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. 2016 May 10;7(19):27185-98.
doi: 10.18632/oncotarget.8448.

The degree of intratumor mutational heterogeneity varies by primary tumor sub-site

Levi G Ledgerwood  1 , Dhruv Kumar  1 , Agda Karina Eterovic  2 , Jo Wick  3 , Ken Chen  4 , Hao Zhao  4 , Loubna Tazi  5 , Pradip Manna  6 , Spencer Kerley  6 , Radhika Joshi  1 , Lin Wang  7 , Simion I Chiosea  7 , James David Garnett  1 , Terance Ted Tsue  1 , Jeremy Chien  8   9 , Gordon B Mills  2 , Jennifer Rubin Grandis  10   11 , Sufi Mary Thomas  1   9   12
Affiliations

The degree of intratumor mutational heterogeneity varies by primary tumor sub-site

Levi G Ledgerwood et al. Oncotarget. .

Abstract

In an era where mutational profiles inform treatment options, it is critical to know the extent to which tumor biopsies represent the molecular profile of the primary and metastatic tumor. Head and neck squamous cell carcinoma (HNSCC) arise primarily in the mucosal lining of oral cavity and oropharynx. Despite aggressive therapy the 5-year survival rate is at 50%. The primary objective of this study is to characterize the degree of intratumor mutational heterogeneity in HNSCC. We used multi-region sequencing of paired primary and metastatic tumor DNA of 24 spatially distinct samples from seven patients with HNSCC of larynx, floor of the mouth (FOM) or oral tongue. Full length, in-depth sequencing of 202 genes implicated in cancer was carried out. Larynx and FOM tumors had more than 69.2% unique SNVs between the paired primary and metastatic lesions. In contrast, the oral tongue HNSCC had only 33.3% unique SNVs across multiple sites. In addition, HNSCC of the oral tongue had fewer mutations than larynx and FOM tumors. These findings were validated on the Affymetrix whole genome 6.0 array platform and were consistent with data from The Cancer Genome Atlas (TCGA). This is the first report demonstrating differences in mutational heterogeneity varying by subsite in HNSCC. The heterogeneity within laryngeal tumor specimens may lead to an underestimation of the genetic abnormalities within tumors and may foster resistance to standard treatment protocols. These findings are relevant to investigators and clinicians developing personalized cancer treatments based on identification of specific mutations in tumor biopsies.

Keywords: HNSCC; HPV; deep sequencing; intratumor heterogeneity; mutation.

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Conflict of interest statement

No potential conflicts of interest were identified by any of the authors.

Figures

Figure 1
Figure 1. Degree of SNV heterogeneity varies based on sub-site
A. Schematic depicting specimen acquisition from the primary (P) tumor or metastatic (M) lymph node. HNSCC specimens were taken from multiple locations separated by a distance of at least 5 mm at the primary tumor site and in most cases, paired metastatic lymph node. Figure demonstrating SNVs present (blue/orange) or absent (white) within primary (P) or metastatic (M) B. oral tongue HNSCC (patient 2, 4, 5 and 6), and C. laryngeal (patient 1 and 7) and FOM (patient 3) HNSCC. D. Functional consequences of SNVs present within tumor specimens are depicted in the bar graph. The majority of mutations from various sites were missense mutations.
Figure 1
Figure 1. Degree of SNV heterogeneity varies based on sub-site
A. Schematic depicting specimen acquisition from the primary (P) tumor or metastatic (M) lymph node. HNSCC specimens were taken from multiple locations separated by a distance of at least 5 mm at the primary tumor site and in most cases, paired metastatic lymph node. Figure demonstrating SNVs present (blue/orange) or absent (white) within primary (P) or metastatic (M) B. oral tongue HNSCC (patient 2, 4, 5 and 6), and C. laryngeal (patient 1 and 7) and FOM (patient 3) HNSCC. D. Functional consequences of SNVs present within tumor specimens are depicted in the bar graph. The majority of mutations from various sites were missense mutations.
Figure 1
Figure 1. Degree of SNV heterogeneity varies based on sub-site
A. Schematic depicting specimen acquisition from the primary (P) tumor or metastatic (M) lymph node. HNSCC specimens were taken from multiple locations separated by a distance of at least 5 mm at the primary tumor site and in most cases, paired metastatic lymph node. Figure demonstrating SNVs present (blue/orange) or absent (white) within primary (P) or metastatic (M) B. oral tongue HNSCC (patient 2, 4, 5 and 6), and C. laryngeal (patient 1 and 7) and FOM (patient 3) HNSCC. D. Functional consequences of SNVs present within tumor specimens are depicted in the bar graph. The majority of mutations from various sites were missense mutations.
Figure 1
Figure 1. Degree of SNV heterogeneity varies based on sub-site
A. Schematic depicting specimen acquisition from the primary (P) tumor or metastatic (M) lymph node. HNSCC specimens were taken from multiple locations separated by a distance of at least 5 mm at the primary tumor site and in most cases, paired metastatic lymph node. Figure demonstrating SNVs present (blue/orange) or absent (white) within primary (P) or metastatic (M) B. oral tongue HNSCC (patient 2, 4, 5 and 6), and C. laryngeal (patient 1 and 7) and FOM (patient 3) HNSCC. D. Functional consequences of SNVs present within tumor specimens are depicted in the bar graph. The majority of mutations from various sites were missense mutations.
Figure 2
Figure 2. TP53 mutations p.I251N and p.Y236C within the primary laryngeal tumor sub-sites developed independent of each other
The primary tumor from patient 7 was assessed for mutations in TP53 at 2 sites separated by a distance of 1 cm. Two specific mutations in TP53 that were separated by 45 bases were analyzed in each sequencing read (gray bars). Across all sequencing reads, tumor A had 6.87% alterations in the amino acid residue p.I251N and 21.86% alterations in p.Y236C. Tumor B had 59.23% alterations in the p.I251N residue and no alteration in p.Y236C. Thus, mutations in p.I251N and p.Y236C residues emerged independently at different sites (A and B) within the primary tumor.
Figure 3
Figure 3. Heterogeneity in copy number varies based on the sub-site or origin
The copy number variability identified within A. laryngeal (patient 1 and 7) and FOM (patient 3) tumors, and B. oral tongue tumors. The key specifies the color code to depict the status of CNVs in each gene. Larynx and FOM tumors demonstrated significantly higher number of CNVs compared to oral tongue tumors.
Figure 3
Figure 3. Heterogeneity in copy number varies based on the sub-site or origin
The copy number variability identified within A. laryngeal (patient 1 and 7) and FOM (patient 3) tumors, and B. oral tongue tumors. The key specifies the color code to depict the status of CNVs in each gene. Larynx and FOM tumors demonstrated significantly higher number of CNVs compared to oral tongue tumors.
Figure 4
Figure 4. Evolutionary analyses of mutated codons in HNSCC from various sites
Panels A., C., E., G., I., K. and M. represent midpoint-rooted phylogenetic trees of the tumors using the maximum likelihood approach. The branch lengths are proportional to the number of nonsynonymous mutations between the branching points. The scale bar represents 0.1 substitutions per site. Panels B., D., F., H., J., L. and N. represent the evolutionary models of tumors in each patient and list the mutated genes in each cluster. The commonly mutated genes are in dark blue and genes with unique mutations are listed below the brown and orange lines for the primary and metastatic tumor, respectively.
Figure 5
Figure 5. Affymetrix SNP 6.0 array and TCGA data analysis demonstrate that oral cavity tumors have fewer mutations than larynx tumors
A. Number of single nucleotide variations (SNV), copy number variations (CNV) and loss of heterozygosity (LOH) in all samples from patient 1 (larynx tumor) and patient 5 (oral tongue tumor) are graphed. Numbers in the stacked bars indicate the number of mutations. B. TCGA analyses of percentage of genes with low (0-200) or high (200-400) number of mutations in tumor from oral cavity or larynx.
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