Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

INTRODUCTION: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. MATERIALS AND METHODS: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. RESULTS: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. CONCLUSION: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

The current state of digital cytology and artificial intelligence (AI): global survey results from the American Society of Cytopathology Digital Cytology Task Force.

INTRODUCTION: The integration of whole slide imaging (WSI) and artificial intelligence (AI) with digital cytology has been growing gradually. Therefore, there is a need to evaluate the current state of digital cytology. This study aimed to determine the current landscape of digital cytology via a survey conducted as part of the American Society of Cytopathology (ASC) Digital Cytology White Paper Task Force. MATERIALS AND METHODS: A survey with 43 questions pertaining to the current practices and experiences of WSI and AI in both surgical pathology and cytology was created. The survey was sent to members of the ASC, the International Academy of Cytology (IAC), and the Papanicolaou Society of Cytopathology (PSC). Responses were recorded and analyzed. RESULTS: In total, 327 individuals participated in the survey, spanning a diverse array of practice settings, roles, and experiences around the globe. The majority of responses indicated there was routine scanning of surgical pathology slides (n = 134; 61%) with fewer respondents scanning cytology slides (n = 150; 46%). The primary challenge for surgical WSI is the need for faster scanning and cost minimization, whereas image quality is the top issue for cytology WSI. AI tools are not widely utilized, with only 16% of participants using AI for surgical pathology samples and 13% for cytology practice. CONCLUSIONS: Utilization of digital pathology is limited in cytology laboratories as compared to surgical pathology. However, as more laboratories are willing to implement digital cytology in the near future, the establishment of practical clinical guidelines is needed.

Digital cytology part 1: digital cytology implementation for practice: a concept paper with review and recommendations from the American Society of Cytopathology Digital Cytology Task Force.

Digital cytology and artificial intelligence (AI) are gaining greater adoption in the cytopathology laboratory. However, peer-reviewed real-world data and literature are lacking regarding the current clinical landscape. The American Society of Cytopathology in conjunction with the International Academy of Cytology and the Digital Pathology Association established a special task force comprising 20 members with expertise and/or interest in digital cytology. The aim of the group was to investigate the feasibility of incorporating digital cytology, specifically cytology whole slide scanning and AI applications, into the workflow of the laboratory. In turn, the impact on cytopathologists, cytologists (cytotechnologists), and cytology departments were also assessed. The task force reviewed existing literature on digital cytology, conducted a worldwide survey, and held a virtual roundtable discussion on digital cytology and AI with multiple industry corporate representatives. This white paper, presented in 2 parts, summarizes the current state of digital cytology and AI practice in global cytology practice. Part 1 of the white paper presented herein is a review and offers best practice recommendations for incorporating digital cytology into practice. Part 2 of the white paper provides a comprehensive review of AI in cytology practice along with best practice recommendations and legal considerations. Additionally, the results of a global survey regarding digital cytology are highlighted.

Digital cytology part 2: artificial intelligence in cytology: a concept paper with review and recommendations from the American Society of Cytopathology Digital Cytology Task Force.

Digital cytology and artificial intelligence (AI) are gaining greater adoption in the cytology laboratory. However, peer-reviewed real-world data and literature are lacking in regard to the current clinical landscape. The American Society of Cytopathology in conjunction with the International Academy of Cytology and the Digital Pathology Association established a special task force comprising 20 members with expertise and/or interest in digital cytology. The aim of the group was to investigate the feasibility of incorporating digital cytology, specifically cytology whole slide scanning and AI applications, into the workflow of the laboratory. In turn, the impact on cytopathologists, cytologists (cytotechnologists), and cytology departments were also assessed. The task force reviewed existing literature on digital cytology, conducted a worldwide survey, and held a virtual roundtable discussion on digital cytology and AI with multiple industry corporate representatives. This white paper, presented in 2 parts, summarizes the current state of digital cytology and AI practice in global cytology practice. Part 1 of the white paper is presented as a separate paper which details a review and best practice recommendations for incorporating digital cytology into practice. Part 2 of the white paper presented here provides a comprehensive review of AI in cytology practice along with best practice recommendations and legal considerations. Additionally, the cytology global survey results highlighting current AI practices by various laboratories, as well as current attitudes, are reported.

Distinct profile of driver mutations and clinical features in immunomarker-defined subsets of pulmonary large-cell carcinoma.

Pulmonary large-cell carcinoma-a diagnostically and clinically controversial entity-is defined as a non-small-cell carcinoma lacking morphologic differentiation of either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large-cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large-cell carcinomas by immunohistochemistry for TTF-1 and ΔNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large-cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%) or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large-cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large-cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.

Comparison of multiplanar reformatted CT lung tumor measurements to axial tumor measurement alone: impact on maximal tumor dimension and T stage.

OBJECTIVE: The purpose of this study was to compare measurements of lung tumor size between axial and multiplanar reformatted CT images, as well as to establish whether the difference between these measurements leads to a change in T stage. MATERIALS AND METHODS: Patients with lung tumors who underwent chest CT up to 31 days before lung resection between December 2010 and March 2012 were included. Axial, sagittal, and coronal CT images were evaluated by two independent readers (1 and 2) who were blinded to clinical data. In 89 patients, lung tumors categorized as T1a (54%), T1b (19%), T2a (24%), or T2b (3%) were analyzed. The longest tumor diameter using multiplanar reformatted CT was compared and correlated with axial CT alone and pathologic T stage. Statistical analysis included a Wilcoxon rank sum test to evaluate differences between measurements, intraclass correlation coefficient (ICC), and kappa statistic to assess agreement. RESULTS: Prediction of T stage using axial CT alone compared with multiplanar reformatted CT agreed in 82% of patients for reader 1 (κ = 0.660 [95% CI, 0.531-0.789]) and 80% of patients for reader 2 (κ = 0.695 [95% CI, 0.572-0.818]). Prediction of T stage using multiplanar reformatted CT resulted in upstaging in 18% and 20% of patients (for readers 1 and 2, respectively). Interobserver agreement (ICC [95% CI]) was 0.900 (0.803-0.954) for axial, 0.874 (0.772-0.946) for sagittal, and 0.754 (0.556-0.921) for coronal planes. CONCLUSION: Radiologic measurement of lung tumor T stage was higher using multiplanar reformatted CT as compared with axial CT alone. When available, multiplanar reformatted CT should be used to measure tumor dimension and thus assign an accurate lung cancer T stage.

Melanotic medullary thyroid carcinoma: A case report with review of the literature.

Melanotic medullary thyroid carcinoma is morphologically defined by the presence of melanin deposits in the cytoplasm of tumor cells. It is an extremely rare variant with only 15 cases described in the literature to date and only one report of diagnosis by fine needle aspiration (FNA) biopsy. A 51-year-old woman presented with neck swelling. An ultrasound examination revealed a single solid nodule in the right thyroid lobe that measured 5.4 × 4.7 × 4.3 cm. Laboratory examination revealed elevated levels of serum calcitonin (8643.0 pg/ml), carcinoembryonic antigen (CEA) (86.2 ng/ml), and chromogranin A (123.2 ng/ml). An FNA biopsy of the thyroid nodule revealed predominantly single plasmacytoid cells with round to oval eccentric nuclei and dark brown intracytoplasmic granules. Immunohistochemical studies with Melan-A performed on a cell block slide confirmed that the granules contained melanin. The tumor cells were also positive for calcitonin, CEA, synaptophysin, AE1/AE3, CAM5.2, and HMB-45(focal); the tumor cells were negative for chromogranin, thyroglobulin, PAX8 and TTF-1. The diagnosis was reported as melanotic variant of medullary thyroid carcinoma. The patient underwent a total thyroidectomy which revealed tumor cell expression of insulinoma-associated protein 1 and confirmed neuroendocrine differentiation. Shortly after she presented with tumor recurrence in the thyroidectomy bed. The tumor cells were positive for only S100, SOX10, and Melan-A. Molecular analysis with the SEMA4 Solid Tumor Panel revealed mutations in the HRAS, PIK3CA, PIK3R1, MYC, and CCND3 genes. The final diagnosis was reported as melanocytic medullary thyroid carcinoma with high grade transformation and loss of epithelial and neuroendocrine expression.

The World Health Organization Reporting System for Lung Cytopathology.

The International Academy of Cytology has joined with the International Agency for Research on Cancer (IARC) to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This WHO System defines five categories for reporting lung cytopathology, that is, "Insufficient"/"Inadequate"/"Non-diagnostic," "Benign," "Atypical," "Suspicious for malignancy," and "Malignant," each with a clear descriptive term for the category, a definition, a risk of malignancy and a suggested management algorithm. The key diagnostic cytopathology features of each of the lesions within each category have been established by consensus and will be presented more fully in a subsequent IARC e-book and published hard cover book.The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and provides a review to guide sampling and processing techniques to optimize the handling and preparation of the cytopathology sample emphasizing the cytomorphological differential diagnosis to aid low-resourced settings. The authors recognize that local medical and pathology resources will vary, particularly in low- and middle-income countries, and have developed the WHO System to make it applicable worldwide based on cytomorphology with options for further diagnostic management of the patient.The online WHO System provides a direct link to the WHO Tumour Classification for Thoracic Tumours 5th Edition. It will raise the profile and use of cytopathology by increasing awareness of its current role and its potential role in the era of personalized medicine based on molecular pathology utilizing "small biopsies." Ultimately, the System will improve patient care and outcomes.This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians and improve patient care. The System is based on the current role of lung cytopathology and synthesizes the existing evidence while highlighting areas requiring further research and the future potential role of lung cytopathology.

A brief review of the WHO reporting system for lung cytopathology.

The International Academy of Cytology has joined with the International Agency for Research on Cancer to bring together a group of experts in lung cytopathology to develop a WHO Reporting System for Lung Cytopathology (WHO System). This System aims to improve and standardize the reporting of cytopathology, facilitate communication between cytopathologists and clinicians, and improve patient care. The WHO System describes 5 categories for reporting lung cytopathology: 'Insufficient/Inadequate/Nondiagnostic', 'Benign', 'Atypical', 'Suspicious for malignancy', and 'Malignant', each one with a clear descriptive term, a definition, a risk of malignancy, and a suggested management algorithm. The key diagnostic cytopathologic features of each of the lesions within each category have been established by consensus through an Expert Editorial Board, who are also the authors of this review and selected for each reporting system and chosen based on their expertise in the field and/or diversity of geographical representation. Many other co-authors from around the world also contributed. The assignment of writing and editing responsibilities used the same model as that used for the WHO Classification of Tumours (https://whobluebooks.iarc.fr/about/faq/). The WHO System provides the best practice application of ancillary testing, including immunocytochemistry and molecular pathology, and guides in sampling and processing techniques to optimize the handling and preparation of specimens. The WHO System was created by the authors to be applicable globally and is based on cytomorphology with possibilities for additional diagnostic management of the patient. The authors are aware that local medical and pathology resources would differ, especially in low- and middle-income countries. The WHO Tumour Classification for Thoracic Tumors, Fifth Edition, is directly accessible through the online WHO System.

Reproducibility of histopathological subtypes and invasion in pulmonary adenocarcinoma. An international interobserver study.

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.

A comparative study of the genotype profiles of high-risk human papillomavirus infection in male and female HIV-positive patients and their correlation with anal cytology and biopsy.

INTRODUCTION: Although anal cancer is more common in women, most of the studies on the role of high-risk human papillomavirus (hrHPV) infection in anal squamous lesions have focused on high-risk male patients. Therefore, we compared the genotype profile and clinicopathologic correlation of hrHPV infection in human immunodeficiency virus-positive (HIV+) men and women. MATERIALS AND METHODS: We retrospectively analyzed 2254 HIV+ patients (1931 men and 323 women) who had undergone anal Papanicolaou tests at our institution; 1189 of them also had follow-up biopsy data available. HPV genotyping was performed using the Roche Cobas system and correlated with the cytologic and histologic diagnosis. RESULTS: Compared with the HIV+ men, the HIV+ women had a significantly lower rate of hrHPV infection (67.5% versus 78.5%; P < 0.0001) but a significantly higher rate of high-grade squamous intraepithelial lesions (HSILs) on anal Papanicolaou tests (4.6% versus 2.5%; P < 0.05). Other high-risk HPV (ohrHPV), as a group, is much more common than HPV16 or HPV18 in both genders. HIV+ women had significantly lower HPV16 and ohrHPV infection rates than did HIV+ men. However, the HPV18 infection rates were similar between HIV+ women and HIV+ men. For both genders, the rates of HSILs or high-grade anal intraepithelial neoplasia (AIN2-3) were significantly increased when coinfection of ohrHPV with either HPV16 or HPV18 was present. CONCLUSIONS: Although both HIV+ men and HIV+ women have an increased risk of hrHPV infection, HIV+ women have different hrHPV genotype profiles and higher rates of high-grade lesions. Coinfection with different genotypes of hrHPV can significantly increase the risk of HSILs or AIN2-3 in both genders and could requires vigilant clinical and laboratory follow-up.

Novel handheld PET probes provide intraoperative localization of PET-avid lymph nodes.

INTRODUCTION: The accurate intraoperative localization of malignant nodes can pose a challenge to the surgical oncologist. Positron emission tomography (PET) scanning has significantly increased our ability to detect suspicious lesions. We investigated the ability of a novel, handheld tool to evaluate suspicious nodes intraoperatively and to correlate its findings with those seen on preoperative PET scan. METHODS: Ten nude rats were inoculated with a lymphogenic mesothelioma tumor line and followed weekly with PET scan studies. When suspicious lymph nodes were found, animals were dissected and the intraoperative amount of tissue radiation was analyzed as "tumor-to-background ratio" (TBR) using the PET probes. RESULTS: The intraoperative probe was used to guide dissections and select high-risk nodes based on their specific radiotracer uptake. A total of 52 nodes were harvested; eight of these were suspicious on preoperative PET scan studies. Using a TBR of 2.5, the intraoperative probes were able to localize all suspicious nodes previously seen on PET scan. Both gamma (sensitivity: 100%; specificity: 86%; positive predictive value (PPV): 57%; negative predictive value (NPV): 100%) and beta (sensitivity: 88%; specificity: 91%; PPV: 64%; NPV: 98%) probes showed an excellent area under the curve (AUC) in the receiver operating characteristic analysis (ROC). Both probes had an AUC of 0.95 for localizing suspicious nodes on PET scan. Furthermore, the AUC for detecting malignancy for the gamma probe was 0.90 (95% confidence interval (CI), 0.83-0.99), and for the beta probe it was 0.97 (95% CI, 0.94-1.0), suggesting a better performance of the beta probe for detecting malignancy. CONCLUSIONS: This novel tool may be used synergistically with the PET scan examination to maximize intraoperative nodal selection and sampling.

Core needle lung biopsy specimens: adequacy for EGFR and KRAS mutational analysis.

OBJECTIVE: The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung. SUBJECTS AND METHODS: The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens. RESULTS: Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy. CONCLUSION: Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients.

Building a smart FNA cart: When Google meets cytology.

BACKGROUND: The appropriate management of a fine needle aspiration (FNA) supply cart and equipment set up is essential to ensure the smooth and optimal operation of a busy FNA clinic. We applied Lean strategies such as value stream mapping (VSM), the 5S method (Sort, Set in order, Shine, Standardize, Sustain), and Kanban to remove waste and improve patient flow in an FNA clinic. METHODS: The workflow analysis suggested that existent problems such as suboptimal inventory management and unavailability of standard operating procedures (SOPs) caused a 10% to 85% increase in total procedure time. To improve inventory management, we created a 2-bin Kanban system. We used the "Scan to Web" app and a Google Drive form to create a cost-effective electronic inventory management system. We distributed the essential SOPs in the format of video clips using our YouTube channel and leveraged barcode technology to access the links. RESULTS: Upon completion of our process improvement project, we succeeded to eliminate the stock-out events and maintain a process cycle efficiency of 87%. The 5S audit checklist result increased from 6% to 100% implementation, which is consistent with focused improvement. The developed inventory system enabled us to track the supply usage, forecast demands, and improve the accuracy of orders. CONCLUSIONS: Lean methods such as VSM, 5S, and Kanban combined with open source technologies can be implemented to ensure material availability, track inventory, and provide immediate access to SOPs on demand. The developed system also led to increased efficiency and improved flow, as well as responsiveness to changes in demand.

Cytological features of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP).

INTRODUCTION: The noninvasive encapsulated follicular variant of papillary carcinoma (EFVPC) was recently renamed a noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) because of its unique genetic alterations and biological behavior. The objective of this report is to help cytopathologists and cytotechnologists improve diagnostic accuracy and determine the need for cytogenetic studies during adequacy evaluation of thyroid fine-needle aspirations. MATERIALS AND METHODS: Fifty-five cases of surgery-proven noninvasive EFVPC with corresponding cytology material were reviewed. These cases were collected over 17 years, from 1999 to 2016. RESULTS: Thirty-four of 55 (61.8%) cases were diagnosed as follicular neoplasm or suspicious for follicular neoplasm on cytology. Eighty to ninety percent of cases showed scant colloid, cellular smears with small clusters of follicular cells with nuclear atypia including enlarged nuclei, oval-shaped nuclei, nuclear grooves, mild chromatin powdering, and rare nuclear pseudo-inclusions. CONCLUSIONS: NIFTP has unique features: cytologically similar to follicular neoplasms, and nuclear atypia falling between atypia of undetermined significance (category III) and suspicious for/and papillary thyroid carcinoma (category V/VI) (The Bethesda System for Reporting Thyroid Cytopathology).

EGFR mutations in lung adenocarcinomas: clinical testing experience and relationship to EGFR gene copy number and immunohistochemical expression.

Lung adenocarcinomas responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors possess EGFR mutations and often increased EGFR copy number. We prospectively studied 334 clinical cases using polymerase chain reaction-based assays to detect deletions within exon 19 and the L858R mutation in exon 21, which together account for approximately 90% of EGFR mutations. Seventy-eight (23%) of these tumors had an EGFR mutation, with 55 (71%) exon 19 deletions and 23 (29%) exon 21 L858R mutations. We were able to compare mutant and normal EGFR alleles and found a preferential amplification of the mutant allele. The association of mutations with EGFR amplification (determined by chromogenic in situ hybridization) and EGFR expression (determined by immunohistochemistry) was further examined in a subset of 60 tumors. EGFR amplification (> or =5 EGFR signals per nucleus) was seen in 15 of 29 (52%) EGFR-mutated tumors but in only five of 31 (6%) non-mutated tumors (P = 0.006). EGFR overexpression was strongly associated with amplification but was statistically independent of EGFR mutation. Most patients with EGFR mutations (17 of 29, 59%) never smoked compared with 13% (four of 31) of patients lacking such mutations (P = 0.0003). The association of amplification with smoking status was marginal and was nonexistent with EGFR expression. Thus, these results indicate that EGFR amplification, preferentially of the mutant allele, often accompanies EGFR mutation, whereas EGFR immunohistochemical staining associates with amplification but cannot predict EGFR mutation status.

Global gene expression profiling of pleural mesotheliomas: overexpression of aurora kinases and P16/CDKN2A deletion as prognostic factors and critical evaluation of microarray-based prognostic prediction.

Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated.

Pulmonary toxicity associated with erlotinib.

Interstitial lung disease (ILD) related to therapy with the drug gefitinib has been well reported. The adverse pulmonary effects of erlotinib are less well known. We report a case of fatal pulmonary toxicity in a patient with advanced non-small cell lung cancer who received erlotinib. He had been found to have pathologic findings of usual interstitial pneumonia (UIP) on the resected lung cancer specimen prior to receiving erlotinib. This case and other published evidence should alert physicians to the possibility of fatal erlotinib-induced ILD. Similar to reports in patients receiving gefitinib, those with pathologic findings of UIP on resected lung specimens or known pulmonary fibrosis may be at particular risk for erlotinib pulmonary toxicity.

Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib.

PURPOSE: In patients with non-small cell lung cancer (NSCLC), mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain have been associated with sensitivity to erlotinib and gefitinib. We undertook this study to explore the relationship between EGFR mutation type and clinical variables, including treatment with gefitinib and erlotinib. EXPERIMENTAL DESIGN: In patients with NSCLC, EGFR exon 19 deletion mutations and EGFR L858R point mutations were analyzed by nonsequencing PCR-based methods from paraffin blocks of tissue obtained before treatment. The results were correlated with clinical information (sex, pathologic subtype, race/ethnicity, treatment, and overall survival). RESULTS: The two most common EGFR mutations were identified in 24% (70 of 291; 95% confidence interval, 26%-38%) of tumors from patients with NSCLC. EGFR mutation was associated with Asian ethnicity (P = 0.0023) and being a "never smoker" (P = 0.0001). Among patients with EGFR mutations, 39% (27 of 70) had EGFR L858R, whereas 61% (43 of 70) had an EGFR exon 19 deletion. After treatment with erlotinib (n = 12) or gefitinib (n = 22), patients with EGFR mutations had a median overall survival of 20 months. After treatment with erlotinib or gefitinib, patients with EGFR exon 19 deletions had significantly longer median survival than patients with EGFR L858R (34 versus 8 months; log-rank P = 0.01). CONCLUSIONS: EGFR mutations in exons 19 or 21 are correlated with clinical factors predictive of response to gefitinib and erlotinib. Those with EGFR exon 19 deletion mutations had a longer median survival than patients with EGFR L858R point mutation. These observations warrant confirmation in a prospective study and exploration of the biological mechanisms of the differences between the two major EGFR mutations.

Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.

PURPOSE: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy. EXPERIMENTAL DESIGN: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses. RESULTS: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion. CONCLUSIONS: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.