer.

Mean and standard deviation values of flow cytometry features.

Correlations between features.

PSA values by group.

Boxplots illustrating the performance of the proposed model using various feature sets.

Flow charts illustrating the process to detect the presence and risk of prostate cancer and patient outcomes.

Each box plot contains 30 points, where each point is the average performance evaluation value (i.e. AUC, FPR, TPR, Accuracy (ACC)) from one 10-fold run during (a) k-fold validation results, and (b) independent testing results (i.e. using 10 patient records).

Representative gating strategy for analyzing the expression of activating and inhibitory receptors on peripheral blood natural killer (NK) cells.

Proposed Ensemble Subspace kNN model.

Descriptive statistics of the dataset.

Tests of normality results.

Results of the Kruskal-Wallis test.

Results of the Genetic Algorithm when searching for the best subset of features.

Naming of the models includes the feature selection method (GA) combined with the proposed Ensemble Subspace kNN classifier.

Comparing the performance of the proposed Ensemble Subspace kNN model against conventional machine learning models when using the GA+STAT feature set.

Ad hoc test results.

Results of the best prediction models created during the 30 runs.

Patient clinical features.

Dataset used for differentiating between patients with L/I and H cancer.

Antibody panels for measuring the phenotype of Natural Killer cells.

Source data 1

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)