Validation of a 15-gene hypoxia classifier in head and neck cancer for prospective use in clinical trials

Figures & data

Table 1. Patient and tumor characteristics in cohorts.

	DAHANCA 5 (n = 323)		DAHANCA 18 (n = 96)		DAHANCA 24 (n = 40)		IAEA HypoX (n = 55)		Normalization cohort (n = 108)^a
	n	(%)	n	(%)	n	(%)	n	(%)	n	(%)
Age (years)
Median	60		57		61		57		58
Range	(24–84)		(33–71)		(44–80)		(31–72)		(33–80)
<60 years	159	49	60	63	16	40	30	55	59	55
≥60 years	164	51	36	38	24	60	25	45	49	45
Sex
Female	92	28	18	19	8	20	18	33	21	19
Male	231	72	78	81	32	80	37	67	87	81
Tumor site
Supraglottic larynx	100	31	8	8	9	23	10	18	16	15
Hypopharynx	44	14	6	6	3	8	9	16	9	8
Oropharynx	141	44	61	64	25	63	15	27	83	77
Rhinopharynx	38	12	5	5	3	8	0	0	0	0
Oral cavity	0	0	9	9	0	0	16	29	0	0
Glottis	0	0	2	2	0	0	5	9	0	0
Subglottis	0	0	1	1	0	0	0	0	0	0
Sinonasal carcinoma	0	0	4	4	0	0	0	0	0	0
Tumor stage
T1–2	155	48	61	64	28	70	24	44	72	67
T3–4	168	52	35	36	12	30	31	56	36	33
Nodal stage
N0	138	43	6	6	10	25	19	35	11	10
N1–3	185	57	90	94	30	75	36	65	97	90
Disease stage
I–II	66	20	2	2	8	20	10	18	8	7
III–IV	257	80	94	98	32	80	45	82	100	93
Tumor differentiation
Well or moderate	124	38	37	39	0	0	44	80	28	26
Poor	199	62	35	36	0	0	5	9	28	26
Unknown	0	0	24	25	40	100	6	11	52	48
p16-status
Negative	239	74	41	43	24	60	5	9	45	42
Positive	84	26	55	57	16	40	46	84	63	58
Unknown	0	0	0	0	0	0	4	7	0	0

^aDAHANCA 18 (n = 75) and DAHANCA 24 (n = 33).

Figure 1. Repeatability and technical variability in the various steps in the qPCR procedures by independent replicate analysis of the same samples and plotted as correlations of delta Ct levels and as mean versus difference (Bland-Altman plot). Includes the 15 hypoxia-responsive genes and the three reference genes. Correlation of delta Ct levels in two different sections from the same sample (A). Delta Ct values from the same sample, repeated from RNA level (B). Delta Ct values from the same sample, repeated from cDNA level (C).

Figure 2. Delta Ct values for the 15 hypoxia-responsive genes from six sections from the same xenograft tumor samples.

Figure 3. Reproducibility of the procedure tested by correlation of delta Ct levels of the 15 hypoxia-responsive genes and the three reference genes from two sections from each half of tumor pairs (each pair consisting of one FaDu_DD and one UTSCC33) with one half of the tumors always fixed, embedded and cut at the central laboratory (ECO). Paired delta Ct levels are plotted against each other and as mean versus difference (Bland-Altman plot). Correlation of delta Ct levels of sections with the second halves fixed, embedded, and cut at ECO (A), at the local pathology department at Aarhus University Hospital (DoP) (B), and at seven different institutions in Europe (C).

Figure 4. Classification of 510 independent samples from DAHANCA 5, 18, 24, and IAEA HypoX. Gene expression levels are shown as heatmap (each gene centered on the mean of the values characterizing ‘more’ and ‘less’ hypoxic tumors).

Table 2. Heterogeneity within classifier genes.

	DAHANCA 18, 24, IAEA Hypo		DAHANCA 5
Gene	Observations (187 patients)	Matching overall classification (%)	Observations (323 patients)	Matching overall classification (%)
ADM	184	76%	277	78%
ALDOA	187	74%	287	62%
ANKRD37	181	70%	208	73%
BNIP3	186	65%	305	72%
BNIP3L	187	64%	312	69%
EGLN3	187	78%	287	77%
FAM162A	187	73%	314	76%
KCTD11	184	65%	276	66%
LOX	185	74%	287	68%
NDRG1	187	80%	318	80%
P4HA1	180	76%	169	76%
P4HA2	185	79%	261	69%
PDK1	185	60%	285	62%
PFKFB3	187	75%	317	73%
SLC2A1	187	68%	320	78%
Mean		72%		72%

Figure 5. Intra-tumor heterogeneity. Classification of 20 patients with multiple biopsies, with shaded area indicating two patients where the biopsy with the highest tumor content did not reveal hypoxia, but another biopsy did (A). Classification of 129 samples from 96 patients plotted against tumor content in biopsy (B).