Genomic Evolution after Chemoradiotherapy in Anal Squamous Cell Carcinoma

Clinical characteristics of pilot and extension ASCC cohorts

To investigate the genomic landscape of ASCC and identify features of treatment response, we reviewed ASCC cases treated in the primary and/or recurrent setting at our institution between 2005 and 2013. All cases with sufficient FFPE primary tumor tissue and germline DNA were included in the pilot cohort (n = 13; Table 1; Supplementary Fig. S1). Seven of the 13 patients in the pilot cohort had a complete clinical response to CRT and remained free of disease for at least 2 years (median follow-up, 6.0 years; range, 2.1–9.8 years), whereas 6 patients developed a recurrence during follow-up (median time to recurrence, 14.1 months; range, 3.2–49.1 months). There were no significant differences in clinical or tumor characteristics at diagnosis between patients who developed a recurrence and those who did not (Supplementary Table S1). As expected, nearly all tumors were HPV positive (12/13 primary tumors, 92%), and there was 100% concordance between p16 IHC and HPV in situ hybridization (Table 1; Supplementary Fig. S2). For each of the recurrent cases, the HPV status of the recurrent tumor was identical to the primary tumor.

To expand our findings from the pilot cohort, we identified an extension cohort consisting of an additional 33 ASCC cases (43 tumors). Cases in the extension cohort were identified from the same institutional pool as the pilot cohort, but lacked a source of matched germline DNA. Compared with the pilot cohort, there were more females and more recurrent cases in the extension cohort (Table 1). Seven of the 33 patients in the extension cohort had no disease recurrence during follow-up (median follow-up, 5.0 years; range, 1.0–6.1 years), whereas 26 patients had disease recurrence (median time to recurrence, 7.0 months; range, 2.2–257.6 months; Table 1). Of the 26 recurrent cases, paired primary and recurrent tumor(s) were available for 8 patients (17 tumors), primary tumor alone was available for 3 patients (3 tumors), and recurrent tumor alone was available for 15 patients (16 tumors; Supplementary Fig. S1). HPV status of the extension cohort was determined by p16 IHC, and the majority of cases were HPV positive (25/31, 81%). For cases with both primary and recurrent tumors, the HPV status was identical within each pair.

Genomic landscape of primary ASCC

For all cases in the pilot cohort, WES of the primary tumor and matched germline DNA was performed, and data were analyzed using published techniques (Materials and Methods). Mean target coverage was 107.3× for tumor and 108.4× for germline DNA (Supplementary Table S2). A total of 2,980 somatic mutations, including 1,875 missense, 170 nonsense, 30 frameshift, and 785 silent mutations, were identified in the pilot cohort. The mean somatic mutational burden was 5.7 mutations/Mb (range, 0.8–35.5 mutations/Mb; Supplementary Table S3), similar to other HPV-associated squamous cell carcinomas (18, 19).

Given the limited size of the cohort, we did not observe highly recurrent alterations in novel genes, and we therefore chose to focus our analyses on genes that are recurrently mutated in other HPV-associated squamous cell cancers (Fig. 1). Among the 13 primary tumors in the pilot cohort, four (31%) had a missense mutation in FBXW7, an E3 ubiquitin ligase that targets known oncoproteins, such as c-Myc and cyclin E, and is significantly mutated in multiple tumor types. All four FBXW7 missense mutations occurred at conserved arginines that play a role in substrate binding and are known mutational hotspots (20).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Overview of the mutational landscape of primary and recurrent ASCC. Clinical characteristics, mutation and copy number data, and overall mutational burden are shown for the pilot cohort (left) and extension cohort (right). Primary tumors from each cohort are shown in the top half of the figure, and within each cohort, patients who were free from recurrence during follow-up are on the left (light purple), and patients who developed recurrent disease are on the right (dark purple). In the bottom half of the figure, similar data are displayed for recurrent tumors. Cases with both primary and recurrent tumors are vertically aligned. Copy number status for chromosome 3q is shown for ≥2-fold gain (red) and ≥2-fold loss (blue). Mutation burden is displayed as mutations/megabase (Mb). Recurrence location is annotated as local (defined as the primary tumor site or a pelvic/inguinal lymph node) or distant, and time to recurrence refers to the time interval between completion of chemoradiotherapy and biopsy-confirmed recurrent disease.

We also noted multiple events in other squamous cell carcinoma genes, including known activating mutations in PIK3CA, alterations in the KEAP-1 binding domain of NFE2L2, and mutations in TP63 and EP300, genes involved in squamous cell differentiation (Fig. 1; ref.21, 22). In addition, activating MAPK1 and inactivating PTEN mutations were noted in individual cases. Interestingly, the single HPV-negative case did not have a TP53 mutation, whereas two of the 11 HPV-positive cases harbored TP53 mutations.

WES was also performed for all primary tumors in the extension cohort, and mutations were nominated after removing putative germline variants (Materials and Methods; Supplementary Fig. S3). The most frequently mutated gene in the extension cohort was PIK3CA, which was mutated in 7 of 18 primary tumors (39%). In the combined (pilot plus extension) cohort, 10 of 31 tumors (32%) harbored a PIK3CA mutation, similar to the rate reported in recent targeted sequencing studies (11, 12). Similarly, the overall mutation frequency of FBXW7 in the pooled cohort (6/31, 19%) is comparable with the rate described in other HPV-associated SCCs (12).

We next compared the mutational characteristics of primary tumors that did not recur following CRT versus those that did. PIK3CA, the most commonly mutated gene among primary tumors, occurred at similar frequencies in the two groups (6/14 vs. 4/17, P = 0.44; Supplementary Fig. S4). There was a trend toward increased frequency of EP300 mutations in tumors that did not recur during follow-up (3/14 vs. 0/17, P = 0.08) and increased frequency of FBXW7 mutations in tumors that did recur (5/17 vs. 1/14, P = 0.18).

In addition to the mutational status of individual genes, we also sought to characterize the landscape of somatic copy number alterations. Analysis of both pilot and extension cohorts revealed frequent chromosome 3q amplification, an event that has previously been reported in HPV-associated malignancies and is associated with progression from dysplasia to invasive disease in both anal and cervical squamous cell carcinomas (Supplementary Fig. S5; refs.19, 23, 24). Chromosome 3q harbors several cancer-related genes, including PIK3CA, SOX2, and TP63; however, the role of individual gene amplifications in ASCC tumorigenesis is unknown. There was no difference in chromosome 3q status in primary tumors that recurred following CRT versus those that did not.

Genomic landscape of recurrent ASCC

Although CRT is associated with high response rates, approximately 1 in 5 ASCC patients will develop recurrent disease. To better understand the genetic features of recurrent ASCC, we performed WES of 30 recurrent ASCC tumors from 28 patients (5 patients from pilot cohort, 23 patients from extension cohort; Fig. 1, Supplementary Fig. S1). Fourteen recurrent tumors (from 13 patients) were analyzed in parallel with the corresponding primary tumor from the same patients, whereas 16 recurrent tumors (from 15 patients) did not have an accompanying primary tumor. As in the primary tumor cohort, PIK3CA was the most commonly mutated gene among recurrent tumors (Fig. 1), followed by TP53 and FBXW7. Six of the 28 recurrent cases were HPV negative, and only one of the seven HPV-negative cases in this study was not associated with recurrence. Four of the six recurrent HPV-negative tumors harbored a TP53 mutation, whereas only one of the 22 HPV-positive recurrent tumors had a TP53 mutation (P = 0.003, Fisher exact test). One TP53-mutated tumor had a nonsense mutation in CDKN2A, an event frequently observed in TP53-mutated, HPV-negative head and neck SCC (18).

Genomic evolution in matched primary and recurrent tumors

For 13 patients, paired pre- and post-CRT tumors were available for analysis. In most cases, primary–recurrent pairs had mutations and copy number alterations that were shared as well as events that were unique to either the primary or recurrent tumor (Supplementary Fig. S6). However, to further characterize genomic evolution in the context of CRT, we used sample purity and local copy number data from ABSOLUTE to calculate the CCF of point mutations and generate sibling models for patient-matched pre- and post-CRT tumors from the pilot cohort (n = 5; Materials and Methods; ref.25). In all but one case (AC-P10), we observed a clonal mutation (CCF = 1) in one or more putative driver cancer genes. For case AC-P10, the purity of the recurrent tumor at the time of sample acquisition was estimated to be less than 5%, possibly explaining the lack of any predicted clonal mutations in this sample.

In three of the remaining four cases, one or more clonal mutations were shared between primary and recurrent tumors. For example, in AC-P09, both the primary and recurrent tumors harbored a clonal FBXW7 R505C mutation (Fig. 2A). Interestingly, AC-P04 lacked any evidence of shared mutations between primary and recurrent tumors (Fig. 2B). In this case, the time interval between completion of CRT and recurrence was more than 4 years, raising the possibility that the disease recurrence may actually represent a second primary tumor. Further supporting this hypothesis, the invasive recurrence arose in the setting of diffuse HPV-associated anal intraepithelial neoplasia in a patient with poorly controlled HIV, underscoring the role of HPV and host immune status in ASCC risk and pathogenesis (26).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Genomic evolution in patient-matched pre- and post-CRT tumors. For each case, the fraction of mutations unique to the primary (blue), unique to the recurrent tumor (red), or shared between primary and recurrent tumors (gray) is shown on the left. CCFs for mutations in the primary (x-axis) and recurrent (y-axis) tumors are compared, and the corresponding phylogenetic tree is shown on the right. Select cancer genes are annotated in the CCF plots and the phylogenetic trees (37). A, The primary and recurrent tumors from case AC-P09 share many mutations, including a clonal FBXW7 R505C mutation. B, There are no shared mutations between the primary and recurrent tumor from case AC-P04, suggesting that the recurrent disease may actually represent a second primary tumor. C, In case AC-P15, the primary tumor has a clonal KRAS G12A mutation. The recurrent tumor shares several mutations with the primary tumor; however, the KRAS G12A mutation is absent, whereas a distinct oncogenic KRAS mutation (L19F) is present. In addition, the recurrent tumor harbors a clonal mutation in KEAP1. D, In case AC-P12, the primary tumor has an oncogenic FBXW7 R465C mutation present in the primary tumor. The recurrent tumor shares several mutations with the primary tumor; however, the FBXW7 R465C mutation is absent from the recurrent tumor, whereas a distinct oncogenic FBXW7 R505C mutation is present.

In two of the primary–recurrent pairs, we observed distinct mutations in a single cancer gene in phylogenetically related primary and recurrent tumors. In one case (AC-P15), a known KRAS-activating mutation (G12A) was clonal in the primary tumor (Fig. 2C). Despite sharing many other mutations with the primary tumor, the recurrent tumor lacked the KRAS G12A mutation and instead possessed a distinct KRAS L19F mutation. The KRAS L19F mutation has been observed previously in sporadic colorectal cancers and has an attenuated phenotype compared with the more common KRAS codon 12 mutations (27, 28). Similarly, in another primary–recurrent tumor pair (AC-P12), one FBXW7 hotspot mutation (R465C) in the primary tumor was replaced by a different FBXW7 hotspot mutation (R505G) in the recurrent tumor (Fig. 2D). In both cases, the primary and recurrent tumors shared many passenger mutations, confirming their sibling relationship.

In addition, there were examples in which a recurrent tumor harbored a putative driver mutation in a gene that was not mutated in the primary tumor. For example, a novel KEAP1 mutation was observed in a recurrent KRAS-mutated tumor (Fig. 2C). Activation of the NRF2 pathway by mutation of NFE2L2 or KEAP1 suppresses reactive oxygen species–mediated cellular damage, particularly in KRAS-mutated tumors and is associated with poor response to chemotherapy in lung squamous cell cancer (21).

Effect of CRT on mutational signatures and neoantigen load

The standard treatment for locally advanced ASCC involves concurrent delivery of the multiple DNA-damaging agents, and we sought to characterize the genomic consequences of DNA damage exposure by comparing mutational features of pre- and post-CRT tumors. Among patient-matched tumor pairs, there was a trend toward increased nonsynonymous mutational burden in recurrent compared with primary tumors (7.1 vs. 3.7 mutations/Mb, P = 0.11, paired t test; Fig. 3A and B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3.

Mutational burden, neoantigen load, and an exceptional response to immune checkpoint blockade in recurrent ASCC. A, Comparison of mutational burden in patient-matched pre- and post-CRT tumors from the pilot cohort. B, Comparison of mutational burden in patient-matched pre- and post-CRT tumors from the extension cohort. Nonsyn., nonsynonymous. C, Mutational burden and predicted neoantigen burden in primary tumors (n = 13) from the pilot cohort. D, Representative axial CT images showing a metastatic ASCC lesion at the time of initiation of anti–PD-1 therapy (left), 2 months after initiation (center), and 6 months after initiation (right). The patient continues on anti–PD-1 therapy.

To further characterize the mutational effects of CRT on ASCC, we performed mutational signature analysis of pre- and post-CRT tumors using a novel NMF approach (29). Analysis of all primary and recurrent tumors from the pilot cohort (n = 18) identified three operating mutational signatures (Supplementary Fig. S7A). Two of the signatures closely resembled previously described signatures: one attributed to activity of APOBEC enzymes and one attributed to aging-related accumulation of C>T mutations at CpG motifs (Supplementary Fig. S7B; ref.30). The third signature identified in the cohort is characterized primarily by C>A transversions and did not resemble any described signature. Interestingly, this signature was primarily active in a single recurrent tumor (AC-P04), but was absent in the primary tumor from the same patient (Supplementary Fig. S7C).

Given that DNA damage–induced somatic mutations can result in novel cancer-specific neoantigens, we used patient-specific nonsynonymous mutations and HLA types to predict putative immunogenic neoantigens for matched primary and recurrent tumors from the pilot cohort (Materials and Methods, Supplementary Information; ref.16). As expected, the predicted tumor neoantigen load was closely correlated with the overall mutation burden (Fig. 3C). In four of five tumor pairs from the pilot cohort, the difference in neoantigen burden between primary and recurrent tumors was modest. However, in one case (AC-P-04), the recurrent tumor harbored significantly more mutations and neoantigens than the primary tumor, and this difference was contributed almost entirely by mutations attributed to the unique mutational signature (Supplementary Fig. S7C). Interestingly, this is the same case in which the recurrent tumor did not share mutations with the primary tumor (Fig. 2B).

Exceptional response to PD-1 blockade in ASCC

Recently, immune checkpoint blockade has emerged as an effective treatment strategy for subsets of patients with malignancies, including ASCC (31, 32). One patient in this study (AC_PD1) developed widespread lymph node metastases 4 months following completion of chemoradiotherapy for an HPV-associated ASCC. The patient began systemic therapy with 5-fluorouracil, oxaliplatin, and leucovorin, but CT after six cycles demonstrated progressive disease, and the patient was subsequently enrolled on a clinical trial of the anti–PD-1 antibody pembrolizumab (Supplementary Methods). CT after 6 weeks of treatment with pembrolizumab showed shrinkage in multiple metastatic foci, and subsequent CTs have continued to demonstrate a durable response (tumor burden decreased 79% by RECIST criteria) that continues to persist 27 months following initiation of PD-1 blockade (Fig. 3D). Analysis of the primary tumor from the patient revealed missense mutations in PIK3CA, PIK3CB, and ERBB2 (Fig. 1). The overall mutational burden and predicted neoantigens were higher than all but one other tumor in the pilot cohort, but this difference was not statistically significant (Fig. 3C).