Table 3.

Overview of HPV DNA and p16^INK4a studies in ESCC tissues

Nr studies	Author	Country	Years of diagnosis	Biopsy type	Genotyping method	N (ESCC patients enrolled)	N (ESCC valid DNA)a	N (HPV DNA+ ESCC)(%)	HPV types identified	VL [viral copies per cell]	N (HPV RNA+ among HPV DNA+)	N (p16INK4a+ among HPV DNA+)	N (p16INK4a+ among HPV DNA−)	Cut off for p16INK4a positivity	Cross- contamination controls
	InterSCOPE study					133	118	10 (8%)	16, 33, 35, 45, 70; 11	0.00	1 (10%)	1 (10%)	6 (6%)
		South Africa	1995–2000	AFPE	BSGP5+6+ PCR & TS-E7-PCR	58	48	7 (6%)	16, 35, 45, 70; 11	0.00	1 (10%)b	1 (10%)c	2 (2%)	≥25%	yes (A, B, C)d
		North China (Shanxi)	1997–2005	FFPE		35	35	1 (1%)	16	0.00	0 (0%)	0 (0%)	3 (3%)
		Iran	2005–2007	FFPE		40	35	2 (2%)	16, 33	0.00	0 (0%)	0 (0%)	1 (1%)
1	*Antonsson et al., 2011	Australia	2002–2005	FFPE	GP5+6+ PCR + seq.	222	222	8 (4%)	16, 35	nt	nt	4 (50%)e	nte	ns	yes (A, B, C)
2	*Bellizzi et al., 2009	USA	1994–2007	FFPE	ISH	31	29	8 (4%)	none	nt	nt	0 (0%)	7 (24%)f	≥25%	yes (C)
3	Cao et al., 2014	North China (Shandong)	2006–2008	FFPE	ISH	105	105	29 (28%)	16	nt	nt	25 (86%)	14 (18%)	≥50%	nsg
4	Castillo et al., 2011					166	166	31 (19%)	16, 18, 35, 45, 51, 68; 6	0.121 (0.05 – 0.27)h		16 (53%)i	42 (33%)i
		Pakistan	1996–2002	FFPE	SPF1/2 InnoLiPA	42	42	11 (26%)	16, 18; 6	0.124h	nt	ns	ns	≥10%	nsj
		Columbia	1996–2001			49	49	9 (18%)	16, 18, 35, 45; 6	0.251h		ns	ns
		Japan	1987–2005			75	75	11 (15%)	16, 18, 51, 68	0.072h		ns	ns
5	Castillo et al., 2006	South America				73	73	21 (29%)	16, 18			12 (57%)	25 (49%)k
		Columbia	1996–2001	FFPE	GP5+6+ PCR & seq. & Southern blot	47	47	16 (34%)	16, 18	nt	nt	ns	ns	≥10%	ns
		Chile	1996–2000			26	26	5 (19%)	18			ns	ns
6	Ding et al., 2010	North China (Henan)	2005–2008	FFPE	HPV16 E6 PCR	17	17	8 (47%)	16	nt	nt	2 (25%)	5 (56%)	≥10%	ns
7	Doxatader et al., 2011	USA	ns	FFPE	ISH	20	20	1 (17%)l	ns	nt	nt	1 (17%)l	5 (83%)l	≥50%	ns
8	Herbster et al., 2012	Brazil	2000–2009	FFPE & DFT	GP5+6+ PCR & ISH & seq.	264	264	34 (13%)	16, 18, 66	nt	nt	7 (21%)	3 (10%)	ns	yes (A, B, C)
9	*Koshiol et al., 2009	North China (Linxian)	2006–2007	FFPE & DFT	PCR (PGMY) & SPF10 LiPA	272	267	3 (1%)	16, 31; 89	nt	nt	0 (0%)	ntm	ns	yes (A, B, C)
10	Lofdahl et al., 2012	Sweden	1999–2006	FFPE	GP5+6+ PCR & Luminex	204	204	20 (10%)	16, 33, 45, 51, 52, 73, 82, 66; 42	nt	nt	4 (24%)n	18 (16%)o	nsp	yes (C)
11	*Malik et al., 2011	USA	2001–2009	FFPE	ISH	25	25	0 (0%)q	none	nt	nt	0 (0%)q	ntq	nsr	yes (C)
12	Shuyama et al., 2007	China				59	59	19 (32%)	16, 18, 51; 6	<0.00001 – 1.7		1 (5%)	10 (25%)
		Gansus	1994–2005	FFPE	SPF10 InnoLiPA & GP5+6+ PCR & Southern blot	26	26	17 (65%)	16	<0.00001	nt	nsu	3 (12%)	≥80%	ns
		Shandogs				33	33	2 (6%)	16, 18	<0.0001 – 1.7		nsu	7 (21%)
13	Teng et al., 2014	East China (Shanghai)	1999–2011	FFPE	PCR + RDB	177	177	6 (3%)	16, 35; 11	nt	nt	0 (0%)	5 (3%)	≥80%	yes (B, C)
14	*Vaiphei et al., 2013	India	ns	DFT	PCR	23	23	20 (87%)	16, 18, 39, 52, 59; 6, 11 + 11 more	nt	nt	nsv	nsv	≥40%	yes (Bw, C)

^*Studies that could not be included in meta-analyses for p16^INK4a due to limited tissue analyses (see specific footnotes).

^aNumber of valid ESCC biopsy equals number of patients enrolled in each of the studies.

^bthe single HPV16 mRNA+ case was HPV16 DNA+ by TS-E7-PCR only, did not show upregulation of p16^INK4a (<10% p16^INK4a+ tumor cells), and was not seropositive for HPV16 antibodies.

^cA single HPV DNA+/p16^INK4a+ case (>75% p16^INK4a+ cells) was HPV16 mRNA-. HPV DNA was identified by TS-E7-PCR only; patient was seropositive for HPV16 E6 antibodies.

^dUse of a new blade for sectioning of each tissue specimen (A), different laboratories for DNA extraction, PCR set up and PCR products analyses (B), use of (pretested) HPV DNA+ and HPV DNA− tissues as a control (C).

^eOnly HPV DNA+ tumors (N=8) were tested for p16^INK4a expression by IHC.

^f6/7 tumors were scored as 25–50%, and 1/7 tumors was scored as 50–75% p16^INK4a+ tumor cells.

^gCSCC was used as a positive control during ISH.

^hGeometric mean of HPV genome copies per cell across HPV16 DNA+ tumors analyzed.

ⁱ158 of 166 samples had enough material for p16^INK4a analysis (30/31 HPV DNA+ and 128/135 HPV DNA− cases).

^jFor 11 ESCC cases from Japan authors additionally analyzed tissue biopsies of tumor adjacent epithelia of which none showed HPV DNA+.

^k72/73 samples had enough material for p16^INK4a analysis (21/21 HPV DNA+ and 51/52 HPV DNA− cases).

^lonly p16^INK4a+ cases (N=6), and the equal number of p16^INK4a− cases, were tested for HPV by ISH. Only 1/6 p16^INK4a+ cases was HPV DNA+.

^mOnly HPV DNA+ ESCC were tested for p16^INK4a.

ⁿ17/20 HPV DNA+ ESCC had enough material for p16^INK4a evaluation.

^op16^INK4a was evaluated in 113/184 HPV DNA− cases (6.5 HPV DNA− controls per HPV DNA+ case).

^pSamples were considered positive if there was homogeneous p16^INK4a staining of a clear majority of tumor cells.

^qOnly p16^INK4a+ tumors (N=11) were tested for HPV DNA by ISH.

^rPercentage of p16^INK4a+ cells was not specified. Authors stated that only biopsies with nuclear or nuclear and cytoplasmic, diffuse and strong staining were considered p16^INK4a+.

^sGansu is a high-incidence ESCC region in NW China and Shandong is a low-incidence ESCC region in E China.

^tHPV51 and HPV6 were present only as a co-infection with HPV16 in two respective cases for which region of origin was not specified.

^uAuthors did not specify if the one p16^INK4a+ case was from Gansu or from Shandong.

^vNumber of p16^INK4a+ tumors was not specified (12 tumors were positive for p16 and p63). Authors concluded that p16^INK4a+ and p63 positivity did not correlate with HPV status.

^wAuthors report using sterile forceps and blades but do not specify changing them for sectioning of each tissue sample.

Abbreviations: AFPE - alcohol-fixed paraffin-embedded, FFPE - formalin-fixed paraffin-embedded, DFT - deep frozen tissue, ISH - in situ hybridization, RDB - reverse dot blot, CSCC – cervical squamous cell carcinoma, seq. – sequencing, nt – not tested, ns - not specified