KRAS-variant is a biomarker of cetuximab response potentially through altered immunity in head and neck cancer: Re-analysis of NRG Oncology/RTOG 0522

Abstract

Importance

There is a significant need to find biomarkers of response to radiation and cetuximab in locally advanced head and neck squamous cell carcinoma (HNSCC), as well as biomarkers predicting altered immunity, thereby enabling personalized treatment.

Objective

To evaluate if the KRAS-variant, a germ-line mutation in a microRNA-binding site in KRAS, is a predictive biomarker of cetuximab response and altered immunity in the setting of radiation and cisplatin treatment. To evaluate the interaction of the KRAS-variant with p16 status and blood-based TGF-β1.

Design

Advanced HNSCC patients from NRG Oncology RTOG 0522, a Phase III trial of cisplatin plus radiation+/−cetuximab, were included in this study, and patients with available samples were genotyped for the KRAS-variant.

Methods

Genomic DNA was tested for the KRAS-variant in a CLIA-certified laboratory. Correlation between the KRAS-variant, p16 positivity, outcome, and TGF-β1 levels was evaluated. Hazard ratios (HR) were estimated by Cox model.

Main Outcomes and Measures

The correlation of KRAS-variant status with cetuximab response and outcome, p16 status, and plasma TGF-β1 levels was tested.

Results

Of 891 RTOG 0522 patients eligible for protocol analyses, 413 had biological samples for KRAS-variant testing, and 376 had plasma for TGF-β1 measurement. Seventy patients (16.9%) had the KRAS-variant. Overall, for KRAS-variant patients, cetuximab improved both progression-free survival (PFS) for the first year (HR 0.31, 95%CI 0.10–0.94, p=0.04) and overall survival (OS) in years 1–2 (HR 0.19, 95%CI 0.04–0.86, p=0.03). There was a significant interaction of the KRAS-variant with p16 status for PFS in patients treated without cetuximab (p=0.04). In p16 positive patients, KRAS-variant patients treated without cetuximab had worse PFS than non-variant patients (HR 2.59, 95%CI 0.91–7.33, p=0.07). There was a significant three-way interaction between the KRAS-variant, p16 status and treatment for OS (p=0.02). KRAS-variant patients had significantly elevated TGF-β1 plasma levels (p=0.03), and a trend for worse treatment-related toxicity.

Conclusion and Relevance

KRAS-variant HNSCC patients significantly benefit from the addition of cetuximab to radiation and cisplatin, and there is a significant interaction between the KRAS-variant and p16 status. Elevated TGF-β1in KRAS-variant patients suggests that cetuximab may help these patients by overcoming TGF-beta induced suppression of antitumor immunity.

Introduction

The current standard of care for the non-surgical management of locally advanced head and neck squamous cell carcinoma (HNSCC) is concurrent platinum-based chemotherapy and radiotherapy. Unfortunately, however, this approach results in an ~ 50% treatment failure rate in unselected patients.^1,2 Cetuximab is an anti-EGFR monoclonal antibody that was previously shown to be beneficial for such patients, improving survival when given with definitive radiation, or with cisplatin+5-fluorouracil in recurrent/metastatic HNSCC.^3,4 However, a randomized phase III clinical trial (NRG Oncology RTOG 0522) failed to show any improvement in survival with the addition of 8 weeks of cetuximab to cisplatin and radiotherapy.² To date, beyond EGFR surface expression,⁵ no predictive biomarker of cetuximab response has been identified for these patients.

Recently, human papillomavirus (HPV), reliably measured by p16 expression, has been established as a prognostic biomarker in HNSCC, with p16-positive patients having a favorable outcome compared to p16-negative patients.^1,6,7 One reason for the better prognosis in p16-positive disease is thought to be due to immune mediated mechanisms, as both cisplatin and radiation expose p16-associated antigens, which are immunogenic,⁸ enabling host-mediated immune tumor killing. Consistent with prior reports, p16-positive patients in RTOG 0522 had improved outcome compared to p16-negative patients.² However, there was no benefit of cetuximab treatment when p16 status was considered in RTOG 0522. One hypothesis put forward is lower EGFR tumor cell staining in p16-positive patients in 0522 compared to p16-negative tumors (22% vs. 30%).²

Beyond inhibition of EGFR signaling however, cetuximab has also been shown to work through immune-mediated mechanisms, by enhancing adaptive immunity via antibody dependent cell-mediated cytotoxicity (ADCC),^9,10 and by better enabling cytotoxic T-lymphocyte (CTL) cross-priming by dendritic cells.¹¹ Recently, cetuximab combined with chemotherapy was further confirmed to trigger immunogenic cell death in mouse models through increased phagocytosis by dendritic cells.¹² These immune-based mechanisms of cetuximab efficacy have not yet been studied in the setting of radiation, although radiation also enables antitumor immunity, with the release of danger signals, and dendritic and T cell activation.^13–15 Recently, a biomarker of altered immune function, FCGR2A, a polymorphism in an Fcy receptor, was shown to impact response rate to cetuximab for patients with KRAS mutant tumors, ¹⁶ indicating that inherited polymorphisms can impact cetuximab response.

In the colon cancer field, response to cetuximab is associated with an inherited biomarker disrupting KRAS, referred to as the KRAS-variant.¹⁷ The KRAS-variant is the first microRNA (miRNA) binding site mutation discovered in cancer, and is a functional germ-line mutation in the 3′ untranslated (3′UTR) region of KRAS. While the KRAS-variant is rare in tumors with acquired KRAS mutations, ¹⁸ it leads to gene expression patterns consistent with KRAS-addiction across tumor types. ^19,20 The KRAS-variant is associated with increased cancer risk,^18–20 and the development of multiple cancers,^21,22 and more recently has been found to be a predictive biomarker of response to cancer treatment irrespective of tumor type or tumor acquired mutations.^23–28 In HNSCC the KRAS-variant is found in 15–32% of patients,^29,30 and is therefore highly relevant. HNSCC patients with the KRAS-variant have previously been shown to have a poor response to platinum therapy, similar to findings for KRAS-variant patients with other cancer types.^23,29–31

While the mechanisms leading to the predictive power of the KRAS-variant in treatment response have not been fully elucidated, one could hypothesize that the KRAS-variant identifies patients with altered immunity, based on the fact that KRAS signaling and let-7 expression, both universally abnormal in KRAS-variant associated tumors,^29,32,33 are both critical in systemic immunity.^34–35 In addition, mutant KRAS has been shown to directly inhibit anti-tumor immunity through induction of TGF-β1 secretion, which is known to lead to conversion of conventional T cells into Tregs or Th17 cells.^{36, 37,38} In the setting of radiation, TGF-β1 is proposed to be a master regulator of treatment response, with high levels preventing radiation induced anti-tumor immunity.³⁹ Interestingly, in cells with high TGF-β1, cetuximab has been found to restore the killing activity of patient NK cells against primary HNSCC cells, via enhanced ADCC, in a dose-dependent manner.⁴⁰ Cetuximab response has also been shown to overcome TGF-β1 expressing Tregs, known to inhibit NK cell killing ⁴¹, providing a potential mechanism of action. The role of altered immunity and TGF-β1 levels as potential mechanisms of cetuximab response in KRAS-variant patients has not been previously evaluated.

Thus, here we sought to test the predictive role of the KRAS-variant for HNSCC patient outcome in the setting of radiation, cisplatin and cetuximab, considering p16 status. We further evaluated immune parameters in KRAS-variant patients through plasma TGF-β1 levels. We show for the first time that the KRAS-variant is a predictive biomarker of cetuximab response for HNSCC patients, and interacts significantly with p16. Furthermore, we find that KRAS-variant patients have significantly elevated plasma TGF-β1 levels, consistent with baseline immune suppression.

Patients and Methods

Protocol and Patients

NRG Oncology RTOG 0522 was a phase III trial testing the addition of cetuximab to radiation therapy with concurrent cisplatin for patients with advanced HNSCC.² The study was approved by all IRBs where patients were enrolled, and oral informed consent was obtained from all study participants. Race and sex was collected and is reported in Supplemental Table 1. Eligible patients had pathologically proven squamous cell carcinoma of the oropharynx, hypopharynx, or larynx, with selected stage III or IV disease (T2 N2-3 M0 or T3-4 any N M0), Zubrod performance status 0–1, age ≥ 18 years, and adequate bone marrow, hepatic, and renal function. HPV status was evaluated by p16 expression as previously described.^1,2 See attached protocol (Supplemental Document 1). Nine-hundred and forty patients were enrolled into NRG Oncology RTOG 0522, of whom 891 (94.8%) were eligible for protocol analyses. 413 of 891 patients had biological samples available for KRAS-variant analysis (46.4%, see Supplemental Figure 1 for a CONSORT Flow Diagram). Patients not tested for the KRAS-variant had significantly lower age (p=0.02) than patients tested for the KRAS-variant, but the difference in medians was only 2 years (56 vs. 58) (Supplemental Table 1). Progression-free survival (PFS) and overall survival (OS) were similar for the patients tested and not tested for the KRAS-variant [PFS hazard ratio 0.92 (95%CI 0.76 to 1.13), p=0.44; OS hazard ratio 0.99 (95%CI 0.78 to 1.25), p=0.93]. At the time of analysis, median follow-up for surviving patients was 4.8 years (range 0.2 to 6.9). Of the 413 study participants tested for the KRAS-variant, 5 (1.2%) were homozygous (GG) and 65 (15.7%) were heterozygous (TG), resulting in an overall prevalence of 16.9%. The prevalence of the KRAS-variant was not significantly different in p16-negative (17.4%) versus p16-positive (16.0%) patients, in agreement with prior studies.⁴²

Of the 70 patients with the KRAS-variant, the cetuximab treated subset had fewer patients of Caucasian race (87.5% vs. 100%, p=0.04). Within the non-variant cohort, the cetuximab treated subset had significantly lower age than the subset not receiving cetuximab, but the difference in medians was only 2 years (59 vs. 57, p=0.05)(Supplemental Table 2).

KRAS-variant testing

Genomic DNA from peripheral blood mononuclear cells or whole blood was isolated as previously described⁴³, and 100ng was analyzed in a CLIA-certified laboratory for the KRAS-variant (Mira Dx). Patients that were homozygous (GG) were grouped with those that were heterozygous (TG) for these analyses.

Statistical methods

Local-regional failure (LRF), distant metastasis (DM), PFS, and OS were as defined in the NRG Oncology RTOG 0522 protocol. LRF and DM rates were estimated by the cumulative incidence method.⁴⁴ PFS and OS rates were estimated by the Kaplan-Meier method.⁴⁵ Hazard ratios were estimated by the Cox model.³⁴ Adverse events were graded by Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. Odds ratios were estimated by logistic regression. Patient characteristics were compared by Fisher’s exact test (categorical variables) or Wilcoxon rank-sum test (ordinal or continuous variables). All analyses were performed using SAS version 9.4.

Plasma TGF-β1 levels

Plasma levels of TGF-β1 from 376 patients enrolled on 0522 were measured using multiplex bead assays utilizing reagents from Millipore and the Luminex 100 system as previously described. ⁴⁶ The assay was run using manufacturer controls to ensure precision. The correlation between KRAS-variant status and plasma TGF-β1 levels was evaluated by Wilcoxon rank-sum test.

Results

The KRAS-variant Predicts Response to Cetuximab

We investigated the association of the KRAS-variant with cetuximab response in HNSCC and found a significant, positive effect of 8 weeks of cetuximab treatment for all KRAS-variant patients. KRAS-variant patients had significantly improved PFS in the first year (hazard ratio 0.31, 95%CI 0.10 to 0.94, p=0.04), but not thereafter (hazard ratio 1.76, 95%CI 0.62 to 4.95, p=0.28)(Figure 1A). The cetuximab treatment effect significantly varied over time (p=0.02 for interaction between treatment and PFS time [>1 year]). In the non-variant group there was no impact of cetuximab on PFS with a treatment effect hazard ratio [cetuximab/no cetuximab] of 1.00 (95%CI 0.72 to 1.38, p=0.98). In multivariate analysis, the interaction between treatment, time (>1 year), and KRAS group remained significant (p=0.02), indicating that there is an interaction between treatment and time in the KRAS-variant group (Table 1).

Figure 1. Progression-free survival (PFS) and overall survival (OS) for HNSCC patients by KRAS-variant status and assigned treatment.

A. PFS In total, 179 of 413 (43.3%) patients experienced a PFS failure: 19 of 38 (50.0%) in the non-cetuximab treated KRAS-variant group, 13 of 32 (40.6%) in the cetuximab treated KRAS-variant group, 74 of 169 (43.8%) in the non-cetuximab treated non-variant group, and 73 of 174 (42.0%) in the cetuximab treated non-variant group. B. OS. In total, 134 of 413 (32.4%) patients have died: 14 of 38 (36.8%) in the non-cetuximab treated KRAS-variant group, 8 of 32 (25.0%) in the cetuximab treated KRAS-variant group, 58 of 169 (34.3%) in the non-variant non-cetuximab treated group, and 54 of 174 (31.0%) in the non- variant cetuximab treated group.

Table 1.

Multivariate analysis of PFS and OS (n=413)

	PFS (179 events)		OS (134 events)
	HR (95%CI)	p-value	HR (95%CI)	p-value
Treatment X time X KRAS	–	0.02	–	0.04
Treatment X time, if KRAS-variant	–	0.10	–	0.05
Treatment X time, if non-variant	–	0.21	–	0.80
Treatment X KRAS, if early [1]	–	0.07	–	0.13
Treatment X KRAS, if late	–	0.36	–	0.29
Early treatment effect [1], if KRAS-variant	0.42 (0.14–1.26)	0.12	0.27 (0.06–1.21)	0.09
Late treatment effect, if KRAS-variant	1.22 (0.53–2.80)	0.64	1.47 (0.53–4.03)	0.46
Early treatment effect [1], if non-variant	1.20 (0.80–1.80)	0.39	0.89 (0.56–1.41)	0.62
Late treatment effect, if non-variant	0.79 (0.47–1.33)	0.38	0.81 (0.45–1.48)	0.49

PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval.

¹First year for PFS, first 2 years for OS.

Hazard ratios estimated from Cox models including treatment (cetuximab vs. no cetuximab), treatment X PFS/OS time interaction, KRAS (variant vs. non-variant), treatment X KRAS interaction, treatment X PFS/OS time X KRAS interaction, age, Zubrod performance status (1 vs. 0), primary site (oropharynx vs. others), T stage (T4 vs. T2-3), and N stage (N2b-3 vs. N0-2a).

The pattern of failure in a multivariate analysis suggested that distant metastases (DM) rather than local regional failure (LRF) may be a more likely contributor to the difference in PFS for KRAS-variant patients: in the KRAS-variant group, the treatment effect for DM is 0.45 (95%CI 0.12 to 1.70), and for LRF is 0.84 (95%CI 0.29 to 2.42)(Supplemental Figure 2 and 3). In the non-variant group, the treatment effects for DM and LRF are 0.90 (95%CI 0.48 to 1.70) and 1.24 (95%CI 0.80 to 1.92), respectively.

KRAS-variant patients also had significantly improved OS in the first 2 years (hazard ratio 0.19, 95%CI 0.04 to 0.86, p=0.03), but not thereafter (hazard ratio 2.34, 95%CI 0.58 to 9.41, p=0.23)(Figure 1B). Again, the cetuximab treatment effect significantly varied over time (p=0.02 for interaction between treatment effect and survival time [>2 years]). In the non-variant group, there was no impact of cetuximab treatment on overall survival (treatment effect hazard ratio [cetuximab/no cetuximab] 0.90 (95%CI 0.62 to 1.30, p=0.56). In a multivariate analysis the interaction between treatment, time (>2 years), and KRAS group remained significant (p=0.04)(Table 1), indicating that there is an interaction between treatment and time in the KRAS-variant group. The treatment effect in the first 2 years for the KRAS-variant group is 0.27 (95%CI 0.06–1.21, p=0.09) and 1.47 (95%CI 0.53–4.03, p=0.46) thereafter (p=0.05 for the interaction).

The KRAS-variant interacts with p16 status for PFS

Because p16, a key cell cycle inhibitor, is a known prognostic biomarker in HNSCC, we evaluated outcome in KRAS-variant patients considering p16 status, without or with cetuximab treatment. For PFS, in patients treated without cetuximab, the two-way interaction between KRAS and p16 was significant (p=0.04, Table 2). KRAS-variant/p16-positive patients treated without cetuximab had worse PFS than non-variant/p16 positive patients, of borderline significance (HR 2.59, 95%CI 0.91–7.33, p=0.07, Figure 2A, left panel). However, in cetuximab treated patients, there was no longer a difference in outcome between KRAS-variant/p16-positive patients and non-variant/p16-positive patients (HR 0.89, 95%CI 0.34–2.35, p= 0.82)(Figure 2A, right).

Table 2.

Multivariate analysis of PFS and OS including p16 (n=271)

	PFS (116 events)		OS (85 events)
	HR (95%CI)	p-value	HR (95%CI)	p-value
Treatment X KRAS X p16	–	0.20	–	0.02
Treatment X KRAS, if p16-positive	–	0.14	–	0.05
Treatment X p16, if KRAS-variant	–	0.51	–	0.15
Treatment X p16, if KRAS-non-variant	–	0.12	–	0.01
KRAS X p16, if cetuximab	–	0.84	–	0.11
KRAS X p16, if no cetuximab	–	0.04	–	0.10
Treatment effect, if KRAS-variant and p16-positive	0.60 (0.17−2.10)	0.42	0.21 (0.02−2.04)	0.18
Treatment effect, if KRAS-non-variant and p16-positive	1.74 (0.88−3.42)	0.11	2.36 (0.98−5.67)	0.05
KRAS effect, if no cetuximab and p16-positive	2.59 (0.91−7.33)	0.07	2.48 (0.64−9.65)	0.19
KRAS effect, if cetuximab and p16-positive	0.89 (0.34−2.35)	0.82	0.22 (0.03−1.66)	0.14
p16 effect, if cetuximab and KRAS-variant	0.73 (0.20−2.73)	0.64	0.12 (0.01−1.11)	0.06
p16 effect, if cetuximab and KRAS-non-variant	0.63 (0.36−1.11)	0.11	0.80 (0.41−1.54)	0.50
p16 effect, if no cetuximab and KRAS-variant	1.34 (0.40−4.44)	0.64	0.86 (0.20−3.64)	0.84
p16 effect, if no cetuximab and KRAS-non-variant	0.32 (0.17−0.61)	<0.001	0.21 (0.09−0.49)	<0.001

PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval.

Hazard ratios estimated from Cox models including treatment (cetuximab vs. no cetuximab), KRAS (variant vs. non-variant), p16 (positive vs. negative), treatment X KRAS interaction, treatment X p16 interaction, KRAS X p16 interaction, treatment X KRAS X p16 interaction, Zubrod performance status (1 vs. 0), T stage (T4 vs. T2-3), and N stage (N2b-3 vs. N0-2a).

Figure 2. Progression Free Survival (PFS) and Overall Survival (OS) by KRAS Genotype and p16 Status for Patients treated without (left) or with (right) Cetuximab Treatment.

Solid lines, KRAS-variant, dashed lines, non-variant. Black lines p16-positive, red lines p16-negative. A. PFS. There is a significant two way interaction between KRAS and p16 in patients treated without cetuximab (p=0.04). B. OS. There is a significant three-way interaction between cetuximab treatment, KRAS-variant status, and p16 status (p=0.02).

The KRAS-variant interacts with p16 and Treatment for Overall Survival

For OS, there is a significant three-way interaction between cetuximab treatment, KRAS-variant status, and p16 status (p=0.02). There is also a significant two-way interaction between treatment and KRAS for p16-positive patients (p=0.05), and between treatment and p16 status for KRAS-non-variant patients (p=0.01)(Table 2). In patients treated without cetuximab, KRAS-variant/p16-positive patients had a non-significant trend for worse OS than non-variant/p16-positive patients (HR 2.48, 95%CI 0.64–9.65, p=0.19), which appeared to be non-significantly reversed with cetuximab treatment (HR 0.22, 95%CI 0.03–1.66, p=0.14, Figure 2B). In p16-positive patients, the cetuximab treatment effect is 0.21 (95%CI 0.02–2.04, p=0.18) for KRAS-variant patients compared to 2.36 (95%CI 0.98–5.67, p=0.05) for non-variant patients.

KRAS-variant patients’ immunological landscape and toxicity

Based on the differential effect of cetuximab, especially in p16-positive patients, we investigated immune parameters in KRAS-variant patients. We measured plasma TGF-β1 (generally a negative regulator of local immune response) levels in patients from 0522, and found that KRAS-variant patients had significantly higher plasma TGF-β1 levels compared to non-variant patients (Median 23,376 pg/ml versus 18,477 pg/ml, respectively; Wilcoxon rank-sum test, p=0.03; Table 3). TGF-β1 levels were also compared by p16 and KRAS-variant status, but the differences were not statistically significant in this smaller cohort (Supplemental Table 3).

Table 3.

Plasma TGFB1 levels in patients with HNSCC grouped by KRAS-variant status. Wilcoxon rank-sum test p=0.03

	n	Min	Q1	Median	Q3	Max
KRAS-non-variant	311	2261.42	8837.00	18476.52	32379.43	123264.70
KRAS-variant	65	5034.35	12574.03	23376.49	44809.10	109759.72

Based on the known role of TGF-β1 in normal tissue radiation toxicity, we evaluated toxicity in KRAS-variant versus non-variant patients. KRAS-variant patients appeared to have elevated levels of Grade 3–4 mucositis even without cetuximab treatment (47.4% without cetuximab vs 50.0% with, OR 1.11 [95%CI 0.43 to 2.85], p=0.83). In contrast, in non-variant patients the addition of cetuximab significantly increased Grade 3–4 mucositis (37.9% vs 50.6%, OR 1.68 [95%CI 1.09 to 2.58], p=0.02) (Supplemental Table 4). However, a test of the interaction between KRAS-variant status and cetuximab treatment was not significant (p=0.43). KRAS-variant patients also appeared to have elevated levels of Grade 3–4 skin reaction inside the portal even without cetuximab treatment (18.4% without cetuximab vs 15.6% with, OR 0.82 [95%CI 0.23 to 2.89], p=0.76). In contrast, in non-variant patients the addition of cetuximab significantly increased Grade 3–4 skin reactions inside the portal (11.2% vs 21.8%, OR 2.21 [95%CI 1.21 to 4.01], p=0.05), but again a test of interaction was not significant (p=0.16)(Supplemental Table 5). As expected, both non-variant and KRAS-variant patients developed increased skin reaction outside the portal with cetuximab (non-variant OR 50.15, p<0.001; KRAS-variant OR 8.54, p=0.05)(Supplemental Table 6).

To further evaluate if KRAS-variant status and TGF-β1 levels together predict toxicity, we evaluated KRAS-variant patients with known TGF-β1 levels (n=65), and looked at Grade 3–4 mucositis or in-field skin toxicity. We only included patients not treated with cetuximab, as cetuximab is known to be a radiosensitizer. We found that KRAS-variant patients with TGF-β1 levels > versus ≤ the median treated without cetuximab appeared to have an increase in risk of Grade 3–4 toxicity (OR 2.31, 95%CI 0.57–9.41). However, the interaction between KRAS and TGF-β1 was not significant (p=0.4). In contrast, non-variant patients with TGF-β1 levels > versus ≤ the median treated without cetuximab showed little correlation of TGF-β1 levels on toxicity (OR 1.19, 95%CI 0.62–2.25).

Discussion

Advanced HNSCC patients with the KRAS-variant significantly benefit from the addition of cetuximab to radiation and cisplatin therapy, resulting in improved PFS and OS. We found that this benefit fades over time, possibly due to the short course of cetuximab delivered in this study. We further show that there is a significant interaction between the KRAS-variant, p16 status, and cetuximab, with KRAS-variant/p16 patients doing poorly compared to non-variant/p16 patients, a deficit that seems to be overcome by cetuximab. Furthermore, we show that KRAS-variant patients have significantly elevated TGF-β1 expression, suggesting that they may have a baseline immune-deficient and/or anti-tumor immune defective state, which cetuximab in combination with radiation may be helping to overcome.

For HNSCC, while p16 has been shown to be an important prognostic biomarker, it appears that knowledge of both p16 status and KRAS-variant status may be important to determine optimal personalized treatment for these patients. It is particularly notable that KRAS-variant/p16-positive patients had the worst outcomes of all subgroups of HNSCC patients in our study, which appeared to be improved with a short course of cetuximab treatment given with radiation and cisplatin. These patients should perhaps be considered a different entity from non-variant/p16-positive patients, who had excellent outcomes with radiation and cisplatin alone. Work is ongoing to further validate these observations.

Our finding that HNSCC patients with the KRAS-variant may have an altered immune state compared to non-variant patients is novel, but perhaps not surprising, considering that the KRAS-variant is germ-line and, thus, present in all cells, including immune cells. This inherited mutation is known to lead to globally altered gene expression profiles consistent with KRAS-addiction,^32,33 as well as altered expression of the let-7 miRNA,⁴³ both of which are known to have significant effects on the immune system.^34,35 In addition, our findings here of elevated TGF-β1 in these cancer patients, which has long been known to be involved in immunity³⁷ and immune suppression,⁴⁷ further supports our hypothesis that KRAS-variant patients are immune suppressed. Baseline differences in immunity could also help explain the increased cancer risk found in KRAS-variant patients. Interestingly, cetuximab has been shown to overcome the impaired NKG2D-dependent functionality of activated natural killer (NK) cells ^40,41 in the presence of TGF-β1,^48,49 potentially explaining the global benefit of cetuximab treatment seen in KRAS-variant patients. Whether the altered TGF-β1 we see in these patients represents baseline perturbations in homeostasis, or altered responses to tumor challenge, and is a finding that will be found for all KRAS-variant cancer patients, remains unclear, and are the subject of important ongoing investigations.

Although the sample size was small in this study, and a larger study would strengthen the results, our current findings are consistent with prior work with patients with recurrent/metastatic HNSCC treated with cisplatin +/− cetuximab, showing that the addition of cetuximab improved median survival to 3.9 months; however, the interaction with p16 status could not be evaluated previously because of the small sample size.⁴² Our findings are also consistent with those found in KRAS-variant colon cancer patients, where cetuximab monotherapy is clearly beneficial.^16,17 What this study has additionally found is the potential importance of cetuximab delivered in conjunction with radiation therapy for KRAS-variant patients, to seemingly enable them to mount appropriate immunologic responses following tumor-targeted irradiation, diminishing metastatic failures. We also find that patients with the KRAS-variant may have baseline normal tissue radiation sensitivity, possibly through their elevated TGF-β1 levels. Although cetuximab is a known radiosensitizer, there did not appear to be worsened radiosensitivity with the addition of cetuximab for KRAS-variant patients. Studies are ongoing to better understand the mechanisms of radiosensitivity in these patients, to further personalize their treatment.

While a germ-line biomarker that can identify individuals with altered immunity that can be used to direct the best therapeutic combinations is a bold, new concept, the KRAS-variant is unfolding as a true candidate. Based on our findings in this study, defining how anti-cancer agents affecting the tumor-host-immune relationship differently impact KRAS-variant patients is a critical, and pressing need, as this knowledge could help in personalizing developing immune based cancer therapies for all cancer patients.