Characteristics of KRAS Mutations in Lung Adenocarcinoma
Characteristics of KRAS Mutations in Lung Adenocarcinoma
In the present study, by combining detailed histopathological analysis with high-sensitivity mutation detection method, we identified a novel association of KRAS mutations with solid growth pattern and tumor-infiltrating leukocytes in non-mucinous lung adenocarcinomas. In addition, we expanded on several previously described histological and clinical associations of KRAS and EGFR mutations.
Although this is the first study to identify a propensity of KRAS+ lung adenocarcinomas for solid growth pattern, several previous studies did hint at this association. First, several studies showed an association of KRAS mutations with poor differentiation. Although there is currently no standardized grading system for lung adenocarcinomas, solid growth pattern is the central parameter in grading of adenocarcinomas system-wide, and it is likely that the presence of solid growth pattern, at least in part, explains the association of KRAS mutations and poor differentiation in those studies. Furthermore, an association of KRAS mutations with a gene expression profile correlating with solid histology was noted in a study by Motoi et al. Lastly, an association of KRAS mutations and 'tumor islands', which, in turn, were associated with solid growth pattern, was recently reported by Onozato et al.
Three potential factors could have contributed to the differences in the reported histological associations of KRAS mutations in lung adenocarcinomas across studies:
Overall, KRAS mutations appear to have a dual histological association in lung adenocarcinomas—one with non-mucinous carcinomas with a solid component, which we found to have KRAS mutations in 55% of cases, and the other with mucinous carcinomas formerly designated mucinous bronchioloalveolar carcinoma ('invasive mucinous adenocarcinoma'), which are reported to harbor KRAS mutations in 30% to >80% of cases. In this study, KRAS mutations were also over-represented in the latter tumors, occurring in 67% (4/6) of cases; although statistical analysis of this association was limited by overall rarity of this tumor type in our unselected patient population. In addition to the dual role of KRAS in invasive adenocarcinomas, KRAS mutations have also been reported to be paradoxically over-represented in pre-invasive glandular lesions - pure bronchioloalveolar carcinomas/adenocarcinomas in situ; these lesions were excluded from the present study to focus the analysis on conventional invasive adenocarcinomas. From the perspective of lung cancer pathogenesis, these pleotropic histological associations may hint at the complex role of KRAS mutations in stem cells. One hypothesis is that KRAS mutations may arise in distinct stem cells, giving rise to neoplasms with divergent histology. Alternatively, KRAS mutations may arise in a common pleuripotent stem cell with a broad differentiation potential. These possibilities are in line with pre-clinical data that KRAS-mediated tumorigenesis is significantly influenced by the cellular context.
Our finding that KRAS mutations are associated with solid histology and tendency for greater necrosis and cytological atypia may represent the underlying link between KRAS+ genotype and aggressive clinical behavior in lung adenocarcinomas. Several recent studies have demonstrated that solid growth pattern is a strong predictor of adverse clinical outcome, whereas lepidic pattern—associated with EGFR mutations—is a predictor of indolent behavior in lung adenocarcinomas. Thus, the distinct association of KRAS and EGFR mutations with aggressive vs indolent histologies, respectively, parallels the differences in prognosis. Because the follow-up available for patients in this series was too short for survival analysis, future studies with survival data and multivariate analysis will be needed to determine whether indeed KRAS and EGFR mutations exert their prognostic effects via a link to distinct histological subsets or whether these effects are histology-independent.
Of interest, the association of KRAS mutations and solid histology in lung adenocarcinomas ties in with our recent description of a high frequency (40%) of KRAS mutations in large cell (undifferentiated) carcinomas showing glandular immunophenotype. We proposed that these clinically aggressive tumors represent a spectrum of adenocarcinomas with an extreme amount of solid growth pattern. The high frequency of KRAS mutations in conventional adenocarcinomas with partial solid histology reported in this study is in line with that proposal, as is the low-frequency of EGFR mutations seen in both adenocarcinomas with solid component and large cell carcinomas with glandular immunoprofile. The propensity for solid growth/poor differentiation of KRAS-mutant tumors is also consistent with the finding of a high rate (38%) of KRAS mutations in sarcomatoid/pleomorphic lung carcinomas.
The finding that KRAS-mutated carcinomas are associated with tumor-infiltrating leukocytes, in addition to representing a potential confounder in molecular testing, may itself have biological and clinical significance. Presence of inflammatory cells has been implicated as both favorable and unfavorable prognostic indicator in several malignances, consistent with the capacity of immunity to exert both anti-tumor and pro-tumor effects depending on both tumor and host factors. Non-small cell lung carcinomas are frequently associated with prominent tumor-infiltrating lymphocytes and other inflammatory cells, but their significance remains controversial. Both adverse and favorable prognostic effects having been reported, which may be related to different subsets of inflammatory cells, scoring criteria, and patient populations. In this study, only the overall extent of inflammatory infiltrate was analyzed, and further study will be needed to evaluate specific leukocyte subsets. Although association of inflammation and KRAS mutations is a novel observation, Dacic et al noted that high level of tumor-infiltrating lymphocytes is uncommon in adenocarcinomas with EGFR mutations; this trend for pan-inflammatory infiltrate was also seen in the present study. We cannot exclude that the degree of inflammation in adenocarcinomas with KRAS vs EGFR mutations reflects tissue response to tumors with more vs less aggressive histology, respectively. Nevertheless, these data raise the possibility that patients with KRAS-mutated lung adenocarcinomas may be an especially attractive subset for clinical trials of immunomodulatory agents aimed at enhancing the anti-tumor activity of tumor-infiltrating lymphocytes, such as therapeutic antibodies to PD-1 and PD-L1.
In addition to describing novel histological associations of KRAS mutations, this study also expanded on the previously recognized histological and clinical association of EGFR mutations. Previous studies consistently reported that EGFR mutations are associated with non-mucinous non-solid histology, as also seen in this study. The association of EGFR mutations with individual histological patterns has significant variability in the literature, and includes lepidic, papillary, micropapillary, and in some studies acinar. In this study, individual patterns associated with EGFR mutations were lepidic, papillary, and, to a lesser degree, acinar. However, the strongest association of EGFR mutations was with hobnail cytological features, which were typically seen in carcinomas with lepidic component and/or tumors with characteristic serrated intra-glandular infoldings, which could be variably described as displaying papillary/micropapillary/acinar patterns (data not shown). Hobnail cytology is proposed as a defining feature of terminal respiratory unit-type adenocarcinomas, and it is possible that several architectural patterns emerging as associated with EGFR mutations in individual studies represent variable annotation of architectural manifestations of this type of adenocarcinoma.
It has been suggested that TTF-1 expression represents a feature of terminal respiratory unit-type adenocarcinomas and that while EGFR+ adenocarcinomas are uniformly TTF-1-positive, carcinomas with KRAS mutations tend to be TTF-1-negative. Here we clarify that the lack of TTF-1 expression in KRAS+ carcinomas applies primarily to mucinous carcinomas, of which 57% in this study were TTF-1-negative, whereas the lack of TTF-1 expression in KRAS+ non-mucinous carcinomas is rare (5% in this series). Similarly, we clarify that despite propensity for solid growth, a subset of KRAS+ non-mucinous carcinomas displays hobnail cytological features in better differentiated areas, suggesting that these tumors do not always belong to a non-terminal respiratory unit lineage, consistent with previous observations.
A notable observation in this study is that KRAS+ adenocarcinomas have a greater propensity for solid growth pattern compared not only with EGFR+ but also with KRAS−/EGFR− carcinomas. It is worth noting, however, that KRAS−/EGFR− is not a molecularly homogenous group but rather a mixture of carcinomas with various low-frequency molecular alterations, including ALK, BRAF, HER2, ROS1, and RET (frequency of each ranging from <1% to 5%), as well as tumors with yet unidentified molecular events. Despite its heterogeneous nature, the pooled clinical outcome for this group was found to be favorable compared with KRAS+ tumors and inferior compared with EGFR+ tumors in clinical studies, which parallels the different propensities of these groups for solid growth pattern identified in this study, although at least some molecular subsets within KRAS−/EGFR− group—namely ALK-rearranged carcinomas—are also known to also show a propensity for solid histology (in addition to the classic association with signet ring cells) and aggressive behavior. Greater pack-year smoking history in patient with KRAS+ carcinomas compared with both the EGFR+ as well as with KRAS−/EGFR− group in this series is in line with a link to never-smokers of the EGFR+ group and several known molecular subsets within the KRAS−/EGFR− group, including ALK, ROS1, and RET.
From a practical perspective on predictive molecular testing, our data support previous conclusion that while both EGFR and KRAS mutations are associated with propensities for distinct histological and clinicopathological characteristics, none of these associations have sufficient predictive value to allow triage of cases for molecular studies, and therefore all lung adenocarcinomas should undergo molecular testing irrespective of histological and clinical features (with possible exception being the exclusion of mucinous carcinomas from testing for EGFR mutations). On the other hand, estimation of pre-test probability of mutations may have value in some clinical settings. For such situations, nomograms, based on clinical +/− histological features, have been recently developed to predict the likelihood of EGFR mutations. The findings in this study, particularly the predictive effect of solid histology on the likelihood of KRAS and EGFR mutations, may be of value for refinement of such nomograms. Although KRAS mutations have thus far evaded therapeutic targeting, and the current value of testing for these mutations in lung carcinomas is to serve as negative predictors for other targetable mutations, it is hoped that effective targeted therapies for mutant KRAS will emerge in the near future.
In summary, we have described here a novel association of KRAS mutations with propensity for solid histology in non-mucinous lung adenocarcinomas, which may explain the adverse clinical outcome portended by KRAS mutations. We also describe an association of KRAS mutations with tumor-infiltrating leukocytes, which raises the possibility that patients with KRAS-mutated adenocarcinomas may benefit from novel immunomodulatory agents.
Discussion
In the present study, by combining detailed histopathological analysis with high-sensitivity mutation detection method, we identified a novel association of KRAS mutations with solid growth pattern and tumor-infiltrating leukocytes in non-mucinous lung adenocarcinomas. In addition, we expanded on several previously described histological and clinical associations of KRAS and EGFR mutations.
Although this is the first study to identify a propensity of KRAS+ lung adenocarcinomas for solid growth pattern, several previous studies did hint at this association. First, several studies showed an association of KRAS mutations with poor differentiation. Although there is currently no standardized grading system for lung adenocarcinomas, solid growth pattern is the central parameter in grading of adenocarcinomas system-wide, and it is likely that the presence of solid growth pattern, at least in part, explains the association of KRAS mutations and poor differentiation in those studies. Furthermore, an association of KRAS mutations with a gene expression profile correlating with solid histology was noted in a study by Motoi et al. Lastly, an association of KRAS mutations and 'tumor islands', which, in turn, were associated with solid growth pattern, was recently reported by Onozato et al.
Three potential factors could have contributed to the differences in the reported histological associations of KRAS mutations in lung adenocarcinomas across studies:
One potential factor is under-detection of KRAS mutations by assays with suboptimal sensitivity, such as Sanger sequencing. The relevance of method sensitivity is particularly supported by our finding that lung carcinomas harboring KRAS mutations are enriched with inflammatory cells. Standard macrodissection of such tumors may fail to enrich for tumor cells due to their intimate association with inflammation, and consequently extracted DNA may be diluted by DNA contributed by inflammatory cells. Thus, KRAS-mutated carcinomas may be particularly prone to false-negative results by standard Sanger sequencing, which has a notoriously low analytical sensitivity, requiring high tumor cell content (40–50%). By contrast, Sequenom platform, used in this study, requires ~10% tumor cell content. The possibility that KRAS mutations may be under-detected by Sanger sequencing is indirectly supported by the data in colorectal carcinomas, where in a matched comparison, Sanger sequencing was found to under-estimate the frequency of exon 2 KRAS mutations by 9% compared with more sensitive methods.
The second factor potentially contributing to the variability in molecular/histological correlation results in individual studies could be the variation in the designation of histological patterns. This is illustrated in a recent inter-observer reproducibility study, which showed significant variability in designation of histological patterns in lung adenocarcinomas among pathologists. Although solid pattern showed one of the highest concordances, a potential source of variability comes from the lack of agreement on the designation of complex glandular patterns (such as cribriform), which are currently variably classified as acinar or solid. These patterns were annotated as a distinct category in this study, and, while over-represented in KRAS+ carcinomas, they did not reach a statistical association with any molecular group. Another potential confounder is recently recommended classification based solely on a single histological pattern, judged to be predominant relative to other patterns, which we found to be difficult to assign objectively in a fair number (29%) of cases due to ≥2 patterns being present in a similar co-dominant amount. In addition, this annotation may exclude tumors in which a pattern is present in a minor amount but is still biologically significant, as illustrated by our finding that both solid and lepidic patterns have a significant effect on the frequency of KRAS and EGFR mutations even when present as a minor component of a tumor.
Lastly, genotype/phenotype associations could be influenced by ethnic factors. In particular, a potential confounding factor is still a largely unexplained significant difference in the rate of KRAS (and EGFR) mutations in lung adenocarcinomas between western and East Asian populations. Specifically, the baseline rate of KRAS mutations is low (5-10%) in East Asian populations, with a substantial (40–60%) proportion of mutations concentrated in mucinous carcinomas. By contrast, KRAS mutations occur in 25–35% of lung adenocarcinomas in western patients, with the majority (89% in this series) of mutations occurring in non-mucinous carcinomas. Thus, both the frequency and histologic correlates of KRAS mutations in non-mucinous adenocarcinomas may have geographic differences.
Overall, KRAS mutations appear to have a dual histological association in lung adenocarcinomas—one with non-mucinous carcinomas with a solid component, which we found to have KRAS mutations in 55% of cases, and the other with mucinous carcinomas formerly designated mucinous bronchioloalveolar carcinoma ('invasive mucinous adenocarcinoma'), which are reported to harbor KRAS mutations in 30% to >80% of cases. In this study, KRAS mutations were also over-represented in the latter tumors, occurring in 67% (4/6) of cases; although statistical analysis of this association was limited by overall rarity of this tumor type in our unselected patient population. In addition to the dual role of KRAS in invasive adenocarcinomas, KRAS mutations have also been reported to be paradoxically over-represented in pre-invasive glandular lesions - pure bronchioloalveolar carcinomas/adenocarcinomas in situ; these lesions were excluded from the present study to focus the analysis on conventional invasive adenocarcinomas. From the perspective of lung cancer pathogenesis, these pleotropic histological associations may hint at the complex role of KRAS mutations in stem cells. One hypothesis is that KRAS mutations may arise in distinct stem cells, giving rise to neoplasms with divergent histology. Alternatively, KRAS mutations may arise in a common pleuripotent stem cell with a broad differentiation potential. These possibilities are in line with pre-clinical data that KRAS-mediated tumorigenesis is significantly influenced by the cellular context.
Our finding that KRAS mutations are associated with solid histology and tendency for greater necrosis and cytological atypia may represent the underlying link between KRAS+ genotype and aggressive clinical behavior in lung adenocarcinomas. Several recent studies have demonstrated that solid growth pattern is a strong predictor of adverse clinical outcome, whereas lepidic pattern—associated with EGFR mutations—is a predictor of indolent behavior in lung adenocarcinomas. Thus, the distinct association of KRAS and EGFR mutations with aggressive vs indolent histologies, respectively, parallels the differences in prognosis. Because the follow-up available for patients in this series was too short for survival analysis, future studies with survival data and multivariate analysis will be needed to determine whether indeed KRAS and EGFR mutations exert their prognostic effects via a link to distinct histological subsets or whether these effects are histology-independent.
Of interest, the association of KRAS mutations and solid histology in lung adenocarcinomas ties in with our recent description of a high frequency (40%) of KRAS mutations in large cell (undifferentiated) carcinomas showing glandular immunophenotype. We proposed that these clinically aggressive tumors represent a spectrum of adenocarcinomas with an extreme amount of solid growth pattern. The high frequency of KRAS mutations in conventional adenocarcinomas with partial solid histology reported in this study is in line with that proposal, as is the low-frequency of EGFR mutations seen in both adenocarcinomas with solid component and large cell carcinomas with glandular immunoprofile. The propensity for solid growth/poor differentiation of KRAS-mutant tumors is also consistent with the finding of a high rate (38%) of KRAS mutations in sarcomatoid/pleomorphic lung carcinomas.
The finding that KRAS-mutated carcinomas are associated with tumor-infiltrating leukocytes, in addition to representing a potential confounder in molecular testing, may itself have biological and clinical significance. Presence of inflammatory cells has been implicated as both favorable and unfavorable prognostic indicator in several malignances, consistent with the capacity of immunity to exert both anti-tumor and pro-tumor effects depending on both tumor and host factors. Non-small cell lung carcinomas are frequently associated with prominent tumor-infiltrating lymphocytes and other inflammatory cells, but their significance remains controversial. Both adverse and favorable prognostic effects having been reported, which may be related to different subsets of inflammatory cells, scoring criteria, and patient populations. In this study, only the overall extent of inflammatory infiltrate was analyzed, and further study will be needed to evaluate specific leukocyte subsets. Although association of inflammation and KRAS mutations is a novel observation, Dacic et al noted that high level of tumor-infiltrating lymphocytes is uncommon in adenocarcinomas with EGFR mutations; this trend for pan-inflammatory infiltrate was also seen in the present study. We cannot exclude that the degree of inflammation in adenocarcinomas with KRAS vs EGFR mutations reflects tissue response to tumors with more vs less aggressive histology, respectively. Nevertheless, these data raise the possibility that patients with KRAS-mutated lung adenocarcinomas may be an especially attractive subset for clinical trials of immunomodulatory agents aimed at enhancing the anti-tumor activity of tumor-infiltrating lymphocytes, such as therapeutic antibodies to PD-1 and PD-L1.
In addition to describing novel histological associations of KRAS mutations, this study also expanded on the previously recognized histological and clinical association of EGFR mutations. Previous studies consistently reported that EGFR mutations are associated with non-mucinous non-solid histology, as also seen in this study. The association of EGFR mutations with individual histological patterns has significant variability in the literature, and includes lepidic, papillary, micropapillary, and in some studies acinar. In this study, individual patterns associated with EGFR mutations were lepidic, papillary, and, to a lesser degree, acinar. However, the strongest association of EGFR mutations was with hobnail cytological features, which were typically seen in carcinomas with lepidic component and/or tumors with characteristic serrated intra-glandular infoldings, which could be variably described as displaying papillary/micropapillary/acinar patterns (data not shown). Hobnail cytology is proposed as a defining feature of terminal respiratory unit-type adenocarcinomas, and it is possible that several architectural patterns emerging as associated with EGFR mutations in individual studies represent variable annotation of architectural manifestations of this type of adenocarcinoma.
It has been suggested that TTF-1 expression represents a feature of terminal respiratory unit-type adenocarcinomas and that while EGFR+ adenocarcinomas are uniformly TTF-1-positive, carcinomas with KRAS mutations tend to be TTF-1-negative. Here we clarify that the lack of TTF-1 expression in KRAS+ carcinomas applies primarily to mucinous carcinomas, of which 57% in this study were TTF-1-negative, whereas the lack of TTF-1 expression in KRAS+ non-mucinous carcinomas is rare (5% in this series). Similarly, we clarify that despite propensity for solid growth, a subset of KRAS+ non-mucinous carcinomas displays hobnail cytological features in better differentiated areas, suggesting that these tumors do not always belong to a non-terminal respiratory unit lineage, consistent with previous observations.
A notable observation in this study is that KRAS+ adenocarcinomas have a greater propensity for solid growth pattern compared not only with EGFR+ but also with KRAS−/EGFR− carcinomas. It is worth noting, however, that KRAS−/EGFR− is not a molecularly homogenous group but rather a mixture of carcinomas with various low-frequency molecular alterations, including ALK, BRAF, HER2, ROS1, and RET (frequency of each ranging from <1% to 5%), as well as tumors with yet unidentified molecular events. Despite its heterogeneous nature, the pooled clinical outcome for this group was found to be favorable compared with KRAS+ tumors and inferior compared with EGFR+ tumors in clinical studies, which parallels the different propensities of these groups for solid growth pattern identified in this study, although at least some molecular subsets within KRAS−/EGFR− group—namely ALK-rearranged carcinomas—are also known to also show a propensity for solid histology (in addition to the classic association with signet ring cells) and aggressive behavior. Greater pack-year smoking history in patient with KRAS+ carcinomas compared with both the EGFR+ as well as with KRAS−/EGFR− group in this series is in line with a link to never-smokers of the EGFR+ group and several known molecular subsets within the KRAS−/EGFR− group, including ALK, ROS1, and RET.
From a practical perspective on predictive molecular testing, our data support previous conclusion that while both EGFR and KRAS mutations are associated with propensities for distinct histological and clinicopathological characteristics, none of these associations have sufficient predictive value to allow triage of cases for molecular studies, and therefore all lung adenocarcinomas should undergo molecular testing irrespective of histological and clinical features (with possible exception being the exclusion of mucinous carcinomas from testing for EGFR mutations). On the other hand, estimation of pre-test probability of mutations may have value in some clinical settings. For such situations, nomograms, based on clinical +/− histological features, have been recently developed to predict the likelihood of EGFR mutations. The findings in this study, particularly the predictive effect of solid histology on the likelihood of KRAS and EGFR mutations, may be of value for refinement of such nomograms. Although KRAS mutations have thus far evaded therapeutic targeting, and the current value of testing for these mutations in lung carcinomas is to serve as negative predictors for other targetable mutations, it is hoped that effective targeted therapies for mutant KRAS will emerge in the near future.
In summary, we have described here a novel association of KRAS mutations with propensity for solid histology in non-mucinous lung adenocarcinomas, which may explain the adverse clinical outcome portended by KRAS mutations. We also describe an association of KRAS mutations with tumor-infiltrating leukocytes, which raises the possibility that patients with KRAS-mutated adenocarcinomas may benefit from novel immunomodulatory agents.
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