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Journal of Gastrointestinal Oncology logoLink to Journal of Gastrointestinal Oncology
. 2021 Oct;12(5):2423–2437. doi: 10.21037/jgo-20-610

Molecular characterization of squamous cell carcinoma of the anal canal

Samantha A Armstrong 1, Rita Malley 1, Hongkun Wang 1, Heinz-Josef Lenz 2, David Arguello 3, Wafik S El-Deiry 4, Joanne Xiu 3, Zoran Gatalica 3, Jimmy J Hwang 5, Philip A Philip 6, Anthony F Shields 6, John L Marshall 1, Mohamed E Salem 5,#, Benjamin A Weinberg 1,#,^,
PMCID: PMC8576238  PMID: 34790403

Abstract

Background

Squamous cell carcinoma of the anal canal (SCCA) is an uncommon malignancy with limited therapeutic options. Nivolumab and pembrolizumab show promising results in patients with SCCA. Human papillomavirus (HPV)-negative tumors are frequently TP53-mutated (TP53-MT) and often resistant to therapy.

Methods

We present a large molecularly-profiled cohort of SCCA, exploring the underlying biology of SCCA, differences between TP53-wild type (TP53-WT) and TP53-MT tumors, and differences between local and metastatic tumors. SCCA specimens (n=311) underwent multiplatform testing with immunohistochemistry (IHC), in situ hybridization (ISH) and next-generation sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chi-square testing was used for comparative analyses.

Results

The most frequently mutated genes included PIK3CA (28.1%), KMT2D (19.5%), FBXW7 (12%), TP53 (12%) and PTEN (10.8%). The expression of PD-1 was seen in 68.8% and PD-L1 in 40.5% of tumors. High TMB was present in 6.7% of specimens. HER2 IHC was positive in 0.9%, amplification by chromogenic in situ hybridization (CISH) was seen 1.3%, and mutations in ERBB2 were present in 1.8% of tumors. The latter mutation has not been previously described in SCCA. When compared with TP53-WT tumors, TP53-MT tumors had higher rates of CDKN2A, EWSR1, JAK1, FGFR1 and BRAF mutations. PD-1 and PD-L1 expression were similar, and high TMB did not correlate with PD-1 (P=0.50) or PD-L1 (P=0.52) expression.

Conclusions

Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

Keywords: Squamous cell carcinoma of the anal canal (SCCA), anal carcinoma, targeted therapy, genomic profiling

Introduction

Anal cancers are relatively rare, with an estimated 8,590 new cases in the US in 2020, although the incidence is rising (1). The most common histological subtype of anal cancer is squamous cell carcinoma of the anal canal (SCCA) (2). Although SCCA occurs more frequently in women than men, its incidence is rising in men who have sex with men and persons infected with HIV (3). Despite this, treatment for locoregional SCCA has not changed in many years and consists of the Nigro protocol, first described in 1974, of definitive concurrent chemoradiation with 5-fluorouracil (5-FU) and mitomycin-C, with surgery reserved for the salvage setting (4). A phase III EORTC study confirmed the use of combined modality therapy with chemoradiation (5-FU plus mitomycin-C), demonstrating improved local control and colostomy-free survival compared to radiation alone (5). Until recently, metastatic SCCA was treated with the same decades-old chemotherapy regimen of 5-FU plus cisplatin (6). This combination was recently challenged by carboplatin plus weekly paclitaxel in the phase II InterACCT trial (n=91), results at a median follow-up of 28.6 months revealed that carboplatin plus paclitaxel had comparable response rates to 5-FU plus cisplatin; the overall response rate (ORR) was 59% (95% CI, 42.1% to 74.4%) for carboplatin plus paclitaxel versus 57% (95% CI, 39.4% to 73.7%) for cisplatin plus 5-FU, with reduced serious events (36% compared to 62% P=0.016) and improved progression-free survival [PFS, 8.1 months (95% CI, 6.6 to 8.8 months) versus 5.7 months (95% CI, 3.3 to 9.0 months)]. Median overall survival (OS) was 20 months (95% CI, 12.7 months to not reached) versus 12.3 months (95% CI, 9.2 to 17.7 months, HR 2.00, 95% CI, 1.15 to 3.47; P=0.014) (7). These data indicate carboplatin plus paclitaxel should be considered as a new standard of care. The persistence of these standards of care is in part due to the rarity of SCCA and the difficulty of completing large, randomized clinical trials. Moreover, patients with local disease have variable responses to chemoradiation. New insights into the molecular biology of SCCA reveal the underlying mechanisms of carcinogenesis and are changing the treatment paradigm.

Human papillomavirus (HPV) is involved in 85–88% of SCCA (8). SCCA that is not related to HPV infection represents a small minority of cases. These cases are notoriously refractory to chemoradiation and frequently harbor TP53 mutations (9). Deeper sequencing techniques on 392 HPV-PCR negative cervival cancer specimens, detected HPV in 43.1% of specimens (n=169) suggesting HPV as the oncogenic driver even if previously testing negative (10). Interestingly, many precursor lesions (anal intraepithelial neoplasia II/III) are also positive for HPV (especially HPV16), suggesting that HPV infection is an early event in potential oncogenesis; however, few of these precursor lesions will actually become malignant (11). It remains unclear why some precursor lesions progress while others do not, but immunosuppression (especially from HIV infection) plays a role via the evasion of HPV-specific CD4+ and CD8+ T cells (12,13). While HPV infection is often transient in immunocompetent individuals, HIV-positive patients often have a persistent HPV infection (9). The HPV viral genes E6 and E7 contribute to oncogenic transformation. The E6 oncoprotein encoded by the HPV genome directly targets and inactivates the p53 protein, which usually functions as a tumor suppressor (14). Consequently, p53 is unable to induce apoptosis. The E7 oncoprotein effectively deregulates the cell-cycle; E7 forms complexes with the phosphorylated retinoblastoma protein (pRb), which is responsible for inhibiting transcriptional activity in the G1 phase of the cell cycle. Once complexed with E7, pRb is degraded, leading to progression of the cell through the S phase (15). Multiple other signaling pathways have been implicated in SCCA carcinogenesis, including epidermal growth factor receptor (EGFR), PI3K/AKT/mTOR, VEGF, hedgehog and Bcl-2 (15). Given the few treatment options for metastatic SCCA, knowledge of the activity of these pathways in individual tumors is vital to effective therapeutic selection.

A previous study of 199 recurrent or metastatic SCCA tumors was notable for frequent mutations in PIK3CA (33%) and TP53 (15%). The tumors also had high immunohistochemistry (IHC) expression of EGFR (89%), TOP2A (85%), TOPO1 (67%) and low ERCC1 (49%), potentially conferring sensitivity to anti-EGFR antibodies, anthracyclines, irinotecan and platinum-based chemotherapies, respectively (16). Few tumors in this study were tested for PD-1 or PD-L1, and PD-L1 expression is known to correlate with a worse prognosis in SCCA (17). Another study preformed comprehensive genomic profiling on 574 SCCA tumors to analyze the prevalence as well as mutational profiling details of tumor suppressor gene CYLD (18). CYLD mutations were seen in 13% of patients, and correlated with lower tumor mutational burden (TMB) and less alterations in PIK3CA. Additional comprehensive molecular profiling of SCCA are necessary to establish clinically useful biomarkers.

We sought to expand on this work through several avenues. First, we analyzed a large known cohort of molecularly-profiled SCCA. Second, by understanding the differences between profiles of primary and metastatic SCCA, pathways affecting metastasis can be identified. Third, there is recent data evaluating the molecular characteristics of the difference between TP53-WT versus TP53-MT tumors and our cohort adds to this growing field (19,20). Thus, continued knowledge expansion of molecular pathways that confer this resistance to standard therapy would help guide the selection of alternative treatments. Finally, the exploding field of immunotherapy holds particular promise in metastatic SCCA. Because SCCA tends to flourish in an immunosuppressed environment, there is significant hope that checkpoint inhibition can be utilized to harness the immune system in attacking SCCA.

In this study, we aimed to establish the true prevalence of PD-1 and PD-L1 expression in a larger repository of SCCA tumors. In addition, we sought to analyze the mutational burden in SCCA tumors because a higher mutational burden may lead to greater diversity in neoantigens and increase the likelihood of an effective immune response (21). Mutational burden has proven to be a useful biomarker for efficacy of immunotherapy in non-small cell lung cancer, urothelial cancer, melanoma and mismatch repair (MMR)-deficient tumors (22-25). We present the following article in accordance with the MDAR reporting checklist (available at https://dx.doi.org/10.21037/jgo-20-610).

Methods

A multi-institutional database was searched to identify patients diagnosed with SCCA from January 1, 2015, through March 31, 2019. All patient data were deidentified, negating the need for patient informed consent. Each archived formalin-fixed, paraffin-embedded tumor sample or newly obtained biopsy sample was assessed by the Caris Life Sciences (Phoenix, AZ, USA) multiplatform profiling service. Tumor samples were accompanied by limited patient demographic and clinical information, and this is shown in Table 1.

Table 1. Patient demographics.

Parameter Number
Total cases (n) 311
Age (years)
   Median age 61
   Interquartile range 31–89
Gender, n (%)
   Female 225 (72.3)
   Male 86 (27.7)
Location of primary tumor, n (%)
   Anus 117 (56.9)
   Anal canal 92 (29.6)
   Anorectum 16 (5.1)
   Overlapping lesion of rectum, anus, anal canal 10 (3.2)
   Rectum 7 (2.3)
   Anorectal junction 6 (1.9)
   Skin of anus 1 (0.3)
   Perianal skin 1 (0.3)
   Cloacagenic zone 1 (0.3)
Specimen site, n (%)
   Local 210 (67.5)
   Distant metastasis 101 (32.5)

Molecular analysis

All 311 SCCA specimens underwent multiplatform testing with the test selection based on the primary oncologist’s recommendations. Testing included protein expression through IHC assay (n=302), gene amplification through chromogenic in situ hybridization (CISH, n=90), copy number alteration (CNA, n=215), fragment analysis (FA, n=15), fusion analysis via RNA sequencing (n=50), transcriptome sequencing (n=10) and next-generation sequencing [NGS, hot spot NGS (n=64) and Illumina NextSeq, 592 gene, NGS (n=226)]. TMB (n=209) was calculated using only somatic nonsynonymous missense mutations, and TMB-high was defined as ≥17 mutations (26). Details of the samples’ testing are shown in Figure 1.

Figure 1.

Figure 1

Specimen testing demographics. SCCA, squamous cell carcinoma of the anal canal; IHC, immunohistochemistry; NGS, next-generation sequencing; CISH, chromogenic in situ hybridization; CNA, copy number alteration; FA, fragment analysis.

IHC was performed on full formalin-fixed paraffin-embedded (FFPE) sections of glass slides, using automated staining techniques, per the manufacturer’s instructions, and were optimized and validated per CLIA/CAO and ISO requirements. Staining was scored for intensity (0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining) and staining percentage (0–100%). Results were categorized as positive or negative by defined thresholds specific to each marker based on published clinical literature that associates biomarker status with patient responses to therapeutic agents. A board-certified pathologist evaluated all IHC results independently. The primary antibody used against PD-L1 was SP142 (Spring Biosciences, Pleasanton, CA, USA). The staining was regarded as positive if its intensity on the membrane of the tumor cells was ≥2+ and the percentage of positively stained cells was >5%. The primary antibody used for PD-1 was MRQ-22 (Ventana) and staining was scored as positive if the number of PD-1+ TIL was >1 cell per high-power field. PD-1 TIL density was evaluated using a hotspot approach. The whole tumor sample was reviewed at a low power (4× objective), and the areas of highest density of TIL in direct contact with malignant cells of the tumor at 400× visual field (40× objective × 10× ocular) were enumerated.

Statistical analysis

The frequency of the protein expression, mutation and amplification data was compared between tumor samples that were TP53-WT vs. TP53-MT and primary vs. metastatic via either Chi-square tests or Fisher’s exact tests when appropriate. SAS software version 9.4 (SAS Ins., Cary, NC, USA) was used for the analysis. P values <0.05 were considered statistically siginificant.

Ethical statement

Informed consent was not required for this study; these analyses were conducted using de-identified patient data obtained from Caris Life Sciences (Phoenix, AZ, USA) database. It was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Results

Baseline patient characteristics

We performed an analysis of 311 SCCA cases. The median patient age was 61 years (interquartile range, 31–89 years). The majority of patients (72.3%) were female, and specimens were examined from both primary and metastatic sites (67.5% and 32.5%, respectively), with the most common metastatic site being the liver (51.5%, n=52). Local specimen lesions include the anus, anal canal, anorectum, overlapping lesion of rectum/anus/anal canal, rectum, anorectal junction, skin of anus, perianal skin, cloacogenic zone, vagina/labia, urethra, skin of thigh, gluteal skin and inguinal lymph node. Distant metastatic specimen sites include liver, small bowel, omental, ovary, brain, paraspinal, lung and pleural lesions as well as neck, cervical, supraclavicular, periaortic, paratracheal, subcarinal, scalene and retroperitoneal lymph nodes. Table 1 provides the details of the patient demographic characteristics.

Recurrent mutation and biomarker incidence

Recurrent mutations were seen in 82 genes (Table 2). In summary, the most frequently mutated genes included PIK3CA (28.1%), KMT2D (19.5%), FBXW7 (12%), TP53 (12%) and PTEN (10.8%). Of the 220 tumors subjected to Illumina NextSeq (592 gene) testing, the most common mutations were PIK3CA (28.1%), KMT2D (19.5%), KMT2C (16.2%), TP53 (12.0%), PTEN (10.8%), FBXW7 (10.3%), ARID1A (4.8%), ATRX (4.1%), APC (3.8%) and NOTCH1 (3.4%). IHC analysis showed frequent expression of EGFR (97.7%) and PTEN (68.1%). MMR deficiency frequency tested by IHC of MLH1, PMS2, MSH2 and MSH6 was 1.1%. Expression of PD-1 was seen in 68.8% (44/64) of tumors, and PD-L1 was seen in 40.5% (119/294). HER2 IHC was positive in 0.9% (1/106) of samples, and amplification by CISH was seen 1.3% (1/78) of samples. Mutations in ERBB2 were present in 1.8% (4/219) of samples.

Table 2. Mutation and biomarker incidence.

Name Incidence (%) Incidence ratio Technique
MSH2 100 92/92 IHC
MSH6 100 90/90 IHC
MLH1 98.9 91/92 IHC
PMS2 98.9 87/88 IHC
EGFR 97.7 43/44 IHC
PD-1 68.8 44/64 IHC
PTEN 68.1 64/94 IHC
PD-L1 40.5 119/294 IHC
PIK3CA 28.1 62/221 NGS
PIK3CA 24.2 15/62 NGS hot spot
KMT2D 19.5 36/185 NGS
KMT2C 16.2 19/177 NGS
FBXW7 12.9 8/62 NGS hot spot
TP53 12. 25/208 NGS
PTEN 10.8 22/204 NGS
FBXW7 10.3 20/195 NGS
TMB 6.7 14/209 NGS
PIK3CA 5.4 11/205 CNA
TP53 5 3/60 NGS hot spot
ARID1A 4.8 4/84 NGS
ATRX 4.1 3/73 NGS
APC 3.8 8/212 NGS
NOTCH1 3.4 7/204 NGS
RB1 3.3 2/61 NGS hot spot
AKT1 3.2 2/62 NGS hot spot
KRAS 3.2 2/62 NGS hot spot
TRK A/B/C 3.1 2/64 IHC
BCL6 2.9 6/205 CNA
NFIB 2.9 6/205 CNA
PBRM1 2.9 6/206 NGS
EP300 2.9 5/174 NGS
CDKN2A 2.8 6/216 NGS
KRAS 2.7 6/221 NGS
FGF19 2.5 5/204 CNA
TFRC 2.4 5/205 CNA
FGF4 2.4 5/208 CNA
FGF3 2.4 5/209 CNA
KDM5C 2.4 3/126 NGS
FGFR3 2.3 5/214 NGS
AKT1 2.3 5/218 NGS
BAP1 2.3 5/219 NGS
CYLD 2.1 4/191 NGS
RB1 2 4/197 NGS
PIK3R1 2 4/204 NGS
KLHL6 2 4/205 CNA
CCND1 1.9 4/210 CNA
BRCA2 1.9 4/215 NGS
MUTYH 1.8 4/217 NGS
ERBB2 1.8 4/219 NGS
ZNF703 1.5 3/203 CNA
CALR 1.5 3/205 CNA
ETV5 1.5 3/205 CNA
LPP 1.5 3/205 CNA
LYL1 1.5 3/205 CNA
PSIP1 1.5 3/205 CNA
SMAD2 1.5 3/205 CNA
EGFR 1.4 3/208 CNA
FH 1.4 3/215 NGS
CTNNB1 1.4 3/216 NGS
HRAS 1.4 3/221 NGS
MSI 1.3 2/156 NGS
CREBBP 1.1 2/188 NGS
NSD1 1 2/198 NGS
ARID2 1 2/200 NGS
MYB 1 2/202 CNA
ADGRA2 1 2/203 CNA
FANCG 1 2/204 CNA
PRRX1 1 2/204 CNA
KEAP1 1 2/205 CNA
KRAS 1 2/205 CNA
MALT1 1 2/205 CNA
NSD3 1 2/205 CNA
SMAD4 1 2/205 CNA
SS18 1 2/205 CNA
FGFR1 1 2/209 CNA
MSH6 1 2/210 NGS
ERCC2 0.9 2/213 NGS
ATM 0.9 2/214 NGS
STK11 0.9 2/216 NGS
BRAF 0.9 2/217 NGS
ERBB3 0.9 2/218 NGS
BRCA1 0.9 2/219 NGS

IHC, immunohistochemistry; NGS, next-generation sequencing; CNA, copy number alteration.

TMB and microsatellite instability (MSI)

Mean TMB was 8.6 mutations per megabase. High TMB, as tested by NGS, was present in 6.7% of tumors (n=209). MSI-high tumors by FA and NGS were present in 6.7% (1/15), and 1.3% (2/156) of specimens, respectively.

Mutational differences between TP53-WT and TP53-MT

A comparison of TP53-MT to TP53-WT samples is summarized in Table 3. The incidence rates of statistically non-significant mutations and biomarkers are summarized in Table S1. CDKN2A mutations, known to encode both p16 (INK4a) and p14 (ARF) genes, were exclusively seen in TP53-MT samples (6/25). Additional mutations seen only in TP53-MT samples were BRAF (2/25, NGS), EWSR1 (1/24, CNA), JAK1 (1/25, NGS) and FGFR1 (2/24, CNA). When comparing biomarkers for immunotherapy, they were slightly less prevalent in TP53-MT compared to TP53-WT samples, although this difference was not statistically significant: IHC for PD-1, 50% (2/4) vs. 70% (42/60), P=0.583; IHC for PD-L1, 32.1% (9/28) vs. 41.4% (110/266), P=0.345; NGS for MSI, 0% (0/25) vs. 1.53% (2/131); and NGS for FA, 0% (0/0) vs. 6.67% (1/15), P=1.000. All HER2-positive samples were from TP53-WT. When testing for APC, TP53-MT samples had a significantly higher incidence of APC than TP53-WT samples by NGS (20%, 5/25 compared to 1.6%, 3/187, P<0.001) and by NGS hotspot (33.33%, 1/3 compared to 0%, 0/59, P=0.048). NOTCH1 incidence was also significantly higher in TP53-MT (12%, 3/25) than TP53-WT (2.23%, 4/179) with P=0.04 for NGS. The incidence of CALR by CNA was significantly higher in TP53-MT (8.33%, 2/24) compared to TP53-WT (0.55%, 1/181) tumors, P=0.037. FGFR1 incidence was significantly higher in TP53-MT (8.33%, 2/24) than in TP53-WT tumors (0%, 0/185), P=0.013, and ZNF703 incidence was higher in TP53-MT (8.33%, 2/24) compared to TP53-WT samples (0.59%, 1/170), P=0.041. PIK3CA incidence by NGS was significantly higher in TP53-WT (30.6%, 60/196) compared to TP53-MT samples (8%, 2/25), P=0.018.

Table 3. Incidence rates of statistically significant mutations and biomarkers in TP53-MT (n=28) tumors versus TP53-WT (n=238).

Alteration Test TP53-MT (n=28) incidence (%) TP53-MT incidence ratio TP53-WT (n=238) incidence (%) TP53-WT incidence ratio P value
Tumor suppressor genes
   CDKN2A NGS 24 6/25 0 0/191 0.0000
Immune
   APC NGS 20 5/25 1.6 3/187 0.0007
   APC NGS hot spot 33.33 1/3 0 0/59 0.0484
RAS/RAF/MEK
   BRAF NGS 8 2/25 0 0/192 0.0128
PI3K related kinases
   PIK3CA NGS 8 2/25 30.6 60/196 0.0178
Other
   NOTCH1 NGS 12 3/25 2.23 4/179 0.0410
   CAL-R CNA 8.33 2/24 0.55 1/181 0.0367
   FGFR1 CNA 8.33 2/24 0 0/185 0.0127
   ZNF703 CNA 8.33 2/24 0.55 1/170 0.0409

TP53-MT, TP53-mutated; TP53-WT, TP53-wild type; NGS, next-generation sequencing; CNA, copy number alteration.

Mutational differences between local and metastatic lesions

The analysis of both local and metastatic lesions is summarized in Table 4, with Figure 2 illustrating the statistically significant differences in incidence rates between local and metastatic tumor samples. The incidence rates of statistically non-significant mutations and biomarkers are summarized in Table S2. Local tumors showed higher incidence of PD-1 80.9% (38/47 IHC) compared to 35.3% (6/17), P<0.001 in metastatic lesions. Local tumors also showed a higher incidence of PTEN 13.8% (19/138 NGS) compared to 4.5% (3/66 NGS) in metastatic lesions, P=0.047. Metastatic lesions had significantly greater incidence of FGFR3 mutations, 6.8% (5/73 NGS) compared to 0% (0/141) in local tumors, P=0.004. All HER2-positive samples were from metastatic lesions.

Table 4. Incidence rates of statistically significant mutations and biomarkers in local versus metastatic locations.

Alteration Test Metastatic incidence (%) Metastatic incidence ratio Local incidence (%) Local incidence ratio P value
Immune
   PD-1 IHC 35.29 6/17 80.85 38/47 0.0005
SMAD pathway
   SMAD2 CNA 4.35 3/69 0 0/137 0.0365
PI3K related kinases
   PTEN NGS 4.55 3/66 13.77 19/138 0.0470
Other
   FGFR3 NGS 6.85 5/73 0 0/141 0.0042

IHC, immunohistochemistry; CNA, copy number alteration; NGS, next-generation sequencing.

Figure 2.

Figure 2

Incidence of mutations in PI3K related kinases in local and metastatic cases of SCCA. *, P<0.05; **, P<0.01; ***, P<0.001. SCCA, squamous cell carcinoma of the anal canal; NGS, next-generation sequencing; CNA, copy number alteration; IHC, immunohistochemistry.

Discussion

Although SCCA is a rare malignancy, its incidence and morbidity are increasing in the United States. Here we report an extensive comprehensive genomic profiling with NGS and gene expression profiling for patients with SCCA. Several tumor suppressor genes and oncogenes were newly identified in our population of SCCA.

Within this study, we were unable to identify HPV related cases by p16 overexpression given the lack of such testing’s availability at the time of our cohort’s analysis, other HPV driven malignancies such as head and neck cancer, cervical cancer and other gynecologic cancers utilize p16 overexpression as a confirmation for HPV infection as well as a prognostic marker related to PFS and OS [8–11]. The prognostic value of HPV-DNA and p16 expression in SCCA has also been explored, with results revealing that patients testing positively for both HPV-DNA and p16 overexpression have longer OS [18–19]. Newer unlike other gastrointestinal malignancies such as colon or pancreatic adenocarcinomas, SCCA rarely harbors KRAS mutations, with an incidence of 3% or less, which is confirmed in this comprehensive analysis (27,28). Similar to smaller studies, PIK3CA was the most common mutation identified. PIK3CA mutations are seen in approximately one-third of all specimens, frequently occurring in TP53-WT tumors (see Figure 3), suggesting that HPV may drive the accumulation of activating mutations in PIK3CA, which have been linked to higher risks of relapse (16,27-30). PIK3CA mutations have been identified in other HPV driven malignancies, including SCC of the head and neck (31). In addition, MLL2/KMT2D and MLL3/KMT2C are important in histone modification and are associated with oncogenesis as well as TP53 expression in preclinical studies. Prior SCCA studies identified mutations in these genes (29,32,33). Our analysis revealed high rates of mutations in both epigenetic regulators MLL2/KMT2D (19.5%) and MLL3/KMT2C (16.2%), the majority of both mutations in TP53-WT specimens.

Figure 3.

Figure 3

Incidence of mutations in TP53-MT (n=28) tumors and TP53-WT (n=238). *, P<0.05. CNA, copy number alteration; NGS, next-generation sequencing; TP53-MT, TP53-mutated; TP53-WT, TP53-wild type.

The tumor suppressor gene TP53, responsible for cell cycle regulation and apoptosis, is known to be overexpressed in SCCA. Many studies have seen a correlation between TP53 mutations and worse locoregional control and reduced disease-free survival in patients with SCCA (34-37). One study (n=119) found 4% of SCCA expressed exon 5 TP53 mutations (30). Another recent study (n=106) confirmed the correlation with HPV-negativity, p53 staining, and TP53 mutation in SCCA are associated with inferior OS as well as reduced recurrence-free survival (19). Our study discovered TP53 mutations in 12% of NGS samples (25/208) and 5% of NGS hot spot samples (3/60).

Rare mutations noted in this study include HRAS, found in 1.4% (3/221 NGS) of samples, all of which were in local TP53-WT specimens. HRAS was first noted in a smaller SCCA whole-exome sequencing cohort, along with ARID1A mutations, and were felt to be driver mutations of SCCA (38). ARID1A was prevalent in 4.8% (4/84 NGS) of our study specimens, all of which were TP53-WT, mostly localized tumors.

Potentially targetable mutations

Outside of immunotherapy, there are currently no clinically established biological markers to guide therapy for patients with SCCA. Table 5 reviews potentially targetable mutations identified in this analysis.

Table 5. Potentially targetable mutations outside of immunotherapy.

Potentially targetable mutation/test Treatment Incidence
DDR pathways Synthetic lethality with agents combined with a PARPi
   BRCA 1/NGS 2/219 (0.9%)
   BRCA 2/NGS 4/215 (1.9%)
   ATM/NGS 2/214 (0.9%)
   ATRX/NGS 3/73 (4.1%)
   PTEN/IHC 64/94 (68.1%)
PIK3CA
   PIK3CA/NGS 62/221 (28.1%)
   PIK3CA/NGS hot spot 15/62 (24.2%)
   PIK3CA/CNA 11/205 (5.4%)
BRAF BRAF/MEK inhibitors
   BRAF NGS 2/217 (0.9%)
HER2 Targeted HER2 agents
   ERBB2 NGS 4/219 (1.8%)
   HER2/IHC 1/106 (0.9%)
   HER2/CISH 1/78 (1.3%)
EGFR EGFR inhibitors
   EGFR/IHC 43/44 (97.7%)
   EGFR/CNA 3/208 (1.4%)
FGFR FGFR inhibitors
   FGFR3/NGS 5/214 (2.3%)
   FGFR2/NGS 1/215 (0.05%)

DDR, DNA damage response; NGS, next-generation sequencing; IHC, immunohistochemistry; CNA, copy number alteration; EGFR, epidermal growth factor receptor; FGFR, Fibroblast growth factor receptor.

PI3KCA

As noted above, PIK3CA was the most common mutation in this collection of SCCA samples, with an incidence of 28.1% (62/221) by NGS, and 24.2% (15/62) by NGS hot spot. PI3K and AKT are located downstream of the EGFR receptor. Following the binding of EGFR to its receptor and subsequent activation of PI3K and AKT, the cell signals to survive and proliferate. Mutations in PIK3CA, a portion of the PI3K kinase, result in constitutive PI3K activity, subsequent activation of AKT, and downstream oncogenic activity (29,30). In one PDX mouse model of SCCA, there was no did response to treatment with a PI3K inhibitor, suggesting that the high PIK3CA mutation rate and abnormally high PI3K activity plays an important role in anal carcinogenesis (29,38). The high prevalence of PIK3CA mutations seen in this analysis invites further analysis of PI3K and mTOR inhibitors as potential therapeutic options.

EGFR

Overexpression of EGFR is believed to contribute to tumor development and unregulated cell proliferation and is seen in many epithelial cancers. Our IHC results showed that 97.7% of SCCA samples express EGFR (43/44), although the rate of EGFR mutations, by CNA, is low at 1.4% (3/208). No EGFR mutations were found in 219 NGS or 62 NGS hot spot samples. Similar combinations of high rates of EGFR expression with low mutational rates have been seen in smaller SCCA studies (39-42). Cetuximab is an IgG1 chimeric monoclonal antibody against EGFR that is utilized in other gastrointestinal malignancies when no downstream K-RAS/N-RAS/BRAF mutations are present. The use of cetuximab in patients with SCCA has been studied without success. The phase II ECOG 3,205 and AMC0 45 trials both evaluated the addition of cetuximab to standard frontline 5-FU plus cisplatin concurrent with radiation (43,44). Unfortunately, substantial patient toxicity was seen with added cetuximab; 26–32% of patients experienced grade 4 toxicities (compared to a historic rate of 20% in the RTOG 98-11 trial) (45). Similar studies utilizing cetuximab to treat SCCA also revealed significant toxicities (46-48). However, it is possible that cetuximab may be useful in the PIK3CA wild-type subgroup.

HER2

In the same tyrosine kinase receptor family as EGFR is HER2/ERBB2, ERBB3 and ERBB4. HER2 expression has been well described and documented in many malignancies. It is prevalent in breast cancer and gastric cancer and seen less commonly in salivary, vaginal, bladder, endometrial, cervical and colorectal cancers (49-56). HER2 has emerged as a successful therapeutic target transforming outcomes for patients with breast and gastric cancers. The MY PATHWAY basket trial studied the efficacy of pertuzumab and trastuzumab in HER2-positive tumors, and responses were seen in 9 different tumor types, giving further indication that HER2 is an actionable mutation across many malignancies (57). Our molecular analysis is the first to identify HER2 expression and ERBB2 mutations in SCCA. HER2 IHC was positive in 0.9%, amplification by CISH was seen 1.3%, and mutations in ERBB2 were present in 1.8% of tumors from our population. These findings could lead to novel therapeutic options for this patient population, and the adoption of widespread tumor molecular profiling would allow patients to participate in clinical trials of molecularly-matched therapies.

BRAF

BRAF is located downstream of EGFR. The BRAF protein is a serine/threonine protein kinase responsible for regulating the MAPK/ERK signaling pathway during cell proliferation, differentiation, growth and apoptosis (58). Mutations in the BRAF gene result in downstream activation of the MAPK pathway via phosphorylation of MEK and ERK. The most common type of BRAF mutation is the V600E missense mutation.

In this population of SCCA, the BRAF mutation rate was relatively low, with an overall incidence of 0.9% (n=2) by NGS. This is consistent with previous reports of SCCA mutational analyses, which found most SCCA to be BRAF wild-type on whole-exome sequencing (28,29).

FGFR

Fibroblast growth factor receptors (FGFRs) are a subset of tyrosine kinases unregulated in cancer cells leading to increased proliferation, angiogenesis and cell survival. FGFR inhibitors are approved in biliary and urothelial cancers with FGFR alterations and appear to have activity in other malignancies with FGFR alterations (59-61). Fusions for FGFR2 and FGFR3 were not identified in the 12 samples checked. FGFR2 mutations were identified in 0.05% of samples (n=215) by NGS samples. FGFR3 mutations had an incidence of 2.3% (n=214) by NGS. The incidence of FGFR mutations and fusions in SCCA is low, but warrants further investigation of efficacy with FGFR inhibitors as an innovative therapeutic strategy to overcome drug resistance in the disease.

Immunotherapy

PD-1 blockade with nivolumab or pembrolizumab has shown promising results in SCCA patients (13,62), and we highlight data on the prevalence of TMB, PD-1 and PD-L1 expression in a larger repository of SCCA tumors. PD-L1 expression was found to be high in SCCA, which is likely related to the immune response to HPV oncoproteins (62). A recently published study preformed PD-L1 staining on 62 SCCA tumors and found expression (CPS ≥1) in 32% of cases (19). Amongst those who were HPV-negative with PD-L1 expression, there was a trend toward shorter OS (9.8 vs. 40.6 months; P=0.064). The first trial to establish the therapeutic benefits of immunotherapy in SCCA was the phase II NCI9673 study. This trial investigated the efficacy of nivolumab (3 mg/kg every 2 weeks) in patients with unresectable/metastatic SCCA, who had progressed on prior chemotherapy. The results revealed an objective response rate of 24% and a disease control rate of 72%, including 2 complete and 7 partial responses (13). Thirty patients underwent pretreatment cell-free DNA analysis which revealed mutations in TP53 (n=8, 27%) and PIK3CA (n=7, 23%) (13). Median PFS was 4.1 months, and median OS was 11.5 months. Responders had a higher percentage of CD3+ and CD8+ T cells and higher expression of granzyme-B, PD-1 and PD-L1 on tumor cells (13). Another study was the multicohort phase 1B KEYNOTE-028 trial for PD-L1 positive (≥1%) tumors, in which patients were treated with pembrolizumab at a dose of 10 mg/kg IV once every 2 weeks. Study investigators treated 24 patients with advanced SCCA and observed an ORR of 17% (95% CI, 5.0–37%) and a disease control rate of 58%. The median PFS was 3.0 months (95% CI, 1.7–7.3 months), with 6- and 12-month PFS rates of 31.6% and 19.7%, respectively (62). Results from these trials led to preferential selection of immunotherapy in the second-line for patients with advanced SCCA (63).

Cancers with high TMB, including melanoma and colorectal cancers, respond well to immune checkpoint inhibition, indicating that mutational load correlates with response to checkpoint inhibitors (62). Data suggest only a moderate mutation rate in SCCA, which is accordant with other HPV-associated cancers (13,62). Hence SCCA response to immune checkpoint inhibition is more likely related to an inflammatory tumor microenvironment than to TMB (13). Further evaluation of the efficacy of immune checkpoint inhibitors is ongoing in several clinical trials, as outlined in Table 6.

Table 6. Review of ongoing clinical trials utilizing immunotherapy or targetable mutations.

Clinical trial Size (n) Molecular target Title Status
NCT03233711 200 PD-1; PD-L1 A Randomized Phase III Study of Nivolumab After Combined Modality Therapy (CMT) in High Risk Anal Cancer Recruiting April 17, 2020
NCT02314169 137 PD-1; CTLA-4 A Multi-Institutional Phase II Study of Nivolumab or Nivolumab in Combination with Ipilimumab in Refractory Metastatic SCCA Recruiting April 10, 2020
NCT03074513 160 PD-L1 A Phase II, Single-Arm Open-Label Study of the Combination of Atezolizumab and Bevacizumab in Rare Solid Tumors Recruiting June 13, 2019
NCT02919969 32 PD-L1; VEGF A Multicenter Phase 2 Clinical Trial of Pembrolizumab in Refractory Metastatic Anal Center Recruiting July 25, 2019
NCT04287868 29 HPV vaccine; PDL1/TGF-b IL-12 Phase I/II Trial of Combination Immunotherapy in Subjects with Advanced HPV Associated Malignancies Recuriting June 11, 2020

SCCA, squamous cell carcinoma of the anal canal; HPV, human papillomavirus.

Limitations

Limitations of this study include the lack of broad HPV-DNA and p16 testing to correlate with HPV infection in the setting of SCCA and lack of clinical treatment and outcomes data to correlate with mutational analysis. The first is due to the lack of such testing’s availability as well as lack of relevance in SCCA at the time of our population’s diagnosis. Other limitations include lack of each patient clinical or pathologic staging, ability to obtain germline DNA testing and lack of details regarding specimen histopathologic findings.

Conclusions

In conclusion, we report on the largest comprehensive molecular profiling study of patient SCCA, which provides insights into the pathogenesis of SCCA and identifies potential therapeutic targets.

Acknowledgments

We would like to thank Marion L. Hartley, PhD, for her valuable edits and suggestions during the composition of the manuscript.

Funding: None.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Informed consent was not required. These analyses were conducted using de-identified patient data obtained from Caris Life Sciences database.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

Footnotes

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://dx.doi.org/10.21037/jgo-20-610

Peer Review File: Available at https://dx.doi.org/10.21037/jgo-20-610

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/jgo-20-610). DA, JX, ZG are employed by Caris Life Sciences. The other authors have no conflicts of interest to declare.

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