Somatic KMT2D loss-of-function mutations in lung squamous cell carcinoma: a single-center cohort study

Introduction

Despite the advent of molecular targeted therapy and immunotherapy, lung cancer remains the leading cause of cancer-related death worldwide (1). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all new lung cancer cases (1). Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the most common NSCLC subtypes. Over the past decade, significant progress has been made in the targeted treatment of LUAD harboring driver mutations, such as epidermal growth factor receptor (EGFR), kirsten rats arcomaviral oncogene homolog (KRAS), V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF), proto-oncogene receptor tyrosine kinase (MET), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), and rearranged during transfection (RET) gene aberrations, which have more frequently genetic alterations in LUAD than in LUSC (2-5). However, a vast majority of patients with NSCLC who receive targeted therapies will eventually develop drug resistance and experience tumor relapse (6,7). Recently, immune checkpoint inhibitors [e.g., anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)] have shown significant clinical benefits for patients with advanced NSCLC and a high level of PD-L1 expression. However, two-third of NSCLC either do not express or express PD-L1 at a low level. The benefit of immunotherapy in this subset of NSCLC is rather modest (8-10).

In current clinical practice, NSCLC driver mutations can be relatively easy to detect using next-generation sequencing (NGS) technology from small tumor biopsy samples or blood by analyzing circulating-free DNA (5). The single driver oncogenes are much more common in LUAD and the prognosis for LUSC is poorer (1,11). Only a few targeted therapeutics have yet been approved for the treatment of LUSC, largely due to complex interplay of co-occurring genetic alterations driving the pathogenesis of LUSC as opposed to a clear single driver gene alteration (<20% in European patients) (12). The Cancer Genome Atlas Research Network (13) identified a higher frequency of alterations in NFE2L2, PTEN, NOTCH, TP53, and Rb1 genes in LUSC samples. Additionally, alterations in FGFR, PIK3CA and DDR2 are also identified as potentially actionable in LUSC (14-18). There is an urgent need to gain insight into the molecular profiles of LUSC to dissect the role of individual gene in carcinogenic process to develop more effective therapeutic strategies.

Histone lysine methylation by lysine methyltransferase (KMT) is a posttranslational modification that plays important roles in the epigenetic regulation of a broad spectrum of biological processes, including development, differentiation, metabolism, and tumor suppression (19,20). Histone-lysine N-methyltransferase 2D (KMT2D), also known as MLL2 or MLL4 in some studies, belongs to a family of mammalian histone H3 lysine 4 (H3K4) methyltransferases (19,20). The human KMT2D gene is located on chromosome 12q13.12 and contains 54 exons, encoding a 5,537 amino acid protein including 7 plant homeodomain (PHD) domains, a high mobility group (HMG)-binding motif, an F/Y-rich C terminus (FYRC), an F/Y-rich N terminus (FYRN) motif, and a C-terminal SET domain. The KMT2D protein is a histone methyltransferase that monomethylates H3K4, a hallmark of an active transcription state. Additionally, the presence of the SET domain is responsible for the methyltransferase activity of the KMT2D protein (19,20). The KMT2D protein is essential for maintaining the level of H3K4 monomethylation via the enzymatic Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain, which is correlated with transcriptionally engaged enhancer elements as an active transcription factor (21,22). Somatic loss-of-function (LoF) mutations in the KMT2D gene have been linked to many types of cancers, including lymphoma, leukemia, gastric cancer, esophageal squamous cell carcinoma (ESCC), lung cancer, prostate cancer, chordoid meningiomas, and adult granulosa cell tumor (22). Recent studies have indicated that the KMT2D protein functions as a tumor suppressor and might play an important role in carcinogenesis of LUSC (23,24), and perhaps may act as a driver alteration. The NCOA6 and KMT2C or KMT2D were revealed to act as coactivators of the tumor suppressor and TF p53 in cell assays and the expression of endogenous p53 target genes needs the coactivators in response to doxorubicin, a DNA damaging agent (25). The KMT2C and KMT2D were demonstrated to act as a tumor suppressor in acute myeloid leukemia, follicular lymphoma, and diffuse large B cell lymphoma in three studies in mice (26-28). An important evidence about KMT2D as a key regulator of LUSC tumorigenesis was obtained in cell organoids of LUSC. Kmt2d loss activated receptor tyrosine kinases (RTKs) to a high level, partly through reprogramming the chromatin landscape to decrease the expression of protein tyrosine phosphatases. The study identified KMT2D functioned as a pivotal epigenetic modulator for LUSC oncogenesis and suggested that KMT2D loss leaded LUSC therapeutically vulnerable to RTK-RAS inhibition (24). However, LoF mutations in KMT2D have only been found in a small portion of the LUAD population. In this study, we examined the somatic genome alterations of patients with NSCLC to clarify the molecular mutation characteristics of KMT2D LoF mutations in patients with LUSC and LUAD. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-134/rc).

Methods

Patient cohorts and clinical characteristics

In this retrospective, single-institution cohort study, patients initially diagnosed with LUSC and LUAD at the Zhujiang Hospital between January 2019 and March 2023 were examined. Patients with lung mixed adenosquamous lung carcinoma were excluded. Before the administration of anti-cancer therapy, all included patients’ samples underwent molecular genetic analysis with a targeted NGS gene panel that evaluated approximately 600 tumor-associated genes. The NGS data were reanalyzed to confirm LoF mutations in the KMT2D gene, while missense variants in this gene were not examined in this study due to the vast majority of variants being of uncertain significance. Our cohort consisted of 53 cases of LUSC and 322 cases of LUAD. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics committee of Zhujiang Hospital (No. 2024-KY-142-01). A waiver of patient consent was granted due to the retrospective, data-collection design of this study. Clinical data were obtained via an electronic medical record query, which included information on age, sex, smoking history, stage, specimen site, tumor histology, dates of diagnosis, and gene mutation analysis. Nonsmokers were defined as patients who had smoked fewer than 100 cigarettes in their lifetime. Smokers included former smokers, who were defined as those who quit >12 months before diagnosis, and current smokers, who were defined as those who quit <12 months before or still smoked at diagnosis.

Sample preparation and target sequencing

DNA extraction from tumor samples and targeted NGS were performed in a third-party laboratory (Mygene Diagnostics Co., Ltd., Guangzhou, China). DNA from either frozen (n=36) or formalin-fixed paraffin-embedded (FFPE) (n=299) tumor samples was extracted using the MagPure FFPE DNA LQ Kit C (Magen Biotechnology, Waltham, MA, USA). Germline DNA was extracted from blood using a Surbiopure Blood Genomic DNA kit (GuangZhou Surbiopure Biotechnology Co., Ltd., Guangzhou, China) as a reference for detecting somatic alterations. DNA quantity and purity were assessed using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). A total of 50 ng of each genomic DNA (gDNA) sample based on Qubit quantification was fragmented and subjected to end repair, A-tailing, and adapter ligation via the Universal Plus DNA Library Prep Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Subsequently, libraries were captured using 2.6 M probes from TargetSeq Hyb & Wash Kit v. 2.0 (iGeneTech, Beijing, China) and finally amplified. After quality control with a Qsep 100 analyzer (BIOptic, New Taipei City, Taiwan) confirmed DNA without degradation and quantification with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), the libraries were sequenced on an MGISEQ-2000 platform (BGI Group, Shenzhen, China).

Data analysis

Clean reads were obtained by filtering adapter, low-quality, and reads with a proportion of N>5 using a fastp v. 0.21.0. Clean reads were aligned to the reference human genome hg19 (GRCh37) using Burrows-Wheeler aligner maximum exact matches (BWA-MEM; v. 0.7.17). Somatic single-nucleotide variants (SNVs) and insertions/deletions were detected using VarDict 1.8.3 based on mapped consensus in binary alignment map (BAM) files for tumor and control tissue. Somatic SNVs and indels were further filtered according to the flowing criteria: read depth ≥100 in tumor samples, mapping quality ≥40 and base quality ≥20, variant allele frequency (VAF) ≥1%, supporting reads ≥3 in the tumor, and VAF in the tumor ≥5 times that of the matched normal VAF. Variant annotation for gene consequence was performed using Ensembl Variant Effect Predictor (VEP) 103.1. Somatic SNVs and indels were excluded when their population allele frequency >0.5% according to the 1000 Genomes Project, the Genome Aggregation Database (gnomAD), and the Exome Aggregation Consortium (ExAC) annotations. The copy number was determined using CNVkit 0.9.9 tool. Copy number homozygous deletion (copy number <0.5 and region of deletion >74%) and amplification (copy number >2.5 and amplification region >60%) were included in the analysis. Candidate structural variants (SVs) were determined using lumpy 0.2.13 under default parameters. Potential false SVs were identified and then excluded based on the following criteria: read depth <100 or supported by fewer than 3 split reads or 15 supported read pairs.

Statistical analysis

Descriptive statistics are presented as the median and range for continuous variables and as the number and percentage for categorical variables. Differences between groups were compared using the Chi-squared test or Fisher exact test. The P value less than 0.05 was considered statistically significant.

Results

Patient cohort description

A total of 335 patients diagnosed with NSCLC were included in this study. Among them, there were 53 cases (15.8%) of LUSC and 282 cases (84.2%) of LUAD. The clinical characteristics of patients with LUSC or LUAD are summarized in Table 1. Briefly, patients with LUSC or LUAD had a median age of 68 years (range, 37–91 years) and 61 years (range, 20–88 years) at diagnosis, respectively. There were significantly more male patients and smokers in the LUSC group than the in LUAD group (P<0.001). The clinical stage did not differ significantly between the two patient groups (P>0.05). Moreover, reanalysis of sequencing data revealed a higher prevalence of somatic LoF mutations (nonsense, frameshift, and splice-site variants) for KMT2D in the LUSC group than in the LUAD group (20.8% vs. 2.1%; P<0.001).

KMT2D LoF mutation pattern in NSCLC

LoF mutations in KMT2D were detected in 11 cases of LUSC and 6 cases of LUAD, respectively (Table 2). Of these 17 patients, there were 10 males and 1 female with LUSC and 5 males and 1 female with LUAD. Double LoF mutations in KMT2D were detected in 3 cases of LUSC (P3, P96, and P136) and 2 cases of LUAD (P253 and P318). The types of LoF mutations included 12 nonsense, 4 frameshift, and 6 splice-site mutations. Classical splice-site mutations (exon-intron junctions) were predicted to disrupt messenger RNA (mRNA) splicing, potentially leading to protein dysfunction (Figure 1A). Nonsense and frameshift mutations were predicted to result in truncated proteins of KMT2D lacking the SET domain (Figure 1B). All the LoF mutations in all types were spread throughout the whole gene, while no recurrent mutations or mutation hotspots were identified. The distribution of KMT2D LoF mutations between LUSC and LUAD was very similar.

Figure 1 The distribution of KMT2D LoF mutations in the LUSC and LUAD cohorts. (A) Exon-intron structure map showing the distribution of KMT2D splice-site mutations (exon-intron junctions) in LUSC and LUAD. (B) Nonsense and frameshift mutations distribution throughout the KMT2D protein. Mutations in LUSC are labeled in red font. Mutations in LUAD are labeled in black font. LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma. PHD, plant homeodomain; HMG-box, high mobility group box; FYRN, F/Y-rich N terminus; FYRC, F/Y-rich C terminus; SET, Su(var)3-9, Enhancer-of-zeste, Trithorax domain.

Association of KMT2D and TP53 co-mutations in NSCLC

TP53 mutations occurred concurrently with KMT2D LoF mutations in 90.9% (10/11) of LUSC and 33.3% (2/6) of LUAD cases, respectively. Notably, the mutation allele fraction (MAF) of KMT2D was very similar to that of TP53 in the co-mutated cases (Figure 2). In a 57-year-old male patient (P76) diagnosed with LUSC, sequencing DNA from FFPE tumor tissue showed that the MAFs of KMT2D mutation (c.7539del, p.Q2514Sfs*29) and TP53 mutation (c.1121del, p.G374Vfs*48) were 35.3% and 31.1%, respectively. Subsequent post-treatment monitoring was performed through sequencing peripheral blood circulating tumor DNA (ctDNA), which detected an MAF of 2.08% in the KMT2D mutation and 1.52% in the TP53 mutation. Similarly, in a 53-year-old male patient with LUSC (P313), sequencing from FFPE tumor tissue indicated MAFs of 25.1% in the KMT2D mutation (c.1468G>T, p.E490*) and 25.8% in the TP53 mutation (c.536A>C, p.H179P), while the MAFs of the KMT2D and TP53 mutations in the plasma ctDNA samples were 2.02% and 1.86%, respectively.

Figure 2 Association of KMT2D and TP53 comutations in NSCLC. (A) Venn diagram of 10 cases with LUSC harboring KMT2D and TP53 comutations; (B) Venn diagram of 2 cases with LUAD harboring KMT2D and TP53 comutations; (C) the MAF of KMT2D was very similar to that of TP53 in the comutated cases. Comutations in LUSC are labeled in red font. Comutations in LUAD are labeled in black font. NSCLC, non-small cell lung cancer; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; MAF, mutation allele fraction.

KMT2D mutation concurrence with other actionable gene alterations in NSCLC

Genomic profiling of driver gene mutations of NSCLC indicated that 9 cases of LUSC with KMT2D LoF mutations also exhibited PIK3CA gene amplification (n=5), FGFR1 gene amplification (n=2), and both PIK3CA and FGFR1 gene amplifications (n=2) (Table 3). The presentation in the table were the ones with identifiable mutations/other alterations of interest. There were two patients with LUSC (P3 and P136) who had both the KMT2D mutation and TP53 mutation that lacked known driver mutations. Moreover, a 68-year-old male patient with LUSC (P229) who harbored a KMT2D nonsense mutation (c.14710C>T, p.R4904*) had no TP53 mutation but did have both the PIK3CA and FGFR1 amplifications.

Of the 6 cases of LUAD with KMT2D LoF mutations, 4 cases without the TP53 mutation had the EGFR mutation (n=2), KRAS mutation (n=1), or EML4-ALK fusion (n=1). A 66-year-old male diagnosed with LUAD (P253) harbored two KMT2D mutations (p.R5027* and p.Q3756*) and the TP53 mutation (p.R158L) and EGFR mutation (p.E746_A750del). The other patient with LUAD (P318) without a known driver gene mutation was a 71-year-old male who had the KMT2D mutation (c.13840-1G>A, c.14000-1G>A) and TP53 mutation (p.H179R).

PIK3CA amplification (18/20) or mutation (2/20) were detected in 20 cases of LUSC, PIK3CA mutation (11/12) or amplification (1/12) in 12 cases of LUAD, respectively (Table 3). Of these 20 cases of LUSC, 14 patients with PIK3CA amplification and TP53 mutation. Of these 12 cases of LUAD, 6 patients with PIK3CA mutation and TP53 mutation, 9 cases co-mutated with EGFR mutations (9/12) or KRAS mutations (3/12). There were only 1 patient with FGFR1 amplification in LUAD cohort and 6 cases in in LUSC cohort (Table 3). Of these 6 patients of LUSC, 4 cases also exhibited FGFR1 gene amplification and mutations, 4 cases co-mutated with PIK3CA gene amplification.

Discussion

In this study, we analyzed targeted sequencing data from a cohort of 335 patients diagnosed with NSCLC. The frequency of KMT2D somatic LoF mutations was found to be 20.8% in LUSC and 2.1% in LUAD. We explored more about the characterization of squamous carcinoma driver genes, especially in terms of co-mutations. High frequency of KMT2D and TP53 co-mutations occur in the LUSC cohort. Notably, the MAF of KMT2D was very similar to that of TP53 in the co-mutated cases which need to be confirmed in larger cohorts. Moreover, genomic profiling of actionable gene mutations of NSCLC showed that PIK3CA and/or FGFR1 gene amplification was detected in 81.8% (9/11) of the patients with LUSC and KMT2D LoF mutations. In a recent study, KMT2D protein was identified as a key regulator of LUSC tumorigenesis, and Kmt2d deletion transformed lung basal cell organoids to LUSC (24). However, the characteristics of the co-occurrence gene with KMT2D gene prompted that KMT2D may play important role and interact with the stronger driver genes in the tumor development.

Numerous studies have shown that the KMT2D mutation is closely related to congenital developmental disorders and various types of tumors (27-31). It is well known that KMT2D or its binding partner KDM6A is the major causative gene for autosomal dominant Kabuki syndrome (KS), although cancer has been reported in several individuals with KS (e.g., neuroblastoma, hepatoblastoma, Wilms tumor, Burkitt lymphoma), there is no clear association between KS and an increased risk for cancer (27-31). Heterozygous germline mutations in KMT2D are detected in 56% to 75% of patients with KS, the majority of which are LoF variants.

Inactivating mutations in the KMT2D have been reported in approximately 11% of patients with NSCLC (32). A comparison of the somatic profiles of LUAD and LUSC based on The Cancer Genome Atlas (TCGA) database showed that KMT2D is one of the most commonly mutated genes in LUSC but not in LUAD (33). In a cohort of 105 Korean patients with LUSC, KMT2D was identified as a high frequent mutation with a mutation rate of 24% (34). In addition to NSCLC, SCLC also exhibits frequent inactivating mutations in the KMT2D gene (35,36). However, the KMT2D mutation is associated with reduced survival in NSCLC but not in SCLC (37). Interestingly, in a previous study of a small number of tumors-normal tissue pairs from patients with NSCLC, KMT2D gene mRNA expression was significantly reduced in tumor tissues compared with adjacent nontumor lung tissues, regardless of the mutation status (32). In the present study, we confirmed that KMT2D LoF mutations occur much more frequently in LUSC, we collected the cohorts retrospectively and many patients didn’t accept the administration in the same hospital, it is difficult to perform survival analysis. Whether there is a link between KMT2D mutations and survival in NSCLC will be explored in a future study, needs further exploration.

In addition to its tumor-suppressing candidates’ genes in various tumors, the KMT2D mutations have been found to be closely associated with the development of squamous cell carcinomas, such as head and neck squamous cell carcinoma, ESCC, cutaneous squamous cell carcinoma, cervical squamous cell carcinoma, and LUSC (38-42). The KMT2D acts as a tumor repressor since KMT2D loss of function modestly increased cell proliferation and colony formation in one disrupted KMT2D study. Cells lacking KMT2D showed increased rates of migration and faster cell cycle progression (41). Similarly, when compared with esophageal adenocarcinoma (EAC), ESCC showed a significantly more frequent mutational rate within KMT2D (11.9% vs. 0.8%; P<0.001). A study on urothelial carcinoma (UC) found that KMT2D mutations occurred frequently in UC with squamous differentiation (UCS) compared to UC (48.4% vs. 0%, P<0.001) (43). Notably, LoF mutations in KMT2D were also reported in a case of histologic transformation of LUAD to LUSC after targeted treatment. The patient with LUAD and EML4-ALK fusion treated in sequence with four different tyrosine kinase inhibitors (TKIs) after drug resistance, and developed a well-known ALK-TKI resistance mutation and underwent a histological transformation from LUAD to LUSC. Upon development of resistance, a resistant mutation in ALK: p.I1171N was detected, as well as two LoF mutations in KMT2D were detected (c.4379dupC, p.L1461Tfs*30; c.1940delC, p.P647Hfs*283) (44). The molecular mechanisms through which this gene contributes to histological differentiation and carcinogenesis are still poorly understood.

H3K4 methylation in mammals occurs via an evolutionarily conserved SET1 family of methyltransferases known as complex proteins associated with SET1 (COMPASS). KMT2D forms a multiprotein complex with other co-actors including WDR5, RbBP5, ASH2L, DPY30, NCOA6, PTIP, PA1, and KDM6A (21). The KMT2D core complex predominantly consists of H3K4 mono-methyltransferases on enhancer regions and displays partial functional redundancy with KMT2C (19). The absence of KMT2D protein leads to the collapse of the multiprotein complex and the destabilization of KMT6A. One study showed that KMT2D knockout in bladder cancer cells reduced the activity of H3K4 monomethylation and effectively decreased PTEN and p53 expressions while suppressing STAG2 expression (45). In other research, KMT2D binding sites were found to be highly overlapped with p53-targeted regions, and a wide range of genes involved in the p53 pathway and cAMP-mediated signaling were significantly downregulated in KMT2D knockout cells (46). It was also reported that KMT2D interacts with the transcription factor TP63 on chromatin and regulates TP63 target enhancers to coordinate epithelial homeostasis (47). Moreover, lung-specific deletion of KMT2D was shown to significantly promote KRAS-driven lung tumorigenesis in mice and to shorten the survival of mice bearing KRAS-driven tumors, suggesting that KMT2D loss cooperates with other oncogenic aberrations (e.g., KRAS activation) to increase LUAD tumorigenicity (20). KMT2D loss has been found to suppress the expression of multiple receptor protein tyrosine phosphatases (RPTPs) and promote activation of EGFR and ERBB2 (21). Here, our results indicated that TP53 mutations occurred concurrently with KMT2D LoF mutations in 90.9% of patients with LUSC, and PIK3CA and/or FGFR1 amplification was detected in 81.8% of the patients with LUSC and KMT2D LoF mutations. However, patients with LUAD and KMT2D LoF mutations usually associate with genes alterations in EGFR, KRAS, and ALK.

Conclusions

Collectively, our results prompted that the frequent occurrence of KMT2D somatic LoF mutations in LUSC, while being uncommon in LUAD. Our study is the first Chinese cohort where frequency of KMT2D in LUSC and LUAD is estimated and explored the different frequency of KMT2D between LUSC and LUAD and hinted KMT2D as tumor-suppressing function in LUSC. Moreover, the KMT2D mutation has the potential to contribute to the pathogenesis of LUSC by working in concert with other commonly mutated genes in LUSC, including TP53 mutation, FGFR1 amplification, and PIK3CA amplification. Our work brought the direct evidence for mutation frequency in Chinese population. Further studies are needed to understand the role of an individually altered genes in LUSC to decipher their contribution towards LUSC carcinogenesis to effectively develop anti-tumor therapies.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-134/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-134/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-134/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-134/coif). C.W., Y.Z., and Q.Z. are from Mygene Diagnostics Co., Ltd. and GuangDong Engineering Technology Research Center of Multiplex PCR & Tumor Diagnostics. A.L.R. reports travel/reimbursement from GT Medical Technologies. The other authors have no conflicts of interest to declare.

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). The study was approved by the ethics committee of Zhujiang Hospital (No. 2024-KY-142-01). A waiver of patient consent was granted since this was a retrospective study from data collection only.

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/.

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