Lung cancer is the second most common cancer worldwide in 2019 and the leading cancer-related cause of death among both men and women.1 While the introduction of immunotherapy into the treatment of advanced and metastatic non-small cell lung cancer (NSCLC) has improved survival when compared with chemotherapy, the 5 years survival rate continues to be as low as 23.2% and 15.5% in the first-line and second-line setting, respectively.2 While programmed death ligand (PDL1) expression on tumor cells is to date the only FDA (Food and Drug Administration) approved biomarker of response to anti-programmed cell death protein-1/programmed death ligand 1 (PD1/PDL1) therapies, the 3-year and 5-year survival remains suboptimal even in patients with PDL1 expression on more than 50% of the viable tumor cells, that is, their tumor proportion score (TPS) is ≥50%.2 3 Therefore, tumor PDL1 expression cannot be used as the only biomarker to predict response and survival in patients treated with anti-PD1/PDL1 therapy. There is ongoing research to identify affordable and easily obtained biomarkers that can be used as adjunct to tumor PDL1 expression for precision medicine.

Human leukocyte antigen (HLA) is expressed on somatic as well as immune cells. HLA molecules introduce internally processed cellular antigens to T lymphocytes to trigger a series of signals to activate or inhibit T cell proliferation and differentiation.4 5 HLA molecules are divided into two types, HLA-I and HLA-II. HLA-I is expressed on all nucleated cells, including somatic, cancer and immune cells and presents cell peptides to activate CD8+T cells. HLA-II, on the other hand, is expressed on antigen-presenting cells and mediates the activation of CD4+ helper or regulatory T cells. HLA molecules are encoded by the HLA gene complex located on the short arm of chromosome 6 at locus 21 (6p21).6 7 The HLA locus is not only polygenic but also highly polymorphic with each locus having multiple alleles.8 9 HLA alleles can be grouped based on their peptide-binding repertoire into supertypes.10 Only HLA-A and HLA-B supertypes are well-characterized, with Sidney et al (2008) describing nine supertypes for HLA-A and HLA-B alleles based on their specificity for the anchor amino acids.10

HLA molecule downregulation in cancer tissue is a well-established mechanism of cancer evading the immune system.11–15 The low expression or low diversity of HLA on the cell surface is directly related to the range of antigens that can be presented to T cells. Many studies have looked at the correlation between expressed HLA-I on tumor cells and its effect on survival and response to immunotherapy.16–22 However, the effect of low HLA genetic diversity (or homozygosity) on response to immunotherapy has not been explored, with the exception of two recent studies.23 24 Chowell et al investigated the relationship between genetic HLA homozygosity and survival in 100 NSCLC and 269 patients with melanoma treated with immunotherapy, either with anti-PD1/PDL1 or anti-CTLA4 or both. Reduced survival was correlated with homozygosity in at least one HLA-I locus. Therefore, it was postulated that maximal heterozygosity of HLAs might be associated with higher diversity of antigens presented to the T cells, compared with an individual with homozygosity at one or more HLA loci.23 Furthermore, they observed that HLA-B62 supertype was strongly associated with reduced survival, while the HLA-B44 supertype associated with longer survival.23 Hence genomic HLA can be explored as an economical and easily obtained non-invasive biomarker that can help guide treatment choice among patients with NSCLC patients treated with immunotherapy.

Here, we aim to assess whether homozygosity in HLA-I or HLA-II is associated with survival outcomes among patients with advanced NSCLC treated with single agent anti-programmed cell death protein-1 (PD1) or anti-PDL1 therapy in the first-line or second-line setting.

Tumor specific markers such as PDL1 expression, tumor mutational burden and mismatch repair deficiency have been described as predictors of long-term benefit to immune checkpoint inhibitor blockade.26 However, variations in outcomes can also be influenced by host-specific factors. Chowell et al 2018 showed in a cohort of melanoma and patients with NSCLC treated with single-agent anti-PD1 or in combination with anti-CTLA-4, that HLA-I homozygosity at one or more loci was associated with shorter survival comparing to those who were heterozygous at all HLA-I loci (HR=1.4). However, the study did not demonstrate the effect of HLA homozygosity in patients with NSCLC specifically. The results of our study indicate that homozygosity at one or more HLA-I loci is associated with poor outcomes in patients with NSCLC treated with single-agent anti-PD1/PDL1 in the first-line or the second-line setting.

HLA-I expression in cancer tissue was found to be a key factor determining increased immune cell infiltration into NSCLC tissue, and independent of PDL1 status.27 While there is enough evidence to suggest that downregulation of HLA-I molecule is one of the mechanisms used by the tumor to escape the immune system,13 14 a coordinated balance needs to be achieved to avoid destruction by natural killer cells (NK cells)28 and this might reduce the impact of HLA-I downregulation as a mechanism to evade the immune system. On the other hand, loss of heterozygosity in cancer tissue was found to be associated with poor treatment outcomes.29 30 However, loss of genomic heterozygosity in tumor occurs only in 11% of cases.23 As shown in our study, germline homozygosity of HLA-I at one or more loci also has a clear impact on survival, underscoring the role that HLA-I molecules play on the immune response against NSCLC.

The effect of HLA-I homozygosity on survival was more pronounced in patients with strong PDL1 expression (≥50%). The expression of PDL1 on tumor tissue, as an immune regulatory mechanism has to be preceded by an interaction between peptide/HLA-I molecule and T cell receptors. This implies that the expressed HLA-I molecule is not downregulated in tumor cells and PDL1 expression is the primary mechanism of immune evasion in these cases.27 Therefore, the HLA-I homozygosity may have a profound impact on survival among patients with PDL1 ≥50%, when treated with PD1/PDL1 inhibitors. This also implies that patients with PDL1 <50% utilize other immune evasion mechanisms, including downregulation of HLA in cancer cells.

Our results are in contrast with the report by Negrao et al,24 which did not find a significant correlation between HLA zygosity and survival in an NSCLC cohort treated with PD-1/PDL1 at the M. D. Anderson Cancer Center. Notably, a large proportion (25%) of their patient cohort received anti-PD-1/PDL1 as third-line therapy, while all our study participants were treated either in the first-line or second-line setting. Moreover, their dichotomisation for PDL1 expression was carried out based on 1% TPS, while our subgroup analysis was based on a 50% PDL1 expression cut-off.

Chowell et al showed that the presence of HLA-B62 and HLA-B44 in patients with melanoma treated with immune checkpoint inhibitors are associated with worse (HR=2.29) and better prognosis (HR 0.61), respectively. However, they did not evaluate the role of HLA supertypes in their cohort of patients with NSCLC. In our cohort of patients with NSCLC treated with anti-PD1/PDL1 therapy, the presence of HLA-A02 supertype appears to positively affect survival. This may reflect differences in cancer biology between melanoma and NSCLC, which is reflected by the degree of response to immunotherapy and in particular to anti-CTLA4 agents. The differential effect of HLA supertypes in melanoma and NSCLC, may also reflect the differences in the type of mutations and therefore neoepitopes between these two cancers.31 HLA-A02 molecule binds small aliphatic and small hydrophobic/aliphatic amino acids in the B and F pockets,10 for example, KRAS G12V neoantigen represent the tumor specific antigen bound to HLA-A*02:01.32 This might help predict the type of cancer neoantigens that are associated with better survival among patients with NSCLC treated with anti-PD1/PDL-1. HLA-B is usually expressed more frequently on the cell surface compared with HLA-A.33 Nevertheless, the beneficial effects in survival outcomes observed for carriers of the HLA-A02 supertype suggest that NSCLC might escape this downregulation mechanism.

Homozygosity at one or more genomic HLA-II loci in patients with NSCLC treated with single agent anti-PD1/PDL1 treatment did not demonstrate a direct influence on survival and may require larger numbers to show its impact. HLA-II molecule is rarely expressed on cancer tissue and more likely expressed on antigen-presenting cells.34 Its expression on tumor infiltrating lymphocytes rather than NSCLC tissue is associated with better PFS and OS.35 HLA-II is responsible for the activation of CD4+ helper T-lymphocytes which4 5 then help to activate B-cells and CD8+T cells, as well as promote the induction of memory CD8+T cell.36 37 The role of CD4+T cells is much less understood than CD8+T cells in cancer biology.34 All these factors potentially contributed to our observation of a lack of statistically significant effect of homozygosity at one or more HLA-II molecule on OS and PFS of patients with NSCLC treated with single agent anti-PD1/PDL1.

A secondary observation from our analysis was that anti-PD1 therapy had longer OS and PFS when compared with anti-PDL1 immunotherapy. This observation is consistent with the meta-analysis conducted by Tartarone et al 2019.38 However, the latter study only carried out an indirect comparison and analyzed PFS only. We also considered that the results might be largely biased by our retrospective cohort, which included a large number of long-term survivors who received nivolumab (anti-PD1). However, in the absence of an interaction between the type of therapy and genomic HLA-I, we believe that this finding is unrelated to the main theme of this research project.

Our study has some limitations that require discussion. We did not incorporate the role of tumor mutation burden (TMB) in this analysis, which is thought to play a role in response to immunotherapy.39 However, Chowell et al 201823 showed that low TMB among patients with melanoma did not predict but that rather had an additive effect in the prognostic value of genomic HLA-I in survival. In addition, we had limited access to cancer tissue and were unable to assess somatic loss of HLA-I heterozygosity or HLA expression, which have been correlated individually with shorter OS and PFS.23 40 For some of our patients, a fine needle aspiration (FNA) was utilized for assessment of PDL1 status and other molecular testing. Limited tissue availability after PDL1 analysis and other molecular testing is a significant roadblock in the field. In fact, Hurkmans et al 40 reported of having to exclude 70% of the patients recruited due to tissue inadequacy to perform HLA immunohistochemistry. Moreover, our study is unable to assess the role of homozygosity at one or more HLA-I loci as a predictive marker for response to anti-PD1/PDL1 among patients with NSCLC. Finally, while our subgroup analysis showed a strong association between HLA and survival in patients with PDL1 ≥50% TPS, the small number of homozygous events resulted in large CIs. Thus, more extensive studies are needed to validate the clinical prognosis value of HLA zygosity in NSCLC treated with anti-PD1/PDL1 therapy.

Our study provides enough evidence that genomic homozygosity is associated with a worse prognosis in patients with NSCLC treated with single-agent immunotherapy. This is of special importance in NSCLC which often relies on FNA for diagnosis, and therefore tumor material can be exhausted after performing mutational profiling and PDL1 staining. We propose that, in addition to PDL1 TPS, HLA-I typing should be considered as a non-invasive and cost-effective biomarker to guide treatment personalisation. Given that patients with HLA-I homozygosity are less likely to derive clinical benefit from single-agent anti-PD1/PDL1 therapy, consideration of a more aggressive treatment combinations may be needed to improve their prognosis, especially among those with PDL1 TPS of 50% or more.