Faculty Bibliography

PURPOSE: There is persistent controversy as to whether EGFR and KRAS mutations occur in pulmonary squamous cell carcinoma (SQCC). We hypothesized that the reported variability may reflect difficulties in the pathologic distinction of true SQCC from adenosquamous carcinoma (AD-SQC) and poorly differentiated adenocarcinoma due to incomplete sampling or morphologic overlap. The recent development of a robust immunohistochemical approach for distinguishing squamous versus glandular differentiation provides an opportunity to reassess EGFR/KRAS and other targetable kinase mutation frequencies in a pathologically homogeneous series of SQCC. EXPERIMENTAL DESIGN: Ninety-five resected SQCCs, verified by immunohistochemistry as DeltaNp63(+)/TTF-1(-), were tested for activating mutations in EGFR, KRAS, BRAF, PIK3CA, NRAS, AKT1, ERBB2/HER2, and MAP2K1/MEK1. In addition, all tissue samples from rare patients with the diagnosis of EGFR/KRAS-mutant "SQCC" encountered during 5 years of routine clinical genotyping were reassessed pathologically. RESULTS: The screen of 95 biomarker-verified SQCCs revealed no EGFR/KRAS [0%; 95% confidence interval (CI), 0%-3.8%], four PIK3CA (4%; 95% CI, 1%-10%), and one AKT1 (1%; 95% CI, 0%-5.7%) mutations. Detailed morphologic and immunohistochemical reevaluation of EGFR/KRAS-mutant "SQCC" identified during clinical genotyping (n = 16) resulted in reclassification of 10 (63%) cases as AD-SQC and five (31%) cases as poorly differentiated adenocarcinoma morphologically mimicking SQCC (i.e., adenocarcinoma with "squamoid" morphology). One (6%) case had no follow-up. CONCLUSIONS: Our findings suggest that EGFR/KRAS mutations do not occur in pure pulmonary SQCC, and occasional detection of these mutations in samples diagnosed as "SQCC" is due to challenges with the diagnosis of AD-SQC and adenocarcinoma, which can be largely resolved by comprehensive pathologic assessment incorporating immunohistochemical biomarkers.

Histological subtyping of pulmonary adenocarcinoma has recently been updated based on predominant pattern, but data on reproducibility are required for validation. This study first assesses reproducibility in subtyping adenocarcinomas and then assesses further the distinction between invasive and non-invasive (wholly lepidic) pattern of adenocarcinoma, among an international group of pulmonary pathologists. Two ring studies were performed using a micro-photographic image-based method, evaluating selected images of lung adenocarcinoma histologic patterns. In the first study, 26 pathologists reviewed representative images of typical and 'difficult' histologic patterns. A total number of scores for the typical patterns combined (n=94) and the difficult cases (n=21) were 2444 and 546, respectively. The mean kappa score (+/-s.d.) for the five typical patterns combined and for difficult cases were 0.77+/-0.07 and 0.38+/-0.14, respectively. Although 70% of the observers identified 12-65% of typical images as single pattern, highest for solid and least for micropapillary, recognizing the predominant pattern was achieved in 92-100%, of the images except for micropapillary pattern (62%). For the second study on invasion, identified as a key problem area from the first study, 28 pathologists submitted and reviewed 64 images representing typical as well as 'difficult' examples. The kappa for typical and difficult cases was 0.55+/-0.06 and 0.08+/-0.02, respectively, with consistent subdivision by the same pathologists into invasive and non-invasive categories, due to differing interpretation of terminology defining invasion. In pulmonary adenocarcinomas with classic morphology, which comprise the majority of cases, there is good reproducibility in identifying a predominant pattern and fair reproducibility distinguishing invasive from in-situ (wholly lepidic) patterns. However, more precise definitions and better education on interpretation of existing terminology are required to improve recognition of purely in-situ disease, this being an area of increasing importance.

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 minimal-marker combination, is not firmly established. We therefore studied whole-tissue sections of resected adenocarcinoma and squamous cell carcinoma (n=315) with markers commonly used to identify adenocarcinoma (TTF-1) and squamous cell carcinoma (p63, CK5/6, 34betaE12), and prospectively validated the devised algorithm in morphologically unclassifiable small biopsy/cytology specimens (n=38). Analysis of whole-tissue sections showed that squamous cell carcinoma had a highly consistent immunoprofile (TTF-1-negative and p63/CK5/6/34betaE12-diffuse) with only rare variation. In contrast, adenocarcinoma showed significant immunoheterogenetity for all 'squamous markers' (p63 (32%), CK5/6 (18%), 34betaE12 (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.

New developments in the field of thoracic oncology have challenged the way pathologists approach the diagnosis of pulmonary carcinoma. Nonsmall cell carcinoma is no longer an adequate diagnostic category. Pathologists are required to further classify tumors into adenocarcinoma and squamous cell carcinoma since specific therapies are now recommended depending on the histological tumor type. This change occurred following the discovery of specific molecular alterations that predict response to certain drugs and now molecular testing of tumor cells is often requested to direct therapy. The vast majority of lung cancer is diagnosed in advanced clinical stages, where cytologic or small biopsy material is the only form of tissue diagnosis, thus placing cytology, especially fine needle aspiration biopsy in the front line for management of patients with lung cancer. In this paper we will review the current concepts in the suitability and accuracy of fine needle aspiration biopsy, including diagnosis, classification, prognostic markers, and use of ancillary techniques.

BACKGROUND: There is growing evidence that lung adenocarcinoma and squamous cell carcinoma (SQCC) have distinct oncogenic mutations and divergent therapeutic responses, which is driving the heightened emphasis on accurate pathologic subtyping of non-small cell lung carcinoma (NSCLC). The relative feasibility and accuracy of NSCLC subtyping by small biopsy versus cytology is not well established, particularly in current practice where immunohistochemistry (IHC) is becoming routinely used to aid in this distinction. METHODS: Concurrent biopsy and cytology specimens obtained during a single procedure and diagnosed as NSCLC during a 2-year period (n = 101) were reviewed. Concordance of diagnoses in the two methods was assessed. Accuracy was determined based on subsequent resection or autopsy diagnosis (n = 21) or IHC for thyroid transcription factor 1 and p63 on a subset of cases (n = 43). RESULTS: The distribution of definitive versus favored versus unclassified categories (reflecting the degree of diagnostic certainty) was similar for biopsy (71% versus 23% versus 6%, respectively) and cytology (69% versus 19% versus 12%, respectively), p = 0.29. When results from paired specimens were combined, the rate of definitive diagnoses by at least one method was increased to 84% and the unclassified rate was decreased to 4%. NSCLC subtype concordance between biopsy and cytology was 93%. Kappa coefficient (95% confidence interval) for agreement between methods was 0.88 (0.60-0.89) for adenocarcinoma and 0.76 (0.63-0.89) for SQCC. In pairs with discordant diagnoses (n = 7) the correct tumor type was identified with a similar frequency by biopsy (n = 4) and cytology (n = 3). Factors contributing to mistyping were poor differentiation, necrosis, low cellularity, and lack of supporting IHC. All concordant diagnoses for which verification was available (n = 57) were correct. IHC was used more frequently to subtype NSCLC in biopsy than cytology (32% versus 6%; p = 0.0001). CONCLUSIONS: Small biopsy and cytology achieve comparable rates of definitive and accurate NSCLC subtyping, and the optimal results are attained when the two modalities are considered jointly. The lower requirement for IHC in cytology highlights the strength of cytomorphology in NSCLC subtyping. Whenever clinically feasible, obtaining parallel biopsy and cytology specimens is encouraged.

INTRODUCTION: The two essential requirements for pathologic specimens in the era of personalized therapies for non-small cell lung carcinoma (NSCLC) are accurate subtyping as adenocarcinoma (ADC) versus squamous cell carcinoma (SqCC) and suitability for EGFR and KRAS molecular testing. The aim of this study was to comprehensively review the performance of cytologic specimens for the above two goals in a high-volume clinical practice. METHODS: Subtyping of primary lung carcinomas by preoperative cytology was correlated with subsequent resection diagnoses during a 1-year period (n = 192). The contribution of various clinicopathologic parameters to subtyping accuracy and utilization of immunohistochemistry (IHC) for NSCLC subtyping were analyzed. In addition, the performance of cytologic specimens submitted for EGFR/KRAS molecular testing during a 1-year period (n = 128) was reviewed. RESULTS: Of the 192 preoperative cytology diagnoses, tumor subtype was definitive versus favored versus unclassified in 169 (88%) versus 15 (8%) versus 8 (4%) cases, respectively. Overall accuracy of cytologic tumor subtyping (concordance with histology) was 93% and accuracy of definitive diagnoses 96%. For a group of patients with ADC and SqCC (n = 165), the rate of unclassified cytologic diagnoses was 3% and overall accuracy 96%. IHC was used for subtyping of 9% of those cases, yielding 100% accuracy. The strongest predictors of difficulty in subtyping of ADC and SqCC were poor differentiation (p = 0.0004), low specimen cellularity (p = 0.019), and squamous histology (p = 0.003). Of 128 cytologic specimens submitted for molecular testing, 126 (98%) were suitable for analysis, revealing EGFR and KRAS mutations in 31 (25%) and 25 (20%) cases, respectively. CONCLUSIONS: Cytologic subtyping of NSCLC is feasible and accurate, particularly when morphologic assessment is combined with IHC. Furthermore, routine cytologic specimens can be successfully used for EGFR/KRAS mutation analysis. Our data strongly support the suitability of cytologic specimens for the new therapeutic paradigms in NSCLC.

OBJECTIVE: : To describe pathologic findings in symptomatic World Trade Center-exposed local workers, residents, and cleanup workers enrolled in a treatment program. METHODS: : Twelve patients underwent surgical lung biopsy for suspected interstitial lung disease (group 1, n = 6) or abnormal pulmonary function tests (group 2, n = 6). High-resolution computed axial tomography and pathologic findings were coded. Scanning electron microscopy with energy-dispersive x-ray spectroscopy was performed. RESULTS: : High-resolution computed axial tomography showed reticular findings (group 1) or normal or airway-related findings (group 2). Pulmonary function tests were predominantly restrictive. Interstitial fibrosis, emphysematous change, and small airway abnormalities were seen. All cases had opaque and birefringent particles within macrophages, and examined particles contained silica, aluminum silicates, titanium dioxide, talc, and metals. CONCLUSIONS: : In symptomatic World Trade Center-exposed individuals, pathologic findings suggest a common exposure resulting in alveolar loss and a diverse response to injury

BACKGROUND: Because associations have been reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled engineered nanoparticles (NPs) may also induce adverse effects on the cardiovascular system. OBJECTIVE: We examined the long-term cardiovascular effects of inhaled nickel hydroxide NPs (nano-NH) using a sensitive mouse model. METHODS: Hyperlipidemic, apoprotein E-deficient (ApoE-/-) mice were exposed to nano-NH at either 0 or 79 mug Ni/m3, via a whole-body inhalation system, for 5 hr/day, 5 days/week, for either 1 week or 5 months. We measured various indicators of oxidative stress and inflammation in the lung and cardiovascular tissue, and we determined plaque formation on the ascending aorta. RESULTS: Inhaled nano-NH induced significant oxidative stress and inflammation in the pulmonary and extrapulmonary organs, indicated by up-regulated mRNA levels of certain antioxidant enzyme and inflammatory cytokine genes; increased mitochondrial DNA damage in the aorta; significant signs of inflammation in bronchoalveolar lavage fluid; changes in lung histopathology; and induction of acute-phase response. In addition, after 5-month exposures, nano-NH exacerbated the progression of atherosclerosis in ApoE-/- mice. CONCLUSIONS: This is the first study to report long-term cardiovascular toxicity of an inhaled nanomaterial. Our results clearly demonstrate that long-term exposure to inhaled nano-NH can induce oxidative stress and inflammation, not only in the lung but also in the cardiovascular system, and that this stress and inflammation can ultimately contribute to progression of atherosclerosis in ApoE-/- mice

The most recent WHO classification of lung cancer defines bronchioloalveolar carcinoma (BAC) as a noninvasive carcinoma or adenocarcinoma in situ. However, the use of this terminology is not uniform and does not reflect standardized criteria. As a result, the diagnosis of BAC has been used in association with small, solitary, and well-differentiated adenocarcinoma as well as tumors with advanced clinical stage. At present, there is a growing consensus among specialists in thoracic oncology that BAC or adenocarcinoma in situ is a rare tumor, and the term should be restricted to adenocarcinomas that show a pure lepidic pattern of growth. The amount of invasive component present in a tumor with a predominant lepidic growth pattern has also been under intense scrutiny. The concept of minimally invasive adenocarcinoma is developing in order to differentiate a pure BAC from an invasive adenocarcinoma that still carries an excellent prognosis.