Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix

Table 1 Cytohistological correlation and HPV E6/E7 deep sequencing results

Cytohistological Correlation
Histology			Total			LSIL				HSIL
Samples, n			72			43				29
Histology (biopsy or excision)^a
Documented, n (%)			51 (71)			26 (60)				25 (86)
Not documented, n (%)			21(29)			17(40)				4(14)
Histological Grade^a
CIN 0, n (%)			4 (8)			4 (15)				0
CIN I, n (%)			23 (45)			19 (73)				4 (14)
CIN II/III, n (%)			24 (47)			3 (12)				21 (84)
Cytohistological agreement^b
Agreement, %			78
Expected Agreement, %			46
Kappa			0.6
Std. Error			0.12
p-value			<0.001
			Deep Sequencing Reads for LSIL/HSIL
	Merged^c					Mapped^d
Read Statistic	Total	Total	HPV#1	HPV#2	HPV#3	HPV#4	HPV#5	HPV#6	HPV#7	HPV#8
Samples, n	72	72	72	44	25	13	8	5	2	1
Median, n	242,665	192,236	178,582	14,483	4,040	2,480	2,403	578	590	304
Minimum, n	11,174	6,022	6,022	367	135	407	412	366	332	-
Maximum, n	760,725	673,342	656,806	147,734	62,768	36,970	4,910	3,356	847	-
25th %tile, n	185,144	155,214	99,651	3,535	1,021	566	729	379	332	-
75th %tile, n	324,210	269,603	243,612	45,484	13,102	6,712	3,717	1,634	847	-
			HPV Genotype Concordance Analysis
					Deep vs. Sanger sequencing^e
Agreement Statistic				LSIL				HSIL
Samples, n				43				29
Agreement, %				93				93
Expected Agreement, %				13				53
Kappa				0.92				0.85
Std. Error				0.054				0.119
p-value				<0.001				<0.001

CIN cervical intraepithelial neoplasia, E6/E7 HPV E6/E7 gene amplified by PCR, HSIL high-grade squamous intraepithelial lesion, HPV human papillomavirus, LSIL low-grade squamous intraepithelial lesion
^aCervical histopathology is based on the highest grade documented on cervical biopsy or therapeutic excisional biopsy, i.e. cold knife conization (CKC) and loop excisional procedure (LEEP). Absence or presence of pathology reports in the DoD electronic health records was categorized as “Documented” or “Not documented,” respectively
^bCytohistological agreement was calculated using samples with documented histopathology
^cThe total number of passed filtered reads was 21 million and merged reads was 18 million
^dThe mapped reads corresponding to the nth HPV genotype(s) found in LSIL and HSIL samples is listed in descending order
^eThe dominant (most abundant) HPV genotype determined by BLAST alignment of deep and dideoxy sequences were paired for concordance analysis

ISSN: 1471-2164