Tumor immune microenvironment and mutational analysis of tracheal adenoid cystic carcinoma.

BACKGROUND: Tracheal adenoid cystic carcinoma (TACC) is the second most common type of cancer in bronchial tumors with poor prognosis. Studies on the genomic profiles and tumor immune microenvironment (TIME) of TACC are still relatively rare.
METHODS: Here, we performed whole-exome sequencing (WES), T cell repertoire (TCR) sequencing, and immunohistochemistry (IHC) on the resected tumors and matched peripheral blood leukocytes (PBLs) samples from 25 TACCs collected from April-2010 to Mar-2019.
RESULTS: WES results revealed that LPAR3 and ALPI were recurrently mutated genes, with no classical lung cancer drivers in TACCs (n=8). The median tumor mutation burden (TMB) was 3.67, lower than other solid tumors. Unexpectedly, one patient showed high microsatellite instability (MSI). Recurrent copy number variations (CNVs) affected genes commonly involved in p53, cell cycle, and PI3K-Akt signaling pathways. For TCR estimators of 13 PBLs, the median clonality and Shannon index was 0.15 and 7.02, respectively. Shannon index showed marginally negative association with age (Pearson r =-0.53, P=0.062). Clonotype number and Shannon index of 7 TACC tissues were significantly lower than those of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (Mann-Whitney test, both P<0.001, both P<0.001). Furthermore, programmed cell death 1 ligand 1 (PD-L1), a vital player in TIME, was negative (tumor proportion score, TPS <1%) in all samples (n=14). Patients with less clonotypes had longer progression-free survival (PFS) than those with more PFS (15.0 vs. 9.5 months, P<0.001, HR 12.5, 95% CI: 0.2-675.7). In particular, the clinical and molecular characteristics of one TACC patient receiving immunotherapy have been explained in detail.
CONCLUSIONS: In summary, despite the existence of one patient with MSI-H and chromosome instability, TACC was characterized by a lack of common drivers of lung cancer, negative PD-L1 expression, and low CD3+ and CD8+ T cell infiltration. 2020 Annals of Translational Medicine. All rights reserved.

Introduction

Tracheal adenoid cystic carcinoma (TACC) originates from the tracheal mucinous epithelium, accounting for about 15% of primary tracheal tumors (1). TACCs can occur at any age with the similar prevalence in males and females (1:1.1) during life. Non-epidemiological relations to smoking have been reported (2). TACCs have an insidious onset and high early misdiagnosis rate (3). There are challenges in the diagnosis and treatment of TACC. The symptoms of TACC include cough, chest tightness, asthma, and dyspnea. These are so non-specific that patients are often misdiagnosed as asthma and chronic bronchitis in the early stage. Thus, the diagnosis of TACCs requires biopsy under tracheoscope and pathological examination of multiple protein biomarkers including SMA, P63, p53, ki-67, and CD117 (4). It takes as long as 20 months or a year for the patient to be diagnosed with TACC since the initial symptoms (5). Moreover, malignant tumours are often developed rapidly, thus, tumor was locally advanced stage when the patients were diagnosed. Therefore, the founding of novel biomarkers may improve the diagnosis for TACC.

Surgical resection and radiotherapy are currently the first-line therapies for TACC. Approximately 90% of patients with early-stage TACC undergo resection survived for 5 years (6). However, owing to the characteristics of occult invasion of surrounding collagen fibers or vascular nerve bundles and enveloping extension for invasive diffusion, the inevitable of incomplete movement of the lesion leads to high-rate recurrence (7). Due to the sensitivity to radiation, radiotherapy is the first-line choice for TACCs patients without radical resection and with postoperative recurrence (8-10). Several studies (11) have shown that TACCs had an inadequate response to chemotherapy compared to other pulmonary tumors. Researchers (12) have found that EGFR, KRAS, BRAF, ALK, PIK3CA, PDGFRA, and DDR2 may not be driver genes in Asian primary TACC using a seven gene panel. Recently, a study of large ACC cohort revealed that significantly mutated genes were involved NOTCH pathway (13). Despite the comprehensive usage of targeted-therapy and immunotherapy in lung cancers, trials of targeted therapy to date have not yet identified an agent with sufficient activity to be deemed standard in the treatment of advanced ACC (14-17). Thus, more comprehensively genomic profiles and tumor immune microenvironment (TIME) of TACC are needed.

Here, we collected tumors and matched peripheral blood leukocytes (PBLs), and performed the comprehensive analysis of genomic profiles, tumor mutation burden (TMB) status, microsatellite instability (MSI) status, PD-L1 expression, CD3 and CD8 infiltration, and TCR repertoire to characterize the potential targets and immune-related biomarkers for TACC.

We present the following article in accordance with the STROBE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-3433).

Methods

We enrolled 25 TACCs identified by IHC in the at the First Affiliated Hospital of Guangzhou Medical University from April-2010 to Mar-2019, All sections were stained with hemagglutinin-eosin according to the World Health Organization criteria for PACC and observed by two experienced pathologists. The patients had received surgery, and the untreated surgical specimens were collected for analysis. The status of disease progression and survival was followed up until Jan-2020. The clinical features, treatment process, and survival data were shown in . Whole-exome sequencing (WES) and TCR sequencing on tumors and matched PBLs were performed. TMB was showing the number of SNV and insertions and deletions (InDels) of the coding regions. The 22C3 antibody examined the PD-L1 expression level. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) somatic mutation data from TCGA and TCR data from the Geneplus database (Geneplus-Beijing, Beijing, China) were used for the comparison with TACC. The details of sequencing and statistical methods were shown in Supplementary file.

Table S1

Clinical characteristics of the enrolled 25 TACC patients

Variables	Results
Sex, n (%)
Female	14 (56.0)
Male	11 (44.0)
Age (years), mean ± SD	45.0±15.6
History of smoke, n (%)	4 (16.0)
Smoking index (pack-years, median)	160
Tumor location, n (%)
Proximal	7 (28.0)
Middle	3 (12.0)
Distal	13 (52.0)
Other	2 (8.0)
Tumor size (cm), mean ± SD	(2.8±1.2) *(2.1±0.8)
Symptoms, n (%)
Cough	24 (96.0)
Dyspnea	19 (76.0)
Hemoptysis	7 (28.0)
Time (months), mean ± SD	17.5±14.3
Lymph node invasion	0 (0.0)
Distant metastases	2 (8.0)

TACC, tracheal adenoid cystic carcinoma.

Table S2

Comparison of median progression-free survival between groups

Comparison	mDFS	Test value	P value
Resection vs. nonsurgical treatment	41.2±11.0 vs. 13.3±2.4	6.226	0.013
R0 resection vs. R1 resection	61.3±21.4 vs. 33.3±4.2	0.688	0.407
R0 resection vs. nonsurgical treatment	61.3±21.4 vs. 13.3±2.4	4.519	0.034
R1 resection vs. nonsurgical treatment	33.3±4.2 vs. 13.3±2.4	3.312	0.069

mDFS, median disease-free survival.

Table S3

Treatment and survival data of enrolled 25 TACC patients

Case	Treatment 1	Types of resection treatment	PFS1	Treatment 2	PFS2	Treatment 3	PFS3	OS
1	Interventional therapeutic bronchoscopy + radiation therapy	–	16*	–	–	–	–	16*
2	Resection + radiation therapy	R1	15*	–	–	–	–	15*
3	Resection	R0	14*	–	–	–	–	14*
4	Resection + TP	R1	15*	–	–	–	–	15*
5	Resection	R1	23*	–	–	–	–	23*
6	Resection	R1	12*	–	–	–	–	12*
7	Interventional therapeutic bronchoscopy + TP		6	Immunotherapy	2	Interventional therapeutic bronchoscopy	3*	11*
8	Resection + GP	R0	8*	–	–	–	–	8*
9	Resection	R0	21*	–	–	–	–	21*
10	Radiation therapy + PP + A		18*	–	–	–	–	18*
11	Resection	R0	108	Radiation therapy	1*	Die	–	109
12	Interventional therapeutic bronchoscopy		18	Interventional therapeutic bronchoscopy	13	Interventional therapeutic bronchoscopy	15*	46*
13	Resection	R0	14	Supportive treatment	–	–	–	16*
14	Resection	R0	13*	–	–	–	–	13*
15	Resection + PP	R0	12*	–	–	–	–	12*
16	TP	–	7	Supportive treatment	–	–	–	12*
17	Resection + TP	R1	38	Interventional therapeutic bronchoscopy	12*	–	–	50*
18	Resection	R0	9*	–	–	–	–	9*
19	Resection + PP	R0	29*	–	–	–	–	29*
20	Interventional therapeutic bronchoscopy + radiation therapy + TP	–	13	Anlotinib + Interventional therapeutic bronchoscopy	6	PP	3*	22*
21	Resection + radiation therapy	R0	19*	–	–	–	–	19*
22	Resection + PP	R0	48	Osimertinib + Interventional therapeutic bronchoscopy	7*	–	–	55*
23	Resection + PP + radiation therapy	R1	36	Support care	–	–	–	38*
24	Resection	R1	15	Radiation therapy	18*	–	–	33*
25	Resection	R0	18	Support care	18*	–	–	36*

*, no disease progression. TACC, tracheal adenoid cystic carcinoma; PFS, progression-free survival; OS, overall survival; TP, paclitaxel + platinum; GP, gemcitabine + platinum; PP, pemetrexed + platinum; A, bevacizumab.

The study was conducted in accordance with the Declaration of Helsinki. The scientific, ethical Committee approved this study of the First Affiliated Hospital Guangzhou Medical University (ChiCTR-IPR-15006164). All patients supplied written informed consent.

Results

Clinical features of TACC

Eleven (44%) TACCs were males. The median age was 45 years (from 23 to 76 years). Current or ever, smokers (n=4) were all males. The period from the onset of symptoms to the diagnosis is with an average period of 17.5 (±14.3) months. The majority were in the main bronchus. All patients had no lymph node metastasis, but 2 had lung metastases (). Four types of resection treatments, including surgical, non-surgical, R0, and R1, were accepted. Median disease-free survival (mDFS) of 12 patients with R0 resection was 61.3±21.4 months, mDFS of 7 patients with R1 resection was 33.3±4.2 months, the difference was not statistically significant. The mPFS was 13.3±2.4 months for the six patients who chose non-surgical treatment in the first line ().

Genomic landscape of TACC

We successfully performed WES on eight tumors (all were stage I) with the median sequencing coverage of 211× (115 to 289). Nine hundred thirty-nine non-synonymous somatic mutations were found included 644 SNVs. and 295 InDels of 825 genes. The mutations were made up of missense (64.8%), frameshift (13.0%), and Indels (17.9%). LPAR3 (50%, 4/8), ALPI (38%, 3/8), ARID1A (38%, 3/8), and BCOR (38%, 3/8) were most often mutated genes (). A median of 110 mutations (40 to 203) were detected, lower than LUSC (median 187 mutations, P=0.011), and equivalent to LUAD (median 158 mutations, P=0.240). Two (25%) TACCs were found as TMB-High (containing ≥200 mutations) (18), which both harbored the POLE mutation (19). Particularly, one case showed high microsatellite instability (MSI-H).

Figure 1

The genomic landscape and clinical characteristics of TACC. TACC, tracheal adenoid cystic carcinoma.

Focal CNVs aberration were detected in 75% (6/8) of TACCs. Recurrent copy gains and loss including 4q12, 9p21.3, 9p24.1 and 11q13.3 (), affected CCND1, CDKN2A, CDKN2B, KDR, KIT, PDGFRA, CD274 (PD-L1), and PDCD1LG2 (PD-L2) genes involved in p53, cell cycle, and PI3K-Akt signaling pathways. Case 7 harbored the highest level of CNVs, containing copy number loss and loss of heterozygosity (LOH), followed by case 1 and case 4, all CNVs of these two patients detected were loss of function (LOF). Also, we identified the copy number loss of TP53, BRCA1, and BRCA2 genes, and some of them involved in DNA damage response pathways (DDR), which was a potential marker in immune checkpoint inhibitor.

Figure S1

Copy number variations profile. LOF, loss of function; LOH, loss of heterozygosity.

We compared the most common genes across four cohorts with adenoid cystic carcinoma (ACC). We included TACC with ACC of salivary gland cancer (n=935), ACC of the lung (n=76), and adenoid cystic breast cancer (n=38) (13). When comparing the differentially mutated genes with other cohorts, eleven genes showed a discrepancy in frequency (). For example, ARID1A and BCOR were more enriched (both 38%) in TACC than others; the CREBBP gene had the highest frequency in adenoid cystic breast cancer with percentiles of 18.9% (7/37) relative to others ACC types, followed by TACC (12.5%, 1/8). In contrast, the NOTCH1 gene, the most often mutated gene in all three ACC types, was not found in our cohort.

Figure 2

Gene frequency contribution in TACC cohort and ACC cohort. TACC, tracheal adenoid cystic carcinoma. ACC, adenoid cystic carcinoma.

The global profile of TCR repertoire

T cells are abundant component in tumor microenvironment and are key players in tumor immunology (20). To investigate the T cell receptor repertoire in tumor and peripheral blood, we successfully performed TCR sequencing on 7 FFPE and 13 PBL samples from 13 patients, excluding samples with low DNA quality (n=12 PBLs and n=18 FFPE). The diversity of the TCR repertoire was measured by the Shannon index (21) and CDR3 clonotypes (22,23). The clonality metric quantitates the degree of mono-clonal or oligo-clonal expansion by measuring the shape of the clone frequency distribution (24). The overlap level of TCR between PBL and the matched tumor was calculated using the sum of frequencies of unique TCR clones of tumor detected in PBL.

The median number of TCR clonotype was 624 (181 to 2,396) in tumors and 11,783 (5,429 to 39,439) in PBLs. The median Shannon index was 3.91 (1.33 to 4.73), and clonality was 0.29 (0.19 to 0.82) of tumors, while 7.02 (3.30 to 9.28) and 0.27 (0.12 to 0.64) of PBLs, respectively (). The median overlap level of TCR between tissue and PBL was 0.12 (0.01 to 0.71) (). These results showed the varied characteristics of TCR between PBL and tumors. When comparing TCR immune parameters of 7 TACC tissues with LUAD and LUSC, we found that clonotype number and clonality of TACCs were significantly lower than those of LUAD (P<0.001, P<0.001) and LUSC (P<0.001, P<0.001).

Figure S2

TCR diversity distribution of 13 peripheral blood samples and seven tissues from 13 patients and distribution of TCR overlap between peripheral blood and tumor in seven patients. TCR, T cell repertoire.

Associations between TCR repertoire and clinical characteristics and outcome

We evaluated the relationship between the clinical characteristics and Shannon’s entropy, clonality, and unique clones in 13 PBL samples. Clonotype number was not associated with age (Figure S3A). Shannon index showed marginally negative association with age (Pearson r=−0.53, P=0.062, Figure S3B), contrary to the adverse trend of clonality (Pearson r=0.53, P=0.062, Figure S3C). There was no significant association between TCR parameters with the other characteristics, including gender, smoking, and recurrence ().

Figure 3

The difference in TCR diversity in different clinical groups. Correlation between clonotype number and gender (A) or smoking history (B) or recurrence (C). Correlation between Shannon index and gender (D) or smoking history (E) or recurrence (F). Correlation between clonality and gender (G) or smoking history (H) or recurrence (I). Statistical analysis was performed using the Wilcoxon rank-sum test. TCR, T cell repertoire.

Next investigated the impact of TCR repertoire on the clinical outcome using Kaplan-Meier curves and the log-rank test. The “surv_cutpoint” function of the “survminer” R package was used to determine the optimal cutoff point for each TCR parameter based on the maximally selected log-rank statistics. Patients with lower clonotype numbers had longer PFS than those with higher PFS (, 15.0 vs. 9.5 months, P<0.001, HR 12.46, 95% CI: 0.23–675.66), while Shannon index and clonality was not significantly associated with PFS ().

Figure 4

The association of patient’s progression-free survival (PFS) with TCR diversity in 13 patients. (A) PFS difference between patients with high and low clonotype numbers; (B) PFS difference between patients with high and low Shannon index; (C) PFS difference between patients with high and low clonality. P value was calculated with the use of the log-rank test.

CD3+ and CD8+ T cell infiltration and PD-L1 expression of TACC

Due to the availability of specimens, we completed IHC on 14 patients of PD-L1 protein expression and seven patients of CD3+ and CD8+ TILs. All of 14 TACCs showed PD-L1 negative (TPS <1%). Two patients (case 4 and case 14) were CD3+ and CD8+ TILs positive (all showed 5%). Typical results were shown in .

Figure S4

Immunohistochemical (IHC) detection of CD3 and CD8 and PD-L1 in TACC. (A) IHC detection of CD3 and CD8; (B) IHC detection of PD-L1 in TACC. IHC, immunohistochemical; TACC, tracheal adenoid cystic carcinoma.

A case receiving ICIs

Case 7, a 23-year-old male smoker with stage I TACC in the distal trachea, had chosen tracheal endoscopic mass resection, followed by paclitaxel combined with platinum as postoperative chemotherapy. Unfortunately, local tumor recurrence was observed after six months. Subsequently, sindilizumab was given to this patient, and the disease still progressed after two cycles of immunotherapy. Many more details were shown in . Although PD-L1 expression together with CD3+ and CD8+ T cell infiltration was low (), we identified high TMB (203 mutations) as well as various copy number loss in the tumor of this patient ().

Discussion

ACC is a rare malignancy occurring in multiple organs and most common in the salivary gland. In the trachea, it arises from the submucosal layer and has a rapid locoregional spread. ACC spreads most commonly by direct extension, submucosal, perineural invasion, or hematogenous metastasis (3), it is difficult to remove by surgery altogether, and there are many local recurrences. Currently, many studies focus on ACC of the salivary gland, but research on TACC is rare. Our study aims to elucidate genomics and T cell landscape and predict clinical biomarker for TACC.

The genomic profiles of the TACCs were different from other ACC origins. NOTCH1 mutation associated with activation of the Notch pathway in ACC of the trachea and a potential target for therapeutic intervention has been previously proved (25). Our study did not detect NOTCH1 mutations, while we detected Notch pathway-related genes, including CREBBP, NOTCH3, ADAM17, and PSEN1. Our findings provided a clue that the targeted therapies for NSCLC might not be suitable for TACC due to the different genomic profiles of TACC patients with those of NSCLC. In our study, LPAR3 and ALPI were frequently mutated genes in TACC. LPAR3 encodes lysophosphatidic acid receptor 3, a member of G-protein coupled receptor family. The LPAR3 gene was associated with tumor aggregation in several cancers including ovarian cancer (26), oral squamous cell carcinoma (27), and breast cancer (28). ALPI is a gene that expresses intestinal alkaline phosphatase, an intestinal cell differentiation marker. ALPI was a responsive indicator of histone deacetylase inhibitor (HDACi) in colon cancer cells (29). However, the function of these two genes in TACC needs further exploration.

We evaluated the TMB level and performed IHC to detect PD-L1 protein expression, CD3+, and CD8+ T cell infiltration in TACC. Although none was positive PD-L1 expression, two cases had a low level of CD3+ and CD8+ T cell infiltration. Besides, TMB-H and MSI-H TACC were found, which shed light on the ICI treatments for TACC. In our cohort, one patient (case 7) with TMB-H and MSI-H and extensive copy number loss (containing genes coding PD-L1 and PD-L2) in his primary tumor received PD-1 inhibitor. However, the earlier study reported that salivary type primary malignant tracheal tumors do not significantly express PD-L1 (30). Unfortunately, rapid disease progression occurred. We did not obtain a second biopsy of the immunotherapy baseline for this retrospective case. Therefore, we were unable to assess whether ICI resistance variants were generated in post-chemotherapy progressed lesion. Mainly, copy number variations (CNVs) has been linked with the outcome of immunotherapy. Thus, it is also necessary to investigate the role of CNVs in response to immunotherapy.

To our knowledge, no earlier study has systematically analyzed the genomic characteristics and clinical significance of the TCR repertoire in the peripheral blood of patients with TACC. Our study is the first analysis of the T cell repertoire in ACC using NGS methodology. Despite the limited sample size, our result showed that the peripheral blood TCR repertoire correlates with several clinical characteristics and immune status of TACC patients. However, we should further confirm the results using expanded cohorts in future studies. Currently, both TMB and PD-L1 are indicators for immunotherapy in multiple tumors. In the future, the changes in the TIME, such as TCR repertoire during anti-cancer treatment, might be a novel and useful biomarker for immunotherapy. In this study, we supplied the combine multi-omics analysis to predict biomarker-related clinical benefits in TACC.

Conclusions

In summary, genomic alteration and TIME characterization supplied the molecular basis for in-depth research on the treatment of advanced TACC.

The article’s supplementary files as