Specimen types, clinical features and treatment modalities
The specimens included 89 (42.3%) core needle biopsies, 68 (32.4%) excisions and 53 (25.2%) cytology specimens with cell blocks (endobronchial ultrasound-guided fine-needle biopsies (EBUS) or pleural fluid aspirates). 140 cases (66.7%) were samples of primary lung tumor and 70 (33.3%) were metastases (mediastinal or distant). The patient cohort consisted of 97 (46.2%) men and 113 (53.8%) women, ages 33 to 91 years (mean: 67.4 years), living in central/eastern and northern regions of the state of Maine. 208 of the 210 patients (99%) were Caucasian, 1 (0.5%) Asian and 1 (0.5%) of native American (Penobscot) ancestry. 14 patients (6.7%) were never smokers, 109 (51.9%) were active smokers at the time of diagnosis and 87 (41.4%) former smokers (quit 1 or more years prior to diagnosis). The mean number of pack years among the smokers was 47 (range: 1-122), with 79 (42.9%) having at least 50-pack year history. 131 (62.4%) patients had limited stage: 71 (33.8%) stage I, 25 (11.9%) stage II and 35 (16.6%) stage III. 79 (37.6%) patients had advanced (stage IV) disease. Treatment was administered according to the NCCN guidelines16, dictated by stage, presence of predictive/therapeutic biomarkers and including: surgery/radiation plus/minus adjuvant chemotherapy for stages I-II, definitive chemo/radiation therapy followed by immunotherapy for stage III, and immunotherapy, combination chemo-immunotherapy, targeted therapy or palliative radiation for stage IV. At a median follow-up of 26 months (range: 1-46 months, SD 13.9), with a minimum of 21 months follow-up for surviving patients, the overall survival (OS) and progression-free survival (PFS) were 44.3% and 39.5%, respectively.
NGS Performance and mutations detected in the studied cases
The tissue sections used for analysis contained a minimum of 500 cancer cells in a micro-dissected area from which 10 consecutive unstained sections, which were obtained during initial sectioning of the tissue for diagnosis. The mean tumor cell content per sample was 40% (range 20-80%). Overall, the variant allelic frequency (VAF) of the mutations detected in this study ranged from 6% to 48% (mean: 32%). Read depths ranged from a minimum of 350X to over 12,000X (mean 2,800X), indicating a satisfactory level of coverage for the regions of interest (ROI).
Overall frequencies of gene mutations detected in at least 1% of the studied cases were: TP53 (44.80%), KRAS (38.1%), EGFR (10%), STK11 (8.6%), BRAF (4.8%), MET (3.8%), ABL-1, ATM, CDKN2A, PIK3CA, (all 2.9%), RB-1 and NRAS (2.4%), APC, ERBB4, PTPN11, SMAD4, (all 1.9%), CTNNB1 and ERBB2 (both 1.4%). Detailed lists of all detected mutations with numbers of patients harboring each mutation are included in the Supplemental Table 1.
Most commonly mutated genes (occurring in greater than 5% of cases)
TP53 mutations.
TP53 mutation was the most commonly detected, occurring in 100 (47.6%) cases. The detected mutations were highly diverse; 77% occurred only in a single case in this cohort. The most common recurring mutations affected residue 273 (p.R273L, p.R273C or p.R273H) seen in 7% of the mutated cases, followed by residue 179 (p.H179R or H179Y), accounting for 5.5% of cases, 248 (p.R248L or R248W; 4.4% of cases), 154 (p.G154V; 3.3% of cases) and 282 (p.R282G; 2.2% of cases). Most missense, frameshift, and nonsense mutations were located in known “hot spots”, annotated as functional protein domains for DNA-binding (amino acid residues 100-292) and tetramerization (residues 325-356). 1718 Such functional domains mutations were seen in 94 (44.8%) cases. The remaining rare mutations were seen outside of these functional domains, including variants located in the transactivation domain (residues 6-29) or splice site variants. Distribution of TP53 mutations in studied cases across protein domains is depicted in Fig. 2.
KRAS mutations.
KRAS mutations were the second most commonly detected, seen in 80 (38.1%) cases. The vast majority of KRAS mutations affected codon 12 (63 cases, 78.8%), with the remaining mutations occurring in codon 13 (9 cases, 11.3%), codon 61 (6 cases, 7.5%), and codons 115 or 146 (one case, 1.3% each). The proportion of KRAS mutations were as follows: p.G12C (28 cases, 35%), p.G12V (19 cases, 23.8%), p.G12D (7 cases, 8.8%), p.Q61H (6 cases, 7.5%), p.G12A and p.G13C (5 cases each, 6.3%), p.G13D (4 cases, 5%), p.G12F (3 cases, 3.8%) and one case each of p.G12S, p.G155E and p.A146T (1.3% each). The spectrum of KRAS mutations detected in the cohort overall is depicted in Fig. 3A.
EGFR mutations.
EGFR mutations were seen in 21 (10%) of cases studied. The most common were exon 19 deletion/insertions and exon 21 p.L858R mutations accounting for 47.6% and 38% of the EGFR mutations, respectively. T790M mutation in exon 20 was identified as a second mutation with a lower allelic frequency in 3 (10.3%) of the EGFR-mutated patients that previously received EGFR inhibitors.
STK11 mutations.
STK11 mutations occurred in 18 (8.6%) cases. The most prevalent mutations were p.Y60Ter (2), p.H168R (2) and p.E256Ter (2) and splice donors (3). The remaining 9 mutations were each identified in single cases (5.6%).
Overall mutational rates per tumor and co-mutation rates of the most commonly mutated genes.
The mean number of mutations per case was 1.41 (range: 0-10, 95% confidence interval: 1.27-1.56). No mutations were detected in 31 (14.7%) cases, 94 (44.8%) had a single mutation, 63 (30%) had two concurrent mutations, 17 (9%) had three and 5 (2.4%) had four or more mutations. TP53, KRAS and STK11 mutations accounted for the majority of mutations, either one of them detected in 152 (72.4%) of cases. TP53 mutation co-occurred with KRAS, STK11 or both of these mutations in 29 (13.8%), 9 (4.3%) and 5 (2.4%) of cases, while KRAS co-occurred with STK11 mutation in 12 (5.7%) cases, respectively.
FISH and IHC results
MET amplification, ALK and ROS1 rearrangement.
MET amplification was seen in 7 cases (3%), with MET/chromosome 7 ratios ranging from 2.17 to 6.78 in these amplified cases (mean 3.8). Only one case had ratio greater than 5.0 (high-level amplification). RET rearrangements were seen in 3 cases (1.4%), ROS1 and ALK rearrangements in 1 case (0.5%) each, respectively.
PD-L1 expression and its correlation with molecular genetic abnormalities.
High PD-L1 expression (>50% TPS) was seen in 74 cases (35%), low PD-L1 expression (1-49% TPS) in additional 59 cases (29%). The remaining 77 patients (37%) were negative for PD-L1 expression (TPS<1). High PD-L1 expression occurred more commonly in tumors with KRAS (p=0.002) or BRAF (p=0.04) mutations, as well as in tumors with MET gene amplification (p=0.011). Both low/high and high PD-L1 expression were less common in tumors with STK11 mutation (p=0.009 and p=0.04, respectively).
Reflexive molecular standing order experience
Utilizing a reflexive standing order for molecular testing ensured completion of testing within 10 working days after the biopsy/excision date in all of the cases studied (mean: 8.5 days; range 7-10 working days after the biopsy date). Obtaining unstained sections for molecular testing at the time of cutting the initial hematoxylin-eosin sections for diagnosis allowed performing molecular studies on tissue from small biopsies and cytology cell blocks, by obtaining unstained ribbons of tissue during the initial diagnostic evaluation, avoiding “refacing” the paraffin blocks, which is necessary when the blocks need to be cut for molecular testing at a later date. During the study period, only two cases (less than 1%) had no residual tumor tissue at diagnosis and could thus not be included in this cohort (never progressed to molecular testing). Prior to introducing the reflexive testing in 2017, our turn-around time exceeded this by up to 5 working days, plus not obtaining sections for molecular testing at diagnosis resulted in an approximately 8% testing failure rate due to lack of residual tissue in the paraffin blocks (data not shown).
Molecular abnormalities, clinical features and survival
Overall survival/progression-free survival and clinical stage.
The overall survival and progression-free survival rates across all stages at the mean follow up of 26 months (with a minimum of 21 months for surviving patients) were 44.3% and 39.5%, respectively. Among patients with stages I, II and III disease (lower stage), 24/71 (33.8%), 8/25 (32%) and 17/34 (50%) died of the disease within the studied time period, resulting in 62.2% overall survival rate in this lower stage category. In contrast, 68/80 (85%) of patients in the advanced stage group (stage IV) died, resulting in a 15% overall survival rate (p<0.001, Kaplan-Meier analysis with log rank; Fig. 4A). Similar findings were seen for progression-free survival. In stages I, II and III, 27/71 (38%), 11/25 (44%) and 16/34 (47%) of patients experienced disease recurrence or progression, with overall 58.5% progression-free survival rate in the lower stage group. This contrasted with 73/80 (91.3%) of patients with stage IV disease, who recurred/progressed, resulting in 8.7% progression-free survival rate (p<0.001, Kaplan-Meier analysis with log rank). The clinical and molecular findings are summarized in Table 1.
Table 1
Clinicopathological parameters and the four most commonly detected mutations
|
TP53 Mutation
|
KRAS Mutation
|
EGFR Mutation
|
STK11 Mutation
|
|
N (%)
|
P
|
N (%)
|
P
|
N (%)
|
P
|
N (%)
|
P
|
Gender
|
|
Male
|
40 (41%)
|
0.4
|
26 (27%)
|
0.003
|
7 (7%)
|
0.25
|
11 (11%)
|
0.22
|
Female
|
54 (48%)
|
54 (48%)
|
14 (12%)
|
7 (6%)
|
Age
|
|
>70 years
|
38 (38%)
|
0.09
|
35 (35%)
|
0.48
|
10 (10%)
|
1.0
|
8 (8%)
|
1.0
|
<70 years
|
56 (50%)
|
45 (41%)
|
11 (10%)
|
10 (9%)
|
Stage
|
|
I-III
|
61 (47%)
|
0.56
|
46 (35%)
|
0.31
|
13 (10%)
|
1.0
|
10 (8%)
|
0.6
|
IV
|
33 (42%)
|
34 (43%)
|
8 (10%)
|
8 (10%)
|
Smoking History
|
|
Smoker
|
90 (46%)
|
0.27
|
77 (39%)
|
0.26
|
14 (7%)
|
<0.001
|
18 (9%)
|
0.6
|
Never smoker
|
4 (29%)
|
3 (21%)
|
7 (50%)
|
0 (0%)
|
Smoking Status
|
|
Current Smoker
|
58 (53%)
|
0.018
|
45 (41%)
|
0.39
|
3 (3%)
|
<0.001
|
10 (9%)
|
0.8
|
Former Smoker
|
36 (36%)
|
35 (35%)
|
18 (18%)
|
8 (8%)
|
Pack Years
|
|
50 or more
|
35 (44%)
|
0.88
|
26 (33%)
|
0.22
|
2 (3%)
|
0.001
|
9 (11%)
|
0.16
|
Less than 50
|
45 (43%)
|
44 (42%)
|
18 (17%)
|
5 (5%)
|
PD-L1 Expression
|
|
Low/High
|
64 (48%)
|
0.25
|
56 (42%)
|
0.14
|
11 (8%)
|
0.34
|
5 (4%)
|
0.002
|
Absent
|
30 (39%)
|
24 (31%)
|
10 (13%)
|
13 (17%)
|
High
|
33 (45%)
|
1.0
|
39 (53%)
|
0.002
|
5 (7%)
|
0.34
|
2 (3%)
|
0.04
|
Low/Absent
|
61 (45%)
|
41 (23%)
|
16 (12%)
|
16 (12%)
|
TP53 mutation and survival.
Among the patients with TP53 mutations affecting the regions encoding the two principal functional domains (DNA-binding and tetramerization), 44% died of disease, compared to 24% of patients without a "hot spot” TP53 gene mutation (p=0.01). By Kaplan-Meier analysis, such TP53-mutated patients showed significantly worse overall survival (p=0.023, Fig. 4B). Controlled for gender, age, stage and smoking history in a multivariate analysis, a stepwise backward elimination Cox regression model showed that clinical stage (p<0.001, HR=1.85, 95% CI 1.57-2.2), TP53 mutation status (p=0.004, HR=1.71, 95% CI 1.18-2.47), male gender (p=0.04; HR 1.47, 95% CI 1.01-2.12) and KRAS/STK11 co-mutation (p=0.05; HR 1.92, 95% CI 1.00-3.69) independently correlated with overall survival (Table 2). Male gender was no longer significant for overall survival when EGFR mutated cases were excluded from analysis, reflecting the higher prevalence of the prognostically favorable EGFR mutation in non-smoking women (p=0.007). Multivariate Cox regression model showed only advanced stage as significant variable for progression-free survival (PFS) (p<0.001, HR=2.62, 95% CI 1.66-4.11). A separate stage IV survival analysis confirmed the adverse effect of TP53 mutation on both OS and PFS in patients with advanced disease (p=0.007 and p=0.003, respectively; Fig 4C).
Table 2
Results of univariate and multivariate analyses: P = P- value, HR = Hazard Risk, CI = Confidence Interval
|
P
|
HR
|
95% CI
|
UNIVARIATE ANALYSIS:
|
|
Stage I (N=64)*:
|
KRAS/TP53 co-mutation
|
0.009
|
3.78
|
1.39 - 10.27
|
|
Stages I-II (N=84)*:
|
KRAS/TP53 co-mutation
|
0.03
|
2.6
|
1.11 - 6.1
|
|
Stage IV (N=71)*:
|
TP53 mutation
|
0.004
|
2.07
|
1.26 – 3.4
|
|
MULTIVARIATE ANALYSIS:
|
|
All stages (N=210):
|
Stage
|
<0.001
|
1.86
|
1.57 - 2.2
|
TP53 mutation
|
0.004
|
1.71
|
1.19 - 2.47
|
Male gender
|
0.04
|
1.47
|
1.01 - 2.12
|
KRAS/STK11 co-mutation
|
0.05
|
1.92
|
1.0 – 3.69
|
|
All stages without EGFR-mutated cases (N=189):
|
Stage
|
<0.001
|
1.82
|
1.53 – 2.16
|
TP53 mutation
|
0.009
|
1.67
|
1.13 – 2.44
|
KRAS/STK11 co-mutation
|
0.03
|
2.1
|
1.08 – 4.07
|
*Without EGFR-mutated cases
|
KRAS mutations and clinical features.
Any KRAS mutation, codon 12 mutations (both p=0.003) and most specifically G12C mutation (p<0.001) tended to occur in women, smokers (both p=0.04) and most prominently in women smokers (p<0.001). No associations of KRAS mutations with outcome were noted.
EGFR mutations and survival.
Among the EGFR mutated cases, shortened survival was seen by Kaplan Meier analysis in advanced stage (p=0.02) and male gender (p=0.025); both independent in multivariate Cox Regression analysis (p=0.005 and 0.01; HR 2.9/95% CI 1.4-6.2 and 8.9, 95% CI 1.7-46.6, respectively).
TP53/KRAS, KRAS/STK11 co-mutations and survival.
KRAS/STK11 co-mutation was associated with worse OS (p=0.018; Fig. 4D). This was independent of stage, gender, or TP53 mutation in the above multivariate model. In stage I, as well as stages I-II, after excluding cases with favorable prognostic effect of EGFR mutation , KRAS/TP53 co-mutation was predictive of adverse OS (p=0.005 and p=0.02; Fig. 4E).
Mutually exclusive mutations.
EGFR mutations were entirely mutually exclusive with BRAF mutations, as well as nearly mutually exclusive with KRAS and NRAS mutations (in only one case, EGFR mutation co-occurred with mutation in either KRAS or NRAS; both p=0.001).
PD-L1 status and survival.
High PD-L1 expression appeared as an adverse factor in both OS and PFS on univariate analysis (p=0.03 and 0.04, respectively) but this was shown to be due to a correlation with advanced stage, where PD-L1 was more often expressed (p=0.012). A multivariate Cox regression model controlled for age, gender, stage and smoking history, showed that PD-L1 status was not independently associated with OS or PFS.
Gene mutations and smoking.
As mentioned earlier, KRAS G12C mutation was more commonly seen in smokers (p=0.04), with G12V being second most common. In non-smokers this order was reversed (Fig. 3B). An inverse association was seen between EGFR mutation and smoking, with 50% of non-smokers harboring an EGFR mutation, in contrast to only 7% of smokers (p<0.001). Among patients with positive smoking history, EGFR mutation occurred in 18% of prior smokers, in contrast to only 2.7% of current smokers (p<0.001), and in 17% of patients with lower than 50 pack-year history, in contrast to only 2.5% of smokers with 50 or greater pack-year history (p=0.001). Considering the positive correlation of male gender with heavy smoking (greater than 50 pack-years; p=0.007) and a female non-smoker status with EGFR mutation (p<0.001), a relative survival advantage of women non-smokers was seen by Kaplan-Meier survival analysis (p=0.05; log rank). However, unlike male gender, such “female non-smoker” status was not an independent survival factor in a multivariate Cox Regression model with stage, TP53 mutation and KRAS/STK11 co-mutation.
PD-L1 expression, gene mutations and survival.
High PD-L1 expression occurred significantly more commonly in tumors with mutations in the MAPK pathway (KRAS, BRAF or NRAS; p<0.001) as well as those with MET gene amplification (p=0.009). Similar trend was seen for tumors with KRAS/TP53 co-mutation, but did not reach significance (p=0.08). In contrast, PD-L1 expression was more commonly absent in tumors with STK11 mutations (p=0.009), even more so in those with KRAS/STK11 co-mutations (p=0.002).