Purpose The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear. Experimental Design We genotyped 3026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical and smoking history data. Results EGFR mutations were found in 43% of never smokers (NS) and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of NS. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. NS were more likely than smokers to have KRAS G>A transition mutation (mostly G12D) (58% vs. 20%, p=0.0001). KRAS G12C, the most common G>T transversion mutation in smokers, was more frequent in women (p=0.007) and these women were younger than men with the same mutation (median 65 vs. 69, p=0.0008) and had smoked less. Conclusions The distinct types of KRAS mutations in smokers vs. NS suggest that most KRAS-mutant lung cancers in NS are not due to secondhand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens.

Immunohistochemistry is increasingly utilized to differentiate lung adenocarcinoma and squamous cell carcinoma. However, detailed analysis of coexpression profiles of commonly used markers in large series of whole-tissue sections is lacking. Furthermore, the optimal diagnostic algorithm, particularly the minimalmarker combination, is not firmly established. We therefore studied whole-tissue sections of resected adenocarcinoma and squamous cell carcinoma (n ¼ 315) with markers commonly used to identify adenocarcinoma (TTF-1) and squamous cell carcinoma (p63, CK5/6, 34bE12), and prospectively validated the devised algorithm in morphologically unclassifiable small biopsy/cytology specimens (n ¼ 38). Analysis of whole-tissue sections showed that squamous cell carcinoma had a highly consistent immunoprofile (TTF-1-negative and p63/CK5/6/34bE12-diffuse) with only rare variation. In contrast, adenocarcinoma showed significant immunoheterogenetity for all 'squamous markers' (p63 (32%), CK5/6 (18%), 34bE12 (82%)) and TTF-1 (89%). As a single marker, only diffuse TTF-1 was specific for adenocarcinoma whereas none of the 'squamous markers,' even if diffuse, were entirely specific for squamous cell carcinoma. In contrast, coexpression profiles of TTF-1/p63 had only minimal overlap between adenocarcinoma and squamous cell carcinoma, and there was no overlap if CK5/6 was added as a third marker. An algorithm was devised in which TTF-1/p63 were used as the first-line panel, and CK5/6 was added for rare indeterminate cases. Prospective validation of this algorithm in small specimens showed 100% accuracy of adenocarcinoma vs squamous cell carcinoma prediction as determined by subsequent resection. In conclusion, although reactivity for 'squamous markers' is common in lung adenocarcinoma, a two-marker panel of TTF-1/p63 is sufficient for subtyping of the majority of tumors as adenocarcinomas vs squamous cell carcinoma, and addition of CK5/6 is needed in only a small subset of cases. This simple algorithm achieves excellent accuracy in small specimens while conserving the tissue for potential predictive marker testing, which is now an essential consideration in advanced lung cancer specimens. Modern Pathology (2011Pathology ( ) 24, 1348Pathology ( -1359 doi:10.1038/modpathol.2011; published online 27 May 2011Keywords: adenocarcinoma; CK5/6; p63; squamous cell carcinoma; TTF-1; 34bE12Adenocarcinoma and squamous cell carcinoma are the two major subtypes of non-small cell lung carcinoma. Until recently, therapeutic approaches to non-small cell lung carcinoma were largely guided by tumor stage, and there was no difference in treatment for adenocarcinoma vs squamous cell carcinoma. This monolithic approach to non-small cell lung carcinoma has dramatically changed in the last few years as a result of three major advances in thoracic medical oncology for advanced disease. These include (1) EGFR-targeted therapies, erlotinib

Background The mutually exclusive pattern of the major driver oncogenes in lung cancer suggests that other mutually exclusive oncogenes exist. We performed a systematic search for tyrosine kinase (TK) fusions by screening all TKs for aberrantly high RNA expression levels of the 3′ kinase domain (KD) exons relative to more 5′ exons. Methods We studied 69 patients (including 5 never smokers and 64 current or former smokers) with lung adenocarcinoma negative for all major mutations in KRAS, EGFR, BRAF, MEK1, and HER2, and for ALK fusions (termed “pan-negative”). A NanoString-based assay was designed to query the transcripts of 90 TKs at two points: 5′ to the KD and within the KD or 3′ to it. Tumor RNAs were hybridized to the NanoString probes and analyzed for outlier 3′ to 5′ expression ratios. Presumed novel fusion events were studied by rapid amplification of cDNA ends (RACE) and confirmatory RT-PCR and FISH. Results We identified 1 case each of aberrant 3′ to 5′ ratios in ROS1 and RET. RACE isolated a GOPC-ROS1 (FIG-ROS1) fusion in the former and a KIF5B-RET fusion in the latter, both confirmed by RT-PCR. The RET rearrangement was also confirmed by FISH. The KIF5B-RET patient was one of only 5 never smokers in this cohort. Conclusion The KIF5B-RET fusion defines an additional subset of lung cancer with a potentially targetable driver oncogene enriched in never smokers with “pan-negative” lung adenocarcinomas. We also report for the first time in lung cancer the GOPC-ROS1 fusion previously characterized in glioma.

Histologic classification of ampullary carcinomas into intestinal, pancreatobiliary, or other subtypes is easily achievable in some cases but difficult in others. Immunohistochemical (IHC) stains may allow distinction between the subtypes; however, their added value to routine hematoxylin and eosin (H&E) evaluation has not been systematically evaluated. Inconsistent histologic subtyping has hampered current clinical research and therapeutic trials. In this study, a consecutive series of 105 ampullary carcinomas was subtyped first by H&E evaluation and then by the evaluation of an IHC panel composed of CK7, CK20, CDX2, MUC1, and MUC2, and the added value of IHC was analyzed. By H&E, a consensus diagnosis, defined as concordant subtyping among at least 3 of the 4 independent study pathologists, was achieved in 81 of the 105 (77%) cases. There was excellent agreement for poorly differentiated and mucinous subtypes (κ=0.72 and 0.89, respectively) but only good agreement for intestinal and pancreatobiliary subtypes (κ=0.57 and 0.48, respectively) and poor agreement for mixed subtype (κ=0.09). By IHC, CK7 showed no informative value (being positive in ≥70% of the cases in both intestinal and pancreatobiliary subtypes), whereas a subtyping schema incorporating the combination staining patterns of CK20, CDX2, MUC1, and MUC2 did. By this schema, "intestinal subtype" was defined as having (1) positive staining for CK20 or CDX2 or MUC2 and negative staining for MUC1, or (2) positive staining for CK20, CDX2, and MUC2, irrespective of the MUC1 result; and "pancreatobiliary subtype" was defined as having positive staining for MUC1 and negative staining for CDX2 and MUC2, irrespective of CK20 results. Cases not fitting one of these 3 categories were regarded as "ambiguous" immunohistochemically. By combining this schema with H&E evaluation, 97 of the 105 cases (92%) could be classified into either intestinal or pancreatobiliary subtype. In particular, immunophenotyping allowed categorization of 75% of poorly differentiated adenocarcinomas and 69% of cases with mixed histologic features as either intestinal or pancreatobiliary subtype. Most mucinous adenocarcinomas (88%) were clearly intestinal subtype by IHC. Thus, our IHC schema enhanced the subtyping of ampullary carcinoma and, in combination with H&E evaluation, allowed a dichotomous classification in 92% of the cases. Should further independent studies reaffirm our findings, this schema may serve as a valuable tool in both diagnostic and research settings.

KRAS mutations define a clinically-distinct subgroup of lung adenocarcinoma patients, characterized by smoking history, resistance to EGFR-targeted therapies, and adverse prognosis. Whether KRAS- mutated lung adenocarcinomas also have distinct histopathologic features is not well established. We tested 180 resected lung adenocarcinomas for KRAS and EGFR mutations by high-sensitivity mass spectrometry-based genotyping (Sequenom) and PCR-based sizing assays. All tumors were assessed for the proportion of standard histologic patterns (lepidic, acinar, papillary, micropapillary, solid and mucinous), several other histologic and clinical parameters, and TTF-1 expression by immunohistochemistry. Among 180 carcinomas, 63 (35%) had KRAS mutations (KRAS+), 35 (19%) had EGFR mutations (EGFR+), and 82 (46%) had neither mutation (KRAS-/EGFR-). Solid growth pattern was significantly over-represented in KRAS+ carcinomas: the mean ± standard deviation for the amount of solid pattern in KRAS+ carcinomas was 27 ± 34% compared to 3 ± 10% in EGFR+ (P<0.001) and 15 ± 27% in KRAS-/EGFR- (P=0.033) tumors. Furthermore, at least focal (>20%) solid component was more common in KRAS+ (28/63; 44%) compared to EGFR+ (2/35; 6%; P<0.001) and KRAS-/EGFR- (21/82; 26%; P=0.012) carcinomas. KRAS mutations were also over-represented in mucinous carcinomas, and were significantly associated with the presence of tumor-infiltrating leukocytes and heavier smoking history. EGFR mutations were associated with non-mucinous non-solid patterns, particularly lepidic and papillary, lack of necrosis, lack of cytologic atypia, hobnail cytology, TTF-1 expression, and never/light smoking history. In conclusion, extended molecular and clinicopathologic analysis of lung adenocarcinomas reveals a novel association of KRAS mutations with solid histology and tumor-infiltrating inflammatory cells, and expands on several previously recognized morphologic and clinical associations of KRAS and EGFR mutations. Solid growth pattern was recently shown to be a strong predictor of aggressive behavior in lung adenocarcinomas, which may underlie the unfavorable prognosis associated with KRAS mutations in these tumors.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of 15 tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and fibroblast growth factor receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

No abstract

Background We previously demonstrated that stage IIIB/IV non-small cell lung cancer (NSCLC) never smokers lived 50% longer than former/current smokers. This observation persisted after adjusting for age, performance status, and gender. We hypothesized that smoking-dependent differences in the distribution of driver mutations might explain differences in prognosis between these subgroups. Methods We reviewed 293 never smokers and 382 former/current smokers with lung adenocarcinoma who underwent testing for EGFR and KRAS mutations and rearrangements in ALK between 2009 and 2010. Clinical outcomes and patient characteristics were collected. Survival probabilities were estimated using the Kaplan-Meier method. Group comparison was performed with log-rank tests and Cox proportional hazards methods. Results While the overall incidence of these mutations was nearly identical (55% never smokers vs. 57% current/former smokers, p=0.48), there were significant differences in the distribution of mutations between these groups: EGFR mutations- 37% never smokers vs. 14% former/current smokers (p<0.0001); KRAS mutations- 4% never smokers vs. 43% former/current smokers (p<0.0001); ALK rearrangements- 12% never smokers vs. 2% former/current smokers (p<0.0001). Among never smokers and former/current smokers, prognosis differed significantly by genotype. Patients harboring KRAS mutations demonstrated the poorest survival. Smoking status, however, had no influence on survival within each genotype. Conclusion Never smokers and former/current smokers with lung adenocarcinomas are not homogeneous subgroups. Each is made up of individuals whose tumors have a unique distribution of driver mutations which are associated with different prognoses, irrespective of smoking history.