Wedding of Molecular Alterations and Immune Checkpoint Blockade

Genomics as a Matchmaker

Elena Fountzilas, MD, PhD; Razelle Kurzrock, MD; Henry Hiep Vo, PhD; Apostolia-Maria Tsimberidou MD, PhD

Disclosures

J Natl Cancer Inst. 2021;113(12):1634-1647. 

In This Article

Investigational Biomarkers Conferring Resistance to Immunotherapy

Several genomic markers, such as loss of beta-2 (β2)-microglobulin and JAK1/2, interfere with the immune response and hence correlate with resistance to immune checkpoint blockade. In addition, loss of Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and alterations in EGFR, KEAP1, STK11/KRAS, and the B-catenin pathway have been associated with immunotherapy resistance, although the mechanisms are unclear. EGFR alterations and murine double minute 2 (MDM2) amplification have been correlated with accelerated progression (also known as hyperprogression) after checkpoint blockade, but the underlying biology has not been elucidated (Table 1, Figure 2).[44,114]

Figure 2.

Investigational biomarkers associated with resistance to immuno-oncology therapy. It is not yet clear if some of these markers, such as KEAP1 and STK11 alterations, are prognostic or predictive. EGFR = epidermal growth factor receptor; JAK = Janus kinase; KEAP1 = Kelch-like ECH associated protein 1; KRAS = v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog; MDM2 = murine double minute 2; PTEN = phosphatase and TENsin homolog deleted on chromosome 10; STK11 = serine/threonine kinase 11.

β2-Microglobulin Mutations

β2-microglobulin is a low-molecular-weight protein that forms MHC-I molecules in combination with the heavy chain. Proper functioning of β2-microglobulin is critical for antigen presentation and assembly of HLA class I complexes. Loss of β2-microglobulin facilitates tumor escape from immune recognition[115–117] and is implicated in resistance to immunotherapy. In one study, a truncated mutation in β2-microglobulin was identified in tissue obtained from a patient with melanoma who progressed on anti–PD-1 therapy.[34] IHC revealed loss of surface expression of MHC-I. In another study, analysis of longitudinal tumor biopsies demonstrated molecular alterations in β2-microglobulin in 5 (29.4%) of 17 patients with advanced melanoma who progressed on immunotherapy.[35] Loss of heterozygosity was noted only in nonresponders, whereas there was no molecular alteration in β2-microglobulin detected in responders.

EGFR Alterations

Immunotherapy provides limited clinical benefit in patients with NSCLC harboring EGFR mutations.[118]EGFR alterations are also associated with hyperprogression after treatment with immunotherapy in some studies, but not in others.[3,37,114,119] In vivo investigation of tumor growth in T-cell–deficient mice, which were injected with EGFR-mutated patient-derived xenografts, showed that nivolumab triggered the accrual of macrophages and led to increased tumor growth and lung dissemination.[120] Investigators have proposed upregulation of PD-1 and PD-L1 through EGFR activation as a mechanism of resistance.[121]

KEAP1 Mutations

KEAP1 regulates cytoprotective responses to oxidative and electrophilic stress by binding the transcription factor NRF2.[122,123] Loss-of-function mutations in KEAP1 result in dissociation and constitutive activation of NRF2, cellular resistance to oxidative stress, and increased tumor cell growth.[124,125] Additionally, NRF2 activates drug efflux pump genes that confer resistance to cytotoxic drugs.[126]KEAP1 mutations occur in 2.7% of patients with cancer, most commonly in patients with NSCLC (15.8%).[38] Studies suggest that KEAP1 mutations confer resistance to immunotherapy because they are associated with a "cold" tumor microenvironment.[38,39] In 1 study, among 1661 patients who received immunotherapy, those with KEAP1 mutations had shorter OS compared with those with wild-type tumors (10 vs 20 months; P = .0029).[38] Other investigators showed that co-mutation of KRAS and KEAP1 (27% of KRAS-mutated tumors) in patients with NSCLC is an independent prognostic factor associated with shorter OS and duration of response to first-line platinum-based chemotherapy in patients who received immunotherapy.[39] In the phase III MYSTIC NSCLC study, KEAP1 mutations correlated with poor outcome across arms (durvalumab alone, durvalumab plus tremelimumab or chemotherapy). Other studies have, however, reported that KEAP1 mutations may be associated with poor prognosis regardless of therapy and are not specifically predictive for checkpoint blockade outcome.[40,41]

Janus Kinase 1 and 2 Loss

Janus kinase (JAK) is a family of intracellular, nonreceptor tyrosine kinases that transduce cytokine signals via the JAK-STAT pathway. Importantly, IFN-γ, critical to immune response, binds to its receptor and recruits and activates JAK1 and JAK2, and subsequently STAT1, thus resulting in immune cell activation.[127–129] Loss of IFN-γ signaling pathway genes has been implicated in immunotherapy resistance.[34,42,43,130,131]

Whole-exome sequencing of paired melanoma tumors (primary tumor at diagnosis and metastatic tumor at the time of recurrence) after disease progression on anti–PD-1 therapy revealed acquired loss-of-function mutations in JAK1 or JAK2, with deletion of the wild-type allele.[34] Functionally, these mutations resulted in lack of response to IFN-γ. In another study, investigators showed that patients with JAK1/2 inactivating mutations did not respond to immunotherapy.[42] Additionally, in vitro exposure to IFN-γ failed to mediate downstream signaling. Finally, in another report, patients with JAK2-mutated advanced melanoma did not respond to anti-CTLA4 treatment, and melanoma cell lines with molecular alterations in IFN-γ pathway genes were refractory to immunotherapy.[130] Together, these data indicate that loss-of-function JAK1/2 mutations can mediate primary or acquired resistance to immunotherapy.

MDM2 Amplification

MDM2 encodes a nuclear-localized E3 ubiquitin ligase, which functions as a negative regulator of the TP53 gene. MDM2 amplification occurred in approximately 4% of more than 100 000 cancers analyzed[44,93,132] and was most commonly noted in patients with liposarcoma (64% of cases), gallbladder adenocarcinoma (11%), sarcoma (11%), and urothelial carcinoma (10%).[44] Most patients (99%) had additional molecular alterations.[44]MDM2 amplification has been linked to hyperprogression after treatment with immunotherapeutic agents.[3,44,133,134] The biologic mechanism by which MDM2 amplification mediates hyperprogression after treatment with immunotherapy is unknown, and it is possible that another gene on the MDM2 amplicon is culpable. One study reported that MDM2 mediates resistance to immunotherapy through degradation of transcription factor NFATc2, leading to reduced T-cell activation.[135]

PTEN Loss

PTEN is the second most commonly mutated tumor suppressor gene. PTEN loss has been described in diverse tumor types, including hepatocellular (57% of patients), prostate (52%), endometrial (49%), and colorectal (48%) cancers.[136] PTEN inhibits PI-3 kinase and AKTsignaling by converting phosphoinositol-(3–5)-trisphosphate to phosphoinositol-(4, 5)-bisphosphate. PTEN loss, through mutations, epigenetic mechanisms, and gene silencing, disrupts its regulatory control of cell proliferation, energy metabolism, angiogenesis, and survival.

PTEN loss has been implicated in immunotherapy resistance in preclinical models, in an immune-suppressive microenvironment in prostate cancer and glioblastoma, and in acquired resistance in the clinic in a patient with metastatic uterine leiomyosarcoma, as well as to poor outcome from checkpoint blockade in melanoma.[45,137–139]

STK11 Mutations With KRAS Alterations

Serine/threonine kinase 11 (STK11) is a tumor suppressor gene that regulates cell proliferation and energy metabolism. Patients with STK11/KRAS-mutated (compared with KRAS-only mutated) tumors have statistically significantly worse clinical outcomes after treatment with immunotherapy.[47] In another study, the presence of STK11 alterations was associated with shorter PFS and OS and lower ORR in patients with NSCLC who received treatment with an anti–PD-1 agent combined with doublet chemotherapy.[52] However, recently published real-world data (Flatiron Health Network) suggest that STK11 mutations confer poor prognosis to patients with NSCLC regardless of treatment and show no specific prediction for immunotherapy outcomes.[41]

TCR Repertoire

The TCR repertoire refers to the multiple possible combinations of TCR sequences and represents a "footprint" for T cells' recognition of tumor neo-antigens. Higher TCR clonality has been associated with response to anti–PD-1 therapy in patients with melanoma.[140,141] In patients with pancreatic cancer, low baseline clonality and higher rates of expanded clones after treatment with ipilimumab were associated with longer OS.[142] Finally, neo-adjuvant treatment with nivolumab in patients with NSCLC induced expansion of neo-antigen-specific T-cell clones.[143] Patients who achieved a major pathological response had higher rates of T-cell clonality.

Wnt/β-Catenin Pathway Alterations

Wnt/β-catenin signaling plays a critical role in proliferation, migration, and division.[144,145] Deregulation of this pathway is a strong "driver" in diverse tumors, mainly CRC, where it occurs in 93% of cases.[146] Many investigators suggest that the Wnt/β-catenin pathway—as a candidate immune-related modulator—correlates with impaired immune cell recruitment in melanoma and other tumor types.[48,137,147–149] An integrative The Cancer Genome Atlas data analysis, based on genomic, transcriptomic, or proteomic approaches, showed that non–T-cell–inflamed tumors (3137 of 9244 tumors, 33.9%) were enriched for WNT/β-catenin pathway activation.[150] Preclinical and clinical models demonstrate that WNT/β-catenin activation leads to suppression of CD8+ T-cell tumor infiltration and evasion of immune elimination.[48,147–149] T-cell suppression through activation of the Wnt/β-catenin pathway has been linked to decreased chemokine levels.[151] In preclinical trials, RNAi-mediated inhibition of the Wnt/β-catenin pathway combined with anti–PD-1/CTLA-4 agents led to T-cell infiltration and tumor growth inhibition.[152]

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