Presented here are the Scientific Abstracts from the Biennial Pulmonary Pathology Society (PPS) Meeting, which took place in June 2013 in Grenoble, France. This meeting of pulmonary specialists from around the world was assembled under the direction and leadership of Elisabeth Brambilla, MD, then president of the PPS. All abstracts were reviewed for scientific content by an abstract review committee prior to their acceptance.

ALK Gene Rearrangement Assessment Comparing Ultrasensitive Immunohistochemistry and Fluorescent In Situ Hybridization Break-Apart Probe Set in 263 Lung Adenocarcinomas

N. Akyürek1 (; P. Bulutay1; Ö. Ekinci1; L. Memiş1; A. S. Yurdakul2; M. Benekli3; I. Taştepe.4 Departments of 1Pathology, 2Pulmonary Medicine, 3Medical Oncology, and 4Thoracic Surgery, Gazi University Medical Faculty, Ankara, Turkey.

Context: Rearrangement of the ALK gene occurs in a subset of lung carcinomas and predicts response to a small-molecule inhibitor of ALK therapy (Crizotinib). Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed, paraffin-embedded tissue for ALK inversions and translocations. The purpose of this study is to evaluate the IHC stain with FISH analysis in the detection of the ALK rearrangement.

Design: Tissue microarrays were constructed from a total of 263 cases of lung adenocarcinoma, including 208 lung resection specimens and 55 biopsies of confirmed extrapulmonary metastases. ALK genetic status was determined by FISH using a break-apart probe set (Vysis, Abbott Molecular). Immunohistochemistry for ALK expression was performed wıth the D5F3 rabbit monoclonal antibody and ultrasensitive OptiView DAB IHC Detection Kit with amplification (Ventana anti-ALK [D5F3] IHC Assay).

Results: Of the 263 lung adenocarcinoma cases, 8 (3.5%) were ALK FISH positive. Of the 8 FISH-positive cases, 7 cases were positive for IHC, whereas 1 case was negative for IHC. Of the FISH-positive cases, male to female ratio was 5:4, and average age was 58.2 years (range, 48–81 years). A predominant solid/cribriform pattern was observed in 5 of the 9 cases (55.5%), and a predominant acinar pattern in 4 of the cases (44.5%). More than half of the cases (5 of 9) showed signet ring cells. No significant relationship was found between ALK rearrangement and patient sex, smoking history, and stage.

Conclusions: Our results demonstrate some discrepancies between ALK testing by FISH and IHC. Currently, FISH appears to be the gold standard for the detection of the ALK rearrangement.

Caprin1 Overexpression in Non–Small Cell Lung Carcinomas

Z. Cetin1; I. H. Ozbudak2 (; S. B. Karauzum1; M. Ozdogan3; G. Ozbilim.2 Departments of 1Medical Biology, 2Pathology, and 3Medical Oncology, School of Medicine, Akdeniz University, Antalya, Turkey.

Context: Caprin1, encoded by the cytoplasmic activation/proliferation-associated protein-1 gene located in the 11p13 chromosome region, has been associated with cell proliferation. The aim of this study was to assess the expression of Caprin1 in non–small cell lung carcinomas.

Design: Caprin1 expression was evaluated by immunohistochemistry in 41 adenocarcinomas (ACs) and 56 squamous cell carcinomas (SCCs). Caprin1 gene copy alterations were analyzed by specific FISH probe in Caprin1-overexpressed cases.

Results: Caprin1 overexpression was observed in 24.39% of ACs and in 26.78% of SCCs. In poorly differentiated SCCs, it was detected in 75% of cases, whereas in well- and moderately-differentiated SCCs it was found in 33.3% and 10%, respectively, of cases. Caprin1 overexpression was more frequent in AC cases with lymph node metastases than those without metastases (35.7% versus 18.5%). Also, in AC cases Caprin1 overexpression was associated with poor outcome, with a median survival time of 13 months in patients with overexpression compared with 22 months in those without overexpression (P = .048). In FISH analyses, among 7 cases of Caprin1-overexpressed SCC, 2 had Caprin1 gene amplification and 4 had polysomy of the chromosome 11. Among 5 cases of Caprin1-overexpressed AC, 4 had polysomy of the chromosome 11.

Conclusions: Caprin1 overexpression was associated with high-grade morphology in SCC and with the presence of lymph node metastasis and reduced survival time in AC. Elevated copy number of 11p13 locus was associated with Caprin1 overexpression in SCC and AC. However, further studies are needed to clarify the prognostic significance.

ALK Gene Copy Number Gains in Non–Small Cell Lung Cancers: Mayo Clinic Experience With 1854 Cases and Correlation with ALK Immunohistochemistry

S. Chang (; B. Larsen; J. Boland; W. Sukov; E. Yi. Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.

Context: In non–small cell lung cancer (NSCLC), the reported prevalence of increased ALK gene copy number in the absence of an ALK translocation is highly variable. Using a large number of cases at a single center, we sought to survey the prevalence of increased ALK copy number and examine its correlation with ALK protein expression.

Design: All cases of NSCLC referred to the Mayo Clinic for ALK rearrangement testing by fluorescence in situ hybridization (FISH) during 2011–2012 were reviewed. Cases with ALK copy number gains without ALK rearrangement were identified. From this subset with increased ALK copy number, ALK immunohistochemistry (IHC) was performed on all cases in which additional sections were available (clone ALK1, Dako, Carpenteria, California).

Results: Of 1854 cases with successful ALK FISH testing, 395 cases (21.3%) demonstrated increased ALK copy number (range, 3–15) without ALK rearrangement. From this subset, ALK IHC was performed on 216 cases and scored from 0 to 3+ as follows: 0 (n = 164; 75.9%), 1+ (n = 37; 17.1%), 2+ (n = 12; 5.6%), and 3+ (n = 3; 1.4%). Although IHC was positive in some cases, no correlation between the magnitude of ALK copy number gain and IHC staining intensity was detected.

Conclusions: ALK copy number gain is present in nearly one-quarter of NSCLC cases. Of these cases, only a small subset has immunohistochemically detectable ALK protein expression, which does not correlate with ALK copy number change, as opposed to the well-documented correlation of IHC results with ALK gene rearrangement status. Additional studies are warranted to determine the clinical significance of ALK copy number gains.

Adequacy of Computed Tomography–Guided Transthoracic Needle Biopsies for Histomolecular Subtyping of Pulmonary Adenocarcinomas: Influence of ATS/ERS/IASLC Guidelines

G. R. Ferretti1; B. Busser2; F. de Fraipont2; E. Reymond1; A. McLeer-Florin3; D. Moro-Sibilot5; A. Jankowski1; E. Brambilla4; S. Lantuejoul4 ( 1Clinique Universitaire de Radiologie et Imagerie Medicale, Centre Hospitalier Universitaire A Michallon, INSERM U 823–Institut A Bonniot–Université J Fourier, Grenoble, France; 2UM Biochimie des cancers et biotherapies, Departement de Biochimie, Toxicologie et de Pharmacologie, Pôle de Biologie et de Pathologie, Centre Hospitalier Universitaire A Michallon, INSERM U 823–Institut A Bonniot–Université J Fourier, Grenoble, France; 3Plateforme de Genetique moleculaire des cancers, Pôle de Biologie et de Pathologie, Centre Hospitalier Universitaire A Michallon–UMR-S 1036–CEA Grenoble–Université J Fourier, Grenoble, France; 4Departement de Pathologie, Pôle de Biologie et de Pathologie, Centre Hospitalier Universitaire A Michallon, INSERM U 823–Institut A Bonniot–Université J Fourier, Grenoble, France; 5Clinique Universitaire de Pneumologie, Pôle Oncologie, Medecine Aigue Communautaire, Centre Hospitalier Universitaire A Michallon, INSERM U 823–Institut A Bonniot–Universite J Fourier, Grenoble, France.

Context: Because metastatic pulmonary adenocarcinomas are investigated for EGFR, K-ras, and ALK mutations/rearrangement, adequacy of computed tomography (CT)–guided transthoracic needle biopsies (TTNBs) needs to be evaluated according to the 2011 ATS/ERS/IASLC guidelines for small specimen.

Design: Two series of 18G TTNBs were retrospectively compared regarding adequacy for histologic subtyping and EGFR/K-ras mutations and ALK rearrangement; the first series included 43 TTNBs collected from January 2010 to February 2011, and the second 48 collected from March 2011 to December 2012.

Results: The two series included 28 women and 63 men (mean age of 65 years); mean size of the lesions was 32.5 mm. Concordance between TTNB and surgical specimens (n = 16) for histology was always reached; by comparing the first to the second series, the number of biopsies increased from 1.6 to 1.85, and length increased from 10.9 to 12.5 mm, whereas the number of stains (TTF1, P63, CK5-6, mucin) per biopsy decreased from 2.6 to 1. Tumor cell percentage was 42% in both, but DNA extracted increased from 2.7 to 3.8 μg; in the first series, 95% of TTNBs were available for EGFR exons 19/21 and K-ras mutations using pyrosequencing, and 76% were available for further analyses, versus 98% and 94%, respectively, in the second series. In the first series, 9 K-ras mutations (20%), 2 EGFR exon 19 deletions (5%), and 1 ALK rearrangement (2%) were detected, versus 13 K-ras mutations (27%), 4 EGFR exon 19/21 mutations (8%), and 2 ALK translocations (4%) in the second series.

Conclusions: Radiologists, biologists, and pathologists have improved their practice since the 2011 ATS/ERS/IASLC guidelines; CT-guided TTNBs enable histologic subtyping and provide a sufficient amount of DNA for genetic analyses.

Application of Web-Based Software to Assist in the Diagnosis of Interstitial Pneumonia

J. Fukuoka1 (; K. Tabata1; T. Tanaka1; Y. Kashima1; M. Kigawa.21Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; 2Department of Biostatistics and Clinical Epidemiology, University of Toyama, Toyama, Japan.

Context: We previously showed that interobserver agreement of pathologic diagnosis for chronic intestinal pneumonia was not high (κ = .13 for general pathologists). To improve this issue, we have created free Web-based software assisting in the pathologic diagnosis of interstitial pneumonia and tested whether the software improved the interobserver agreement.

Design: A total of 4 pathologists independently scored 20 findings into 0 to 3 for 20 chronic interstitial pneumonia cases. Among the 20 findings, 12 findings were selected for statistical analysis. Discriminate analysis was performed using the scores of 12 findings to reach the pathologic diagnosis given after clinical radiologic and pathologic discussion. We created the Web-based software using the calculation formula given from the analysis and published on the Web ( Three pathologists—one general pathologist and two residents—tested the software for 38 consecutive interstitial pneumonia cases without clinical and radiologic data, and the histologic patterns of their tests were compared to those of original pathologic reports given by pulmonary pathologists.

Results: Pathologic patterns seen in the original pathology reports were 24 UIP, 12 NSIP, and 2 DAD. The κ coefficients between 3 pathologists and the original pathology reports were .59, .53, and .44, respectively. The interobserver agreements were significantly increased.

Conclusions: We have created Web-based software to assist in the pathologic diagnosis of interstitial pneumonia, and it may be useful for general pathologists.

A Clinicopathologic Study of the Lung in 72 Individuals With Birt-Hogg-Dubé Syndrome

M. Furuya1 (; Y. Nakatani.2 1Department of Pathology, Yokohama City University, Yokohama Japan; 2Department of Diagnostic Pathology, Chiba University Graduate School of Medicine, Chiba, Japan.

Context: Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant inherited disorder characterized by multiple pulmonary cysts and repeated pneumothorax. The gene responsible for BHD is named folliculin (FLCN). The affected patients have a risk of developing multiple renal cell carcinomas. It is very important to correctly diagnose the patients with BHD when they visit hospitals with pneumothorax.

Design: We investigated clinicopathologic features of the lung in 72 individuals from 33 Asian BHD families. Genetic testing was performed in all probands. Microscopic analysis of the lung specimens of BHD patients who received thoracic surgery (n = 12) was performed.

Results: Genetic testing revealed 10 different mutations, including 6 unique patterns that were not reported in Western BHD families. Radiologic study revealed that all of the individuals who received thoracic CT had radiologically identifiable multiple pulmonary cysts, except for 3 who had microcystic lesions. Microscopically, the cells making up the pulmonary cysts stained positive for phospho-mTOR, phospho-S6, HIF-1α, and VEGF, suggesting deranged mTOR and angiogenic signaling.

Conclusions: Asian BHD families tend to have distinct FLCN mutation patterns. The histopathology of pulmonary cysts in BHD patients are unique, and they can be microscopically distinguishable from other cystic lung diseases.

Pill Filler Angiopathy: Recognition and Investigation of Murder by Oral Pharmaceutical Injection

M. Graham ( Department of Pathology, St Louis University School of Medicine and the Office of the Medical Examiner, City of St Louis, Missouri.

Context: Pulmonary lesions commonly have implications in determining cause of death, explaining clinical observations, and addressing issues in civil litigation, but they are infrequently the centerpiece in the investigation and adjudication of felonious acts. Moreover, it is exceedingly rare for the examination of lung tissue to help in the identification of the perpetrator of felonious assaults. In this case study, lung findings led to substantiation, elucidation of pathophysiology, documentation of route of administration, and recognition of an illicitly administered agent that resulted in significant morbidity in one patient and the death of another.

Design: A case report design was used.

Results: Herein are described 2 siblings (a 4-year-old boy and 5-year-old girl) who were hospitalized because of viral gastroenteritis with dehydration. While in the hospital the younger child suddenly and unexpectedly became difficult to arouse, followed approximately 8 hours later by the development of severe pulmonary dysfunction that led to the child's death a few hours later. Hospital personnel were suspicious of some actions of the children's mother. The death was reported to the medical examiner (ME), and a medicolegal death investigation, including an autopsy, was conducted. Microscopic examination of lung tissue sampled during the autopsy demonstrated recent/acute thrombi within the pulmonary microvasculature. The thrombi were formed on foreign material. Light and electron microscopy (SEM/EDX) coupled with toxicology testing and information obtained from a pharmaceutical manufacturer led to the identification of the causative agent as an oral clonidine preparation consistent with the formulation that a pharmacy had used to fill the mother's prescription. Clinicopathologic correlation indicated that the mental status changes of the child were caused by the direct chemical effects of the clonidine and the respiratory dysfunction was due to the effects of the insoluble thrombogenic pill filler material. Exacerbation of the pulmonary dysfunction in the child who died was attributed to the ongoing pill filler–induced vascular effects and not to the readministration of any substance. Thus, postmortem findings allowed explanations for the initial mental status changes, delayed pulmonary dysfunction, cause of death, method of administration, physical evidence, source of the agent, and corroboration of witness' statements.

Conclusions: The death of a hospital patient may result from the covert administration of an illicit or licit agent. The clinical effects of the administration will be dictated by a variety of parameters, including the components of the injected material—the intended active agent(s) and accompanying substances, such as insoluble filler material. Evaluation of lung tissue may be critical in identifying the nature of the injected material, route of administration, consequent pathophysiology, the source of the material and, in some cases, the perpetrator.

Relationship Between Histologic Subtype and Effusion Cytology of Malignant Epithelioid Mesothelioma

K. Hiroshima1 (; A. Suezawa1; N. Sumi1; M. Takahashi1; D. Wu1; S. Kawamura1; Y. Sekine2; D. Ozaki3; T. Yusa.4 Departments of 1Pathology and 2Thoracic Surgery, Tokyo Women's Medical University, Yachiyo Medical Center, Yachiyo, Japan; Departments of 3Pathology and 4Thoracic Surgery, Chiba Rosai Hospital, Ichihara, Japan.

Context: Subtyping of epithelioid malignant pleural mesothelioma (MPM) according to morphologic features and nuclear grade predicts patient survival. The aim of this study was to predict the prognosis of the patients with MPM from cytologic findings of pleural effusion (PE).

Design: We analyzed the cytologic findings of PE for 6 patients with epithelioid MPM whose diagnosis was confirmed histologically with thoracoscopic biopsy or extrapleural pneumonectomy (EPP).

Results: Numerous atypical cells with large nuclei and macronucleoli with lymphocytes and histiocytes were observed in the PE patients studied. Cell-in-cell engulfment and hump formation on the cytoplasm were frequently observed. Immunocytochemistry on cell blocks supported that these cells were mesothelioma cells. Numerous 3-dimensional large clusters of atypical cells were observed in 3 patients. Cytologic grade was 2 or 3, and histologic subtype of these patients was papillary or solid. Homozygous deletion of p16 gene was detected in the tumor of these patients. In 3 other patients, solitary atypical mesothelial cells with flat ball-like or papillary clusters were detected. Cytologic grade was 1 or 2, and histologic subtype of these patients was acinar or trabecular. Homozygous deletion of p16 gene was not detected. Two patients with cytologic grade 1 have lived more than 2 years after EPP, and 1 patient with cytologic grade 2 died 17 months after EPP, but the cause of his death was proliferation of pancreatic carcinoma.

Conclusions: Presence of solitary atypical mesothelial cells with low-grade atypia and absence of numerous 3-dimensional large clusters in PE predicts a good prognosis for patients with MPM.

CDX-2 Expression in Primary Lung Adenocarcinoma

P. B. Illei (; Q. K. Li. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland.

Context: Primary lung adenocarcinoma (PLA) can exhibit enteric differentiation and is recognized in the new IASLC classification of PLA. Variable expression of PLA markers and intestinal differentiation has been described, making the differential diagnosis between PLA and a metastasis from an occult gastrointestinal primary problematic. Our aim was to determine the rate of CDX-2 expression in PLA without histologic evidence of enteric differentiation.

Design: Tissue microarray sections of 61 PLAs were subjected to routine immunohistochemistry for CDX-2, CK7, CK20, TTF-1, Napsin A, and p40. The cohort included 10 well-, 38 moderately-, and 13 poorly-differentiated PLA patients (31 female, 30 male; age: 45–86 years).

Results: All tumors were strongly CK7 positive, whereas CK20 staining was seen in 4 tumors (2 strong, 1 moderate, and 1 focal). Both TTF-1 and Napsin A were positive in 56 tumors, with only 2 tumors negative for both markers. Focal p40 staining was seen in 1 tumor. A total of 10 tumors were CDX-2 positive (3 strong, 3 moderate, and 1 weak diffuse; 3 focal). A total of 4 were CK20 positive (1 focal), 8 TTF-1 positive, and 9 Napsin A positive, and all were p40 negative. Of the 3 tumors with known EGFR and KRAS mutation status, 1 had an exon 19 (A746_750) EGFR mutation (CDX-2, CK7, and CK20 positive).

Conclusions: CDX-2 expression occurs in primary lung adenocarcinoma lacking intestinal/enteric differentiation, and therefore it should not be used as the single criterion to make this diagnosis. All CDX-2–positive tumors expressed CK7, with a subset also coexpressing CK20. TTF-1 and Napsin A were positive in most tumors, whereas p40 was negative.

Expressions of TTF-1, Napsin A, p40, p63, CK5/6, and Desmocollin-3 in Non–Small Cell Lung Cancer (NSCLC), as Revealed by Imprint Cytology

T. Kawai1 (; S. Tominaga1; S. Hiroi1; S. Ogata1; H. Nakashima.21Departments of Pathology and Laboratory Medicine, and 2Preventive Medicine and Public Health, National Defense Medical College, Tokorozawa, Japan.

Context: New therapies have been introduced that have therapeutic or adverse effects that differ among histologic types, and it is important to differentiate between adenocarcinoma (AC) and squamous cell carcinoma (SCC). We tried transferring cells from a given smear to multiple slides to allow for the use of various immunocytochemical stains on limited materials. Using touch-preparation samples of 215 surgically resected NSCLCs of confirmed histologic classification (AC, n = 101; SCC, n = 114), we performed immunocytochemistry for TTF-1, Napsin A, p40, p63, CK5/6, and desmocollin-3 (DSC-3), and compared cytologic staining results with the corresponding resection.

Design: We examined (1) the expressions of the above 6 antibodies on cells transferred from touch imprints of resected specimens, the extent of staining being positive if more than 5% of the area was stained; and (2) sensitivity (SEN), specificity (SPEC), positive predictive value (PPV), and negative predictive value (NPV) for each antibody.

Results: The histologic correspondence rate with Papanicolaou staining was only 73%. In AC, TTF-1 and Napsin A had PPVs of 67% and 83%, and NPVs of 76% and 86%, respectively. In SCC, p40, p63, and CK5/6 had PPVs of 82%, 85%, and 100%, and NPVs of 98%, 94%, and 83%, respectively (Table).

Conclusions: The immunocytochemical expressions of Napsin A and p40 in imprint cytology seem to be of great utility for accurate histologic differentiation of lung cancers.

Immunohistochemistry for EGFR Mutation Detection

I. Kern (; N. Turnšek Hitij; M. Rot; T. Čufer. University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia.

Context: Mutational analysis of epidermal growth factor receptor (EGFR) is recommended for primary lung adenocarcinoma. Immunohistochemical (IHC) detection with mutation-specific antibodies for common types of EGFR mutations could be a reliable screening method.

Design: A highly selected study group consisted of 84 cases of formalin-fixed tissue specimens of lung carcinoma with predetermined EGFR mutations. EGFR mutations were detected with DNA sequencing and/or amplified refractory mutation system method. Paraffin blocks were cut for IHC after sections were submitted for molecular methods. IHC was performed using mutation-specific antibodies of E746-A750 deletion in exon 19 and L858R point mutation in exon 21. IHC staining reaction was assessed as positive (1+, 2+, 3+) or negative.

Results: There were 76 adenocarcinomas, 6 non–small cell lung carcinomas not otherwise specified, 1 large cell carcinoma, and 1 squamous cell carcinoma. Predominant types of tissue specimen were small biopsies (endoscopic, needle), and 9 were surgical specimens. In 7 cases no tumor was identified in IHC slides. The frequencies of both common types of EGFR mutations together were 71% detected by molecular methods and 57% by IHC detection. Specificity and positive predictive value for both antibodies were 100%. IHC determined exon 19 deletions showed lower sensitivity and negative predictive value (61% and 84%, respectively).

Conclusions: IHC could be used as a reliable, well-established first-step method of detection for the most prevalent EGFR mutation types. IHC-negative cases should be submitted for further molecular testing. IHC might be a valuable method for EGFR mutation detection in tumor-scant samples.

Diffuse Pulmonary Hemangiomatosis Presenting as a Large Intrapulmonary Vascular Shunt: A Case Report of an Exceedingly Rare Pulmonary Vascular Proliferation

B. T. Larsen1 (; S. C. Tanner2; T. V. Colby3; H. D. Tazelaar.3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; 2Department of Pathology, University of Mississippi, Jackson; 3Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, Arizona.

Context: Solitary capillary hemangiomas in the lung are very uncommon, and examples of multifocal/diffuse hemangiomas (diffuse pulmonary hemangiomatosis [DPH]) are exceptionally rare.

Design: Study design was a case report of unsuspected DPH in a 20-year-old woman with a remote history of craniopharyngioma, who presented with weakness, episodic dizziness, and hypoxia.

Results: Thoracic computed tomographic imaging demonstrated several subpleural nodules, predominantly in the right lung along a major fissure. Lung perfusion imaging demonstrated extrapulmonary systolic activity, suggestive of a right-to-left shunt. Transthoracic echocardiography with a bubble study showed normal cardiac function, normal pulmonary arterial and capillary wedge pressures, and a large intrapulmonary shunt. Pulmonary angiography failed to demonstrate an arteriovenous malformation, and she was referred for surgical lung biopsy. Morphologic evaluation of the wedge biopsy revealed multifocal, dense capillary proliferations in a patchy distribution—particularly involving the pleura, interlobular septa, and bronchovascular bundles—that were immunoreactive for CD31 and muscle-specific actin but not D2-40 or pankeratin, consistent with capillary differentiation. Unlike the characteristic capillary distribution in pulmonary capillary hemangiomatosis (PCH), involvement of alveolar septa was minimal, and features of pulmonary veno-occlusive disease (PVOD) were absent.

Conclusions: In light of the unusual distribution of the lesions, lack of PVOD-like changes, and absence of pulmonary hypertension and cardiac disease, this case is distinct from PCH and we believe it represents an extremely rare example of DPH. In the absence of an angiographically demonstrable arteriovenous malformation, a large intrapulmonary shunt should prompt consideration of a microvascular malformation, and DPH should be included in the differential diagnosis.

A Comparison of FISH and Immunohistochemistry in the Detection of ALK Rearrangement in Lung Adenocarcinoma

J. Le Quesne1; M. Maurya2; D. Gonzalez de Castro2; S. Popat3; A. C. Wotherspoon4; A. G. Nicholson1 ( 1Department of Histopathology, The Royal Brompton Hospital, London, United Kingdom; 2Centre for Molecular Pathology, The Royal Marsden Hospital, Sutton, United Kingdom; 3Department of Oncology, The Royal Marsden Hospital, London, United Kingdom; 4Department of Histopathology, Royal Marsden Hospital, London, United Kingdom.

Context: Detection of the ALK rearrangement is crucial to the personalized treatment of patients with crizotinib. The only currently approved test is FISH, but various cheaper and more convenient immunohistochemical assays have been proposed as alternatives. We compared 3 immunohistochemical assays to FISH in a set of archival lung tumor specimens.

Design: Eighteen FISH+ cases were obtained from local archives, with accompanying data on crizotinib therapy and clinical response. A further 14 FISH cases were also retrieved. A total of 17 specimens were excisions, and 15 were biopsies/cytologic. Three antibodies were optimized for immunohistochemical detection of ALK: D5F3, marketed by Ventana and using its proprietary automated system; ALK1 (Dako); and 5A4 (Abcam). All 3 antibodies were applied to all cases. Antibodies were semiquantitatively scored on intensity, and the proportion of malignant cells was stained. Cutoffs for positivity were set by ROC analysis to optimize for correct classification.

Results: The D5F3 assay was more intense but showed relatively high background. Intensity was the most discriminating measure overall. Combination of proportion and intensity did not improve the test, so intensity score alone was used. All antibodies were specific (100%) and sensitive (59%–71%) overall, with only rare discordance between antibodies. No FISHIHC+ cases were seen. Sensitivity was better in excision specimens (83%) than in biopsies and cytology (46%–64%).

Conclusions: IHC is highly specific (100%) and sensitive (87%) in excision specimens. Occasional cases are FISH+ but IHC, particularly when cellular material is scarce, suggesting inaccuracy of FISH or IHC due to inadequate material or suboptimal specimen handling.

Dr. Nicholson has received payment for consultancy from Pfizer in relation to crizotinib therapy in the United Kingdom.

Evaluation of Matrix Metalloproteinases and Their Tissue Inhibitors in Bleomycin-Induced Pulmonary Fibrosis Before and After GSK-3 Treatment

F. Lunardi1; N. Nannini1; B. Montini2; F. Cinetto2; M. Gnoato2; M. Facco2; C. Agostini2; F. Calabrese1 ( Departments of 1Cardiac, Thoracic and Vascular Sciences, and 2Medicine, Hematology and Clinical Immunology, University of Padua, Padua, Italy.

Context: Lung fibrosis is characterized by extensive tissue remodeling, mainly due to aberrant tissue repair related to an imbalance between metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The anti-inflammatory and antifibrotic effects throughout the pharmacologic inhibition of the kinase GSK-3 in a mouse model of bleomycin (BLM)–induced pulmonary fibrosis have been demonstrated previously. The goals of this study were to assess the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the inflammatory and fibrotic (7 and 14 days after BLM treatment) phases of a BLM mouse model and to study whether in vivo inhibition of GSK-3 with SB216763 could modulate MMP activity and MMPs' balance with TIMPs.

Design: MMP gelatinolytic activity was assessed by zymographic analysis in supernatant of bronchoalveolar lavages (BALs) and in lung homogenates. MMP and TIMP expression was evaluated by real-time PCR and Western blotting in BAL cells and in lung homogenates. Their tissue localization was evaluated by immunostaining.

Results: Strong MMP-9 and MMP-2 activity was detected in BALs during the inflammatory phase. In particular, interstitial alveolar macrophages showed very high expression levels of MMP-9 and MMP-2 and their TIMPs, whereas in lung parenchyma, only epithelial injured cells were positive for these markers. During the fibrotic phase, a very high positivity for MMP-2 was detected in lung parenchyma. SB216763 downmodulated MMP-9 and MMP-2 expression in inflammatory and epithelial cells during inflammation and fibrosis.

Conclusions: GSK3 inhibition could play an important role in restoring the extracellular matrix turnover altered in response to lung injury.

Clinical and Morphologic Characterization of IPF Phenotypes

F. Lunardi; N. Nannini; E. Balestro; E. Rossi; F. Rea; M. Saetta; F. Calabrese ( Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy.

Context: Idiopathic pulmonary fibrosis (IPF/UIP) is a progressive disease of unknown etiology, with irreversible scarring in the lung. Recently several studies have demonstrated the existence of different clinical phenotypes: (1) IPF with stable or slowly progressive course (S-IPF); (2) IPF with rapid functional decline (R-IPF); and (3) IPF with acute exacerbations (AE-IPF). Morphologic changes have been considered similar in R-IPF and S-IPF, and diffuse alveolar damage (DAD) superimposed to UIP has been reported as a common finding in AE-IPF.

Design: The aim of the study was to analyze the 3 different phenotypes in order to determine the frequency in our lung transplant center and carefully evaluate lung morphology in explanted lungs. The study examined 59 IPF patients referred to our institution for lung transplantation (LT), followed up for a mean of 37 months. A total of 27 of them underwent LT: different morphologic changes (DAD; vasculitis, honeycomb area, normal/mild remodeled parenchyma, and inflammation) were quantified in lung sections from upper and lower lobes of all explanted lungs.

Results: Considering all of the 59 patients: 63% were S-IPF and 37% were R-IPF; a high percentage of them (23%) developed AE. Among 27 IPF recipients: 30% were S-IPF; 30% were R-IPF; and 40% were AE-IPF. The extension of morphologic changes differed in the 3 groups: S-IPF patients had a significantly higher proportion of normal/mild remodeled lung parenchyma than R-IPF and AE-IPF patients (P < .05), whereas R-IPF and AE-IPF patients had more honeycomb areas (P = .06). DAD was present in all R-IPF patients, in 82% of AE-IPF patients, and in 12% of S-IPF patients (P < .005). Vasculitis was equally present in R-IPF and AE-IPF patients but not detected in S-IPF patients (P < .005). Extensive inflammatory infiltration was seen in R-IPF and AE-IPF patients but not in S-IPF patients (P < .001).

Conclusions: R-IPF showed a morphologic pattern different from S-IPF and more similar to AE-IPF. The identification of R-IPF should be taken into consideration for inclusion in trials and the interpretation of responses to drug therapies.

Pulmonary Vascular Changes Associated With Left-Sided Heart Disease (LHD) and Pulmonary Veno-occlusive Disease (PVOD): A Comparative Morphometric Study

J. Maleszewski1 (; B. Larsen1; S. Jenkins2; P. Pellikka3; J. Ryu4; E. Yi.1 1Division of Anatomic Pathology, 2Department of Health Sciences Research, 3Division of Cardiovascular Diseases, and 4Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, Minnesota

Context: LHD patients may have clinical and morphologic manifestations reminiscent of PVOD. To date, no study has systematically quantified these changes with attention to the variable and distinguishing characteristics of these entities.

Design: As a retrospective pilot review, 3 age- and sex-matched cohorts were evaluated: healthy, LHD, and PVOD (n = 5 for each group). In each case, 10 vessels (2 pulmonary arteries 50–150 μm in diameter, 2 pulmonary veins 50–150 μm in diameter, and 6 small pulmonary vessels <50 μm in diameter) were assessed. Percent medial thickness (%MT), percent intimal thickness (%IT), and adventitial thickness (AT) were measured. Hemodynamic data were obtained from the clinical record. Measures were compared via regression models using generalized estimating equations (multiple vessels per patient).

Results: The mean %ITs in small vessels in the LHD and PVOD groups were 30.4% and 36.6%, respectively, higher than in the healthy group (16.8%; P < .001). The mean %IT in pulmonary arteries was higher in the PVOD group (15.4%) than in the LHD (10.8%) and healthy (6.9%) groups. The %IT in pulmonary veins was higher in the PVOD group (49.2%) than in the LHD (36.7%) and healthy (17.3%) groups. Mean %MT was similar across all cohorts in small vessels and pulmonary veins, but not pulmonary arteries: PVOD (23.2%), LHD (18%), and healthy (13.2%) groups. Mean AT of pulmonary veins was greater in the PVOD group than in the LHD group (9.63 versus 6.75 μm; P = .02).

Conclusions: Our pilot study demonstrated greater mean %IT and mean AT in PVOD patients than in LHD and healthy patients in pulmonary vessels of all sizes. Further study with more patients is ongoing to verify the observed trends.

Molecular Diagnosis of ROS1 Rearranged Pulmonary Adenocarcinomas

L. Mescam-Mancini1,2 (; B. Burroni1; C. Combaz-Lair1; G. Fiandrino1; J. Gervasoni1; D. Moro-Sibilot3; E. Brambilla1; S. Lantuejoul1,2 ; A. McLeer-Florin.1,2 1Department of Pathology, 2Platform of Genetics and Molecular Pathology of Cancers, Pôle de Biologie et de Pathologie, and 3Department of Specialized Acute Medicine, CHU Grenoble, France.

Context: ROS1-rearranged non–small cell lung carcinomas (NSCLCs) have been recently identified as a new molecular subtype of NSCLC. The ROS1 proto-oncogene translocation leads to a constitutive activation of a tyrosine kinase sensitive to MET and ALK inhibitor crizotinib. ROS1 rearrangements are found in 1.2% to 1.7% of NSCLCs, exclusively in native EGFR, KRAS, and ALK adenocarcinomas (triple-negative adenocarcinomas).

Design: A total of 80 triple-negative pulmonary adenocarcinomas were screened for ROS1 rearrangements by both immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). IHC was performed with an antibody directed against the ROS1 chimeric protein, and the break-apart FISH assay was realized to detect ROS1 rearrangements, without any preconception of the fusion partner.

Results: We diagnosed 4 ROS1-positive adenocarcinomas with both a positive FISH result (from 66% to 87% of rearranged nuclei) and positive IHC staining (2+/3+ membrane and cytoplasmic staining). Only one of the rearranged FISH cases was characterized by a classical split pattern; the others showed a variant pattern, with a loss of the 5′ telomeric probe. All of the ROS1 cases were classified as advanced stage (III or IV), arose in nonsmokers or light smokers, and were solid and acinar adenocarcinomas.

Conclusions: A screening algorithm based on an IHC detection of ROS1 chimeric protein, confirmed as positive or doubtful by a ROS1 break-apart FISH assay, could be proposed in triple-negative adenocarcinomas, because the prevalence of ROS1 cases in this selected population reaches 5% in our series.

Usefulness of Liquid-Based Cytology for Immunohistochemical Diagnosis of Lung Cancers

Y. Minami1 (; N. Itoguchi2; M. Noguchi.1 1Department of Diagnostic Pathology, Faculty of Medicine, and 2Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.

Context: Brushing or washing cytology employing bronchoscopy is a standard diagnostic procedure for lung cancer. The present study evaluated the sensitivity of immunohistological diagnosis of lung cancer using bronchial washing materials.

Design: We prospectively studied samples from 49 cases of lung cancer and 17 cases of nonneoplastic pulmonary disease collected using bronchoscopic procedures. We collected exfoliated cells using a thin-layer advanced cytology assay system (TACAS) and performed cytologic diagnosis using Papanicolaou staining. Using the same materials, we also examined the expression of stratifin, OCIAD2 (OCIA domain containing 2), and IGBP1 (immunoglobulin [CD79A]–binding protein 1) by immunohistochemistry, because they are considered to be immunomarkers of early-stage adenocarcinoma.

Results: Twenty-three of the lung cancers were positive for at least 1 of the 3 antigens. The positive cases included not only 19 of 27 class IV/V materials (70.4%), but also 1 of 17 class II materials (5.9%) and 3 of 5 class III materials (60%). Lung cancers were resected from all 23 immunopositive cases. Meanwhile, all 17 materials without neoplasia were negative for all 3 antigens. Very few but certainly atypical cells were detected in the classes II and III materials after reexamination with Papanicolaou staining.

Conclusions: Liquid-based cytology is very useful in the diagnosis of cytologic materials by immunohistochemical staining, because it allows simultaneous screening with both Papanicolaou and immunohistochemical staining. Stratifin, OCIAD2, and IGBP1 are useful immunomarkers of lung cancer and suitable targets for immunohistochemical staining of cytologic materials.

Phenytoin as a Cause of Chronic Eosinophilic Pneumonia

M. J. Misialek ( Department of Pathology, Newton-Wellesley Hospital, Newton, Massachusetts.

Context: Eosinophilic lung diseases comprise a wide spectrum of disorders with overlapping characteristics. Almost every category of drug has been implicated in these diseases and should be considered in the differential diagnoses for patients presenting with such symptoms. It is only with careful clinicopathologic correlation that an accurate diagnosis can be made.

Design: Study design was a case report.

Results: Herein we describe a 56-year-old male smoker with past medical history of seizure disorder, hypothyroidism, and distant alcohol and drug abuse who presented to his primary care physician with complaints of 6 weeks of cough, shortness of breath, dyspnea on exertion, and 16-pound weight loss. On examination his oxygen saturation was 82% on room air. A chest x-ray revealed marked, diffuse bibasilar mixed airspace and interstitial ground-glass opacities. A chest computed tomography scan showed diffuse bilateral lower lobe reticular opacities with areas of more confluent airspace opacity and without significant honeycombing. Considerations included infection, hypersensitivity pneumonitis, cryptogenic organizing pneumonia, or other interstitial lung disease. Bronchoscopy was unremarkable and revealed bronchial epithelial cells, macrophages, and scattered inflammatory cells, including increased eosinophils. He was referred for diagnostic wedge resection of the left lower lobe. Microscopic examination revealed prominent intra-alveolar fibrin and foci of organizing pneumonia, suggesting a diagnosis of acute fibrinous and organizing pneumonia (AFOP), but conspicuous eosinophils and macrophages were more consistent with eosinophilic pneumonia. Intravenous Solu-Medrol was started and phenytoin was switched to levetiracetam. His clinical and radiographic status improved with the disappearance of peripheral eosinophilia, allowing for a switch to oral prednisone and subsequent discharge.

Conclusions: This is the first reported case of chronic eosinophilic pneumonia showing unusual overlap features with AFOP, thought to be caused by phenytoin. The importance of integration of clinical information, imaging, and laboratory and pathologic findings in arriving at a diagnosis of drug-induced respiratory disease is underscored. The literature on phenytoin-related lung disease and pulmonary eosinophilia is reviewed.

Histopathology of Restrictive Allograft Syndrome in Lung Transplantation

M. A. Montero1 (; A. Navarro2; V. Monforte.3 1Department of Histopathology, Royal Brompton and Harefield Foundation Trust, Biomedical Research Unit, Imperial College, London, United Kingdom; Departments of 2Histopathology and 3Respirology, University Hospital Vall d'Hebron, Barcelona, Spain.

Context: Restrictive allograft syndrome (RAS) has been described as one form of allograft dysfunction in lung transplantation. Although it is well known clinically and radiologically, its histopathologic features had been described recently as pulmonary fibroelastosis. The present study describes the histopathologic vascular features of RAS at autopsy.

Design: We reviewed 12 autopsies from patients who died of allograft dysfunction. We divided them into 3 groups depending on clinical and radiologic parameters. These 3 groups corresponded to patients who died of BOS (obstructive ventilatory pattern and air entrapment on HRCT), RAS (restrictive ventilatory pattern and peripheral interstitial infiltrates on HRCT), and a mixed pattern of disease (obstructive ventilatory pattern and peripheral interstitial infiltrates on HRCT). Histopathologic features were described, blinding the clinical and radiologic features of each patient.

Results: A total of 6 of the deceased demonstrated BOS, 4 showed RAS, and 2 displayed a mixed pattern of disease (Table). Interstitial fibroelastosis was seen in all cases diagnosed with RAS and mixed patterns, but in none with BOS. Loss of the interstitial capillary network, intimal fibrosis, and medial wall inflammation involving medium-size vessels (arteries and veins) was seen in all patients diagnosed as RAS and mixed pattern, but only 1 BOS patient showed these findings. Bronchiolitis obliterans was seen in all autopsies where BOS and mixed patterns were identified antemortem, and in 50% of patients with RAS.

Conclusions: RAS histopathology consisted of pulmonary fibroelastosis and a distinctive fibroinflammatory vascular lesion and interstitial capillary loss that was often associated with subpleural fibroelastosis and bronchiolitis. Although interstitial fibroelastosis has already been described in this type of syndrome, we describe a new vascular lesion, which may allow further understanding of the pathogenesis of RAS.

Morphologic and Molecular Characterization of Adenocarcinoma Associated With Chronic Obstructive Pulmonary Disease (COPD)

N. Nannini; F. Lunardi; M. Schiavon; S. Baraldo; E. Balestro; M. Saetta; F. Rea; F. Calabrese ( Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy.

Context: COPD and lung cancer are strictly related, and COPD-associated cancers seem to have specific pathogenetic and morphologic features that are different from tumors arising in smokers without COPD. The aim of the study was to identify the presence of specific morphologic and molecular cancer phenotypes in COPD patients compared with control groups (healthy smokers and never-smokers).

Design: From 2010 to 2012, 43 patients with peripheral adenocarcinoma (10 COPD, 23 smokers, 10 never-smokers) were prospectively enrolled. Different clinical-biohumoral data were collected in all cases. Tissue morphometric analysis of growth pattern (lepidic, solid, acinar, papillary), cell proliferation (Ki-67 expression), intratumoral and peritumoral remodeling (inflammation, fibrosis, and necrosis), and tumoral detection of IL-17 expression was done. Genetic analysis of EGFR and KRAS mutations was also performed.

Results: The most important clinical difference was a higher number of basophils and lower SUV in PET-CT for COPD patients compared with smokers. Concerning morphologic evaluations, tumors detected in smokers showed an increase of solid component in comparison with COPD and never-smoker cancers (P = .05), a reduction of lepidic pattern (P = .01), and a higher Ki-67 value (P = .01). More extensive intratumoral necrosis was found in smokers and COPD patients compared with never-smokers (P = .03 and P = .05, respectively). Increased IL-17 expression was observed in COPD patients compared with smokers and never-smokers. Moreover, KRAS mutation presented a significantly higher percentage in smokers compared with COPD patients and never-smokers (P < .001).

Conclusions: COPD-related cancer presents molecular and morphologic features of lower aggressiveness (increased lepidic component, reduced solid pattern, lower cell proliferation, and less frequent KRAS mutation) compared with smokers.

Basaloid Squamous Cell Carcinoma of the Lung With Low-Grade Features Presenting as an Endobronchial Tumor

S. Ota1; A. Ishii2; Y. Yonemori2; J. Morimoto2; I. Yoshino1,2; M. M. Kenudson3; E. J. Mark3; Y. Nakatani1,2 ( 1Department of Pathology, Chiba University Hospital, Chiba, Japan; 2Department of Diagnostic Pathology, Chiba University Graduate School of Medicine Chiba, Japan; 3Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston.

Context: Basaloid squamous cell carcinoma is a high-grade neoplasm, and no low-grade counterpart has been reported, to the best of our knowledge.

Design/Results: A 56-year-old man presented with bloody sputum. The chest x-ray revealed an abnormal shadow in the left upper lung field. The tumor showed FDG uptake activity on a PET scan. A transbronchial biopsy from the tumor mass filling the B1+2a bronchus revealed a monotonous and uniform basaloid cell proliferation with pseudopapillary and peripheral palisading growth patterns. The patient underwent left upper lobectomy and is currently free of disease 7 months postoperatively. Macroscopically, the resected specimen showed a 2.7-cm, whitish solid tumor fully occupying the bronchial lumen, further extending from B1+2a to its peripheral bronchi, bronchioles, and some alveolar walls. Microscopically, the tumor cells were small to medium sized, with high N:C ratios, and had round to oval nuclei with a condensed chromatin pattern, resembling basal cells. The cells were arranged in a pseudopapillary pattern with well-developed vasculature and a lobular pattern with prominent peripheral palisading. Some areas showed tumor cells with eosinophilic cytoplasm and intercellular bridges. No keratin pearls were observed. Mitotic activity was low, and tumor necrosis was not seen. Immunohistochemically, the tumor cells stained positive for basal/squamous cell markers (p63, p40, and 34βE12), and negative for TTF-1, chromogranin A, synaptophysin, and HPV-ISH.

Conclusions: This low-grade basaloid squamous tumor with endobronchial presentation expands the clinical and pathologic spectrum of basaloid squamous tumors in the lung.

Pathologic Features of Lung Dominant Connective Tissue Disease

K. Otani1; T. Tanaka2; N. Omote3; K. Kataoka3; Y. Kondoh3; H. Taniguchi3; K. Tabata2; S. Nunomura2; T. Itoh1; J. Fukuoka2 ( 1Department of Diagnostic Pathology, Kobe University Hospital, Kobe, Japan; 2Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; 3Department of Respiratory Medicine and Allergy, Tosei General Hospital, Seto, Japan.

Context: “Lung dominant connective tissue disease (LD-CTD)” was recently proposed by Fischer et al for cases that do not fulfill the criteria of CTD but have interstitial lung disease (ILD) with positive autoimmune antibodies or histologic findings suggestive of CTD. Their histologic inclusion criteria—2 or more of 4 findings—have never been validated. We evaluated their histologic criteria to see whether they fit with cases of clinically detected LD-CTD.

Design: A total of 30 cases with VATS biopsy showing interstitial pneumonia detected to fulfill LD-CTD by autoimmune antibody were selected, and 4 histologic criteria—plasmacytic infiltration, lymphoid aggregates with germinal center, pleuritis, and perivascular collagen—were evaluated. Then, using autoimmune antibody, the LD-CTD cases showing UIP pattern (UIP/LD-CTD) were compared with 16 IPF cases without features of LD-CTD. Chi-square test was used for statistical analysis.

Results: A total of 18 of 30 cases (60%) showed UIP pattern. When compared with IPF, plasmacytic infiltration and pleuritis were seen more frequently in UIP/LD-CTD cases (72.2% versus 31.3%, P = .02, and 55.6% versus 12.5%, P = .009, respectively). However, lymphoid aggregates and perivascular collagen did not show significant differences. A total of 10 of 18 cases (56%) fulfilled the histologic criteria of LD-CTD proposed by Fischer et al. On the other hand, 4 cases (25%) of IPF also fulfilled the criteria.

Conclusions: Plasmacytic infiltration and pleuritis were statistically associated with UIP/LD-CTD. However, there may be some overlap between UIP/LD-CTD and IPF, so that the proposed 4 findings may not be the best criteria for detecting LD-CTD. Prospective study for the clinical behavior of histologic LD-CTD is also needed.

Identification of Epidermal Growth Factor Receptor (EGFR) Mutations and Anaplastic Lymphoma Kinase (ALK) Gene Rearrangement in Lung Adenocarcinomas

I. H. Ozbudak (; M. Ozcan; G. Ozbilim. Department of Pathology, School of Medicine, Akdeniz University, Antalya, Turkey.

Context: The identification of molecular alterations has an important therapeutic implication in patients with lung adenocarcinomas. In this study, we presented our experience with the identification of EGFR and ALK mutations using tissue specimens of primary and metastatic lung adenocarcinomas.

Design: EGFR mutations in exons 18, 19, 20, and 21 were evaluated by pyrosequencing. A total of 101 cases of lung adenocarcinomas, including 64 primary and 37 metastatic tumors (brain, lymph node, liver, pleura, bone, and thyroid), were studied. Mutation analyses were performed on 6 cell blocks obtained from fine-needle aspiration, 64 small biopsies, and 31 resection materials. For ALK gene rearrangement, 23 of 101 cases with no EGFR mutation (15 primary and 8 metastatic tumors) were evaluated by fluorescence in situ hybridization using an ALK break-apart probe.

Results: The median age of the 101 patients was 61 years (range, 33–85 years). Among the patient population, 19 patients were female. EGFR mutations were found in 12 of 101 patients (11.8%), with 5 primary tumors (41.7%) and 7 metastatic tumors (lymph node, pleura, brain, liver; 58.3%), and 4 of these 12 patients (33.3%) were female. A total of 6 patients with delE746-A750 (exon 19), 3 patients with delE747-A750insP (exon 19), 2 patients with L858R (exon 21), and 1 patient with Gly719Ser; L861Q (exon 18; 21) were noted. ALK gene rearrangement was evident in 4 of 23 patients (17.4%): 1 (25%) was female and had primary tumor, and 3 (75%) were male and had metastatic tumors.

Conclusions: Metastatic adenocarcinomas demonstrated EGFR mutation more than primary tumors; therefore, repeating molecular testing in metastatic lung adenocarcinomas may uncover newly acquired molecular alterations.

Histology: A Reliable Tool to Classify NSCLC

S. Petkiewicz; H. Sekhon; M. Gomes ( Department of Pathology and Laboratory Medicine, The Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada.

Context: Because of recent advances in treatment, the diagnosis of a non–small cell lung cancer is no longer sufficient in a biopsy, and a subtype must now be specified, if possible. This can be accomplished by careful study of the histologic features and judicious use of immunohistochemistry (IHC). A great deal of effort has been put into developing immunohistochemical profiles for classification of non–small cell lung cancers. However, the gold standard is still histology, and saving tissue for molecular studies is paramount.

Design: Two pulmonary pathologists blinded to the original biopsy diagnosis independently examined 37 biopsy specimens and paired resection specimen diagnosis in order to assess the accuracy of morphologic interpretation alone.

Results: Individually, the pathologists were able to confidently make a diagnosis on H&E sections in 86% and 92% of cases, with only one diagnosis not corresponding to the final resection diagnosis. There was disagreement in 4 cases, and this was resolved by reaching a consensus, which resulted in a final diagnosis in 3 cases and a request for IHC in 1 case. After consensus, only 3 cases required IHC for diagnosis, and all other biopsies were given diagnoses that concurred with the final resections (92% accuracy).

Conclusions: Careful histologic examination is an accurate tool for subtyping non–small cell lung carcinomas in most cases. Immunohistochemistry should be applied only in equivocal cases in order to preserve tissue for molecular tests.

This study was financed by the PALM Academic Enhancement Funds grant, Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Canada.

Glut-1 Expression in Mesothelial Proliferations: A Comparison Study Between Malignant Mesothelioma and Reactive Hyperplasia Describing Intensity and the Extension of Staining

A. Piottante (; A. Garmendia; L. Contreras. Department of Pathology, Clinica Las Condes, Santiago, Chile.

Context: Glut-1 is a glucose transporter usually expressed in red blood cells. It is largely undetectable by immunostains in normal epithelial tissues and benign tumors, but it is expressed in a variety of malignancies, so it has been used to discriminate between benign and malignant lesions. The utility of this marker was confirmed in several studies for mesothelial proliferations, but a characterization of intensity and an extension of staining have never been reported.

Design: We reviewed 8 cases of proven malignant mesothelioma (6 from pleura and 2 from peritoneum) and 6 cases of reactive pleuritis. All of them were surgical specimens. Glut-1 results were graded as follows: negative, positive +1 (weakly positive or moderate incomplete membrane stain), positive +2 (moderate complete or strong incomplete), or positive +3 (strong and complete). The highest score and the extension were recorded for each case.

Results: All pleural and peritoneal mesotheliomas were positive, with +2 and +3 staining in about 5% to 90% of the tumor area. Only 1 of 6 cases of reactive pleural proliferations was focally positive, with +1 staining in less than 5% of the lesion.

Conclusions: Grading intensity of Glut-1 stain appears to be a useful method to discriminate reactive mesothelial cells from malignant pleural and peritoneal mesothelioma. Moderate to strong staining is highly suggestive of malignancy. A negative stain cannot rule out malignancy in small specimens, and an accurate sample is always needed.

Lycoperdonosis, Hypersensitivity Pneumonitis to Wild Funghi Spores or a True Infectious Disease: Report of a Case

A. Piottante1 (; L. Contreras1; R. Gonzalez.2 Departments of 1Pathology and 2Pediatrics, Clinica Las Condes, Santiago, Chile.

Context: Lycoperdonosis is an infrequent infectious disease resulting from inhalation of spores from puffball, a wild mushroom. Reports in humans are extremely rare. Usually considered as a hypersensitivity pneumonitis, we will discuss its probable infectious behavior and nature.

Design: We describe a case of a 6-year-old boy with clinical and histopathologic findings, including special stains and immunostains.

Results: A 6-year-old boy with a diffuse interstitial bilateral pneumonitis developed peripheral nodules after clarithromycin and steroid therapy. Anamnesis highlighted exposure to wild fungal spores by inhalation; the spores were identified as Lycoperdon perlatum. A 2-month treatment with itraconazole and steroids was indicated. One month after suspending therapy, the nodules increased in size. A wedge biopsy was taken. Small white-yellow nodules were identified. Histology showed granulomatous necrotizing irregular nodules with Langhans giant cells, central necrosis, and foci of neutrophils. Grocott stain demonstrated numerous oval spores of 0.4 to 0.6 μm, smooth surface, and a characteristic central dot. No budding was observed. PAS stain was weakly positive. Mucicarmine, Alcian blue, and immunostains for Cryptococcus and Pneumocystis jiroveci were negative. CD4:CD8 ratio was estimated by immunostains as 2:1. The presence of spores inside necrotizing granulomas and the CD4:CD8 ratio favor an infectious nature. He received therapy during 4 additional months, and the nodules finally vanished.

Conclusions: Although lycoperdonosis has been previously considered under the HP spectrum, we believe there are enough data to consider this unusual illness as an infectious disease, and antifungal therapy must be required.

When Coccidioides Infects the Pleura: A 13-Year, Multi-Institutional Experience With Clinical and Pathologic Review of 36 Cases of Primary and Secondary Coccidioidomycotic Pleuritis

R. W. Ricciotti1; T. A. Shekhel2; J. E. Blair3; T. V. Colby4; R. E. Sobonya1; B. T. Larsen5 ( Departments of 1Pathology and 2Internal Medicine, University of Arizona, Tucson; Departments of 3Internal Medicine and 4Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, Arizona; 5Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.

Context: Most pulmonary Coccidioides infections are intraparenchymal; the pleura is rarely involved. Pleuritis is a recognized complication of ruptured cavitary infections and may occur in other settings, but this phenomenon has not been fully characterized.

Design: All cases of coccidioidomycotic pleuritis (2000–2012) were retrieved from the pathology files of the University of Arizona and the Mayo Clinic in Arizona. Clinical history, imaging, and histology were reviewed.

Results: Among 36 patients (median age, 32 years; 22 men), modes of presentation included ruptured cavities (n = 24), pneumonia (n = 10), and primary pleural coccidioidomycosis without evidence of intrapulmonary disease (n = 2). Risk factors included immunosuppression (n = 15) and occupational exposure to soil (n = 6). Symptoms (median, 5 weeks) included cough (n = 19), chest pain (n = 18), dyspnea (n = 17), fever (n = 12), and hemoptysis (n = 7). Imaging showed effusions (n = 21) and pleural thickening (n = 7). Treatment included lobectomy or decortication, with antifungal medications. Histologic review revealed granulomatous pleuritis in all cases, similar for all 3 modes of presentation, including the composition of inflammatory infiltrates, degree of fibrosis, and extent of necrosis. Spherules were present in all cases but were always rare (mean density, <1 per 10 high-power fields). Three deaths occurred (all with ruptured cavities); the remaining patients recovered.

Conclusions: Coccidioidomycotic pleuritis occurs most commonly with ruptured cavitary infections, but it also occurs with concomitant pneumonia or, rarely, as a primary infection. Coccidioidomycosis should be included in the differential diagnosis of pleural effusions, even without evidence of intrapulmonary disease, particularly in immunocompromised individuals. Spherules are rare in pleural tissue, and cultures or serologic studies may be required to confirm the diagnosis.

Prognostic Role of Tumor/Stromal Caveolin-1 Expression in Malignant Pleural Mesothelioma

L. Righi1 (; M. C. Cavallo1; G. Gatti1; V. Monica2; I. Rapa1; S. Busso2; C. Albera3; G. V. Scagliotti.2 1Pathology Unit, Department of Oncology, 2Thoracic Oncology Unit, Department of Oncology, and 3Pulmonary Pathology Unit, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Context: Malignant pleural mesothelioma is a highly aggressive and lethal disease whose adverse prognostic factors include male sex, older age, and platelet count, but the discovery of new prognostic and predictive biomarkers is needed. Caveolin-1 (CAV1) is the most important member of a membrane protein family that includes the major coating proteins of caveolae and was recently described as specifically deregulated in MPM at the genomic level, but a detailed histologic distribution and data on its prognostic or predictive role are still lacking.

Design: A series of 131 malignant pleural mesothelioma tissues (91 epithelial, 17 biphasic, and 23 sarcomatoid) were investigated for CAV1 immunohistochemical expression and correlated with patient clinicopathologic variables and outcome.

Results: CAV1 was expressed in the neoplastic cells of 70 of 91 epithelial (77%), 17 of 17 biphasic, and 23 of 23 sarcomatoid mesothelioma cases, with increasing semiquantitative H-score levels. Furthermore, CAV1 expression was detected in the surrounding spindle-shaped stromal cells of 61 of 91 epithelial mesothelioma cases (67%). The presence of stromal CAV1 expression showed a negative prognostic role (patient mean survival, 17 months; HR, 0.27; CI, 0.14–0.5) with respect to its absence (patient mean survival, 37.8 months) in the epithelial mesothelioma histotype, and is thus comparable to biphasic and sarcomatoid patient overall survivals.

Conclusions: CAV1 expression in malignant pleural mesothelioma is distributed with increasing levels according to well- to poorly-differentiated histotypes. Furthermore, in the epithelial histotype, stromal expression identified a patient subgroup with a worse outcome.

BRAF V600E Expression in Langerhans Cell Histiocytosis (LCH): Clinical and Immunohistochemical Study on 25 Pulmonary and 54 Extrapulmonary Cases

A. C. Roden1 (; X. Hu2; R. Vassallo2; J. H. Ryu2; E. S. Yi.1 Divisions of 1Anatomic Pathology and 2Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, Minnesota.

Context: A recent genomic study demonstrated BRAF V600E mutation in 57% of LCH cases. Subsequently, Yousem et al reported BRAF V600E mutation in 2 of 5 pulmonary LCH (PLCH) cases. BRAF V600E mutant protein expression has been shown to correlate well with genetic mutation status. We evaluated BRAF V600E expression by IHC in adult PLCH and extrapulmonary LCH cases and correlated with demographic and smoking status in PLCH patients.

Design: An electronic search for LCH at the Mayo Clinic in Rochester during 1991–2012 was performed. Glass slides were reviewed to confirm the diagnosis and select the blocks for IHC. Review of electronic records was performed for smoking habits, pack-years, demographic information, and correlation with BRAF V600E expression.

Results: A total of 79 LCH cases included 25 pulmonary (age, 42 ± 11 years; 10 men) and 54 extrapulmonary (age, 27 ± 22 years; 37 men) cases. A total of 7 of 25 PLCH cases (28%) were positive for BRAF V600E (age, 45.3 ± 7.5 years; 2 men); 1 of these 7 also had extrapulmonary involvement. A total of 20 of 54 extrapulmonary LCH cases (37%) were positive for BRAF V600E (age, 29.1 ± 21.9 years; 14 men). All PLCH cases were current or former smokers at the time of diagnosis, whereas 28 of 54 extrapulmonary LCH patients were never-smokers. The cumulative tobacco exposure at the time of diagnosis was significantly higher in BRAF V600E–positive than in BRAF V600E–negative PLCH patients (mean pack years, 48.3 versus 23.7; 2-tailed t test, P = .01).

Conclusions: A subset of PLCH with BRAF V600E expression may be a clonal proliferative process, in which cigarette smoke might play a role.

Feasibility of Combined Cytology and Cell Block on Transthoracic Needle Aspirates for Non–Small Cell Lung Cancer Subtyping and Molecular Testing

M. S. Roh1 (; C. Son2; E. J. Kang.3 Departments of 1Pathology, 2Internal Medicine, and 3Radiology, Dong-A University College of Medicine, Busan, Korea.

Context: Lung cancer therapy is personalized based on histologic subtype and molecular status. Because 70% of lung cancer patients present in advanced stages and are diagnosed on small biopsy or cytology specimens, an accurate but tissue-sparing approach is necessary. This study aims to demonstrate the use of combined cytology and cell block (CB) on transthoracic needle aspirates (TNAs) for non–small cell lung cancer (NSCLC) subtyping and EGFR mutation testing.

Design: The 20-gauge TNA specimens from the 74 lung masses were divided into 3 groups: liquid-based cytology (LBC), CB, and EGFR mutation analysis using peptide nucleic acid–clamp PCR.

Results: Of 74 cytology specimens, benign and malignant diagnoses were given for 10 (13.5%) and 64 (86.5%), respectively. Of 64 malignancies, diagnoses of definitive versus favored versus unclassified carcinoma subtypes were given for 52 (81.3%) versus 7 (10.9%) versus 5 (7.8%) with LBC. With a combination of LBC and CB (with or without immunohistochemistry), the tumors were more likely to have a definitive subtype in 61 cases (95.3%): 36 adenocarcinomas, 16 squamous cell carcinomas, 9 small cell carcinomas, with 3 unclassified NSCLCs. EGFR mutation was found in 16 cancers (15 adenocarcinomas and 1 unclassified cancer), but no EGFR mutation was found in the 10 benign lesions. Of the 15 adenocarcinomas with EGFR mutation, 6 cases (4 favored adenocarcinomas and 2 unclassified) were not definitively diagnosed as adenocarcinoma by LBC alone.

Conclusions: The combination of cytology and CB on TNA allows for a higher diagnostic yield and reliable molecular testing, by which rebiopsy and unnecessary molecular study may be avoided.

EGFR Mutations, MET/EGFR Amplification, and ALK Rearrangement Simultaneously in Five Bronchial-Pulmonary Adenocarcinomas

M. R. Silva1,2,3; A. Alarcao1,2,3; A. F. Ladeirinha1; M. J. Dâ Aguiar1; T. Ferreira1; V. Sousa1,2,3,4; L. Carvalho1,2,3,4 ( 1Institute of Anatomical and Molecular Pathology, 2CIMAGO Research Center for Environment, Genetics and Oncobiology, and 3Centre of Pulmonology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 4Service of Pathology, University Hospital of Coimbra, Coimbra, Portugal.

Context: ALK rearrangement was found in 2% to 7% of lung carcinomas, resulting in a constitutively active and oncogenic protein reported as exclusive of EGFR and KRAS mutations and associated with resistance to EGFR inhibitors but with sensitivity to crizotinib.

Design: Sections of 126 bronchial-pulmonary carcinomas of all histologic types, obtained from surgical specimens, were screened for ALK positivity by FISH with break-apart dual-color probe (Abbott) and immunohistochemistry (IHC) monoclonal antibody clone 5A4 (Leica); EGFR and KRAS mutations were determined by DNA direct sequencing, and EGFR and MET gene amplifications were explored by FISH (Abbott).

Results: IHC was applied in FISH-ALK+ cases, and of the 126 screened tumors, 9 adenocarcinomas (7%) were FISH-ALK+, 3+ or 2+ score in IHQ, corresponding to patients ages 42 to 79. Among these 9 FISH-ALK+ cases, 3 had EGFR mutations and 2 had both EGFR and MET gene amplifications in FISH; KRAS was wild type. IHQ correlates with FISH for ALK gene rearrangement.

Conclusions: EGFR and MET alterations concomitant with ALK rearrangement have to be tested in advanced bronchial-pulmonary adenocarcinoma in order to validate the TKI resistance of EGFR-mutated tumors that benefit from ALK-targeted agents. Searching for EGFR mutations continues to be necessary in this context of targeted therapy.

Different Thyroid Transcription Factor-1 Antibodies in the Diagnosis of Lung Cancer: The Importance of Choosing the Appropriate Cutoff Values

A. J. J. Smits1,2 (; A. Vink2; G. Tolenaars1; G. M. Herder3; J. A. Kummer.1 1Department of Pathology, St Antonius Hospital, Nieuwegein, The Netherlands; 2Department of Pathology, University Medical Center, Utrecht, The Netherlands; 3Department of Pulmonary Diseases, St Antonius Hospital, Nieuwegein, The Netherlands.

Context: With the use of more specific treatments and targeted therapies for non–small cell lung carcinoma, distinction between adenocarcinoma (ADC) or squamous cell carcinoma (SCC) is becoming increasingly important. For this reason an immunohistochemical panel is used in which thyroid transcription factor-1 (TTF-1) is an essential antibody. However, 2 different TTF-1 clones (8G7G3/1 and SPT24) are used in daily practice, which have been suggested to have different sensitivities and specificities. The aim of this study was to assess the differences between these 2 clones and to choose the optimal cutoff value for positivity of each clone for correctly diagnosing primary and metastatic lung ADC.

Design: A total of 182 pulmonary (109 lung ADCs, 62 lung SCCs, and 11 tumors metastatic to the lung) and 115 extrapulmonary (36 metastatic lung ADCs and 79 nonpulmonary tumors) samples were stained using both TTF-1 antibodies. The percentage of tumor cells with nuclear staining was scored in categories of <1%, 1% to 5%, 6% to 25%, 26% to 50%, 51% to 75%, and 76% to 100% weak or strong staining.

Results: Applying the same cutoff value for positivity to both clones resulted in a statistically significant difference between the clones at all cutoff values (P < .001), with a lower sensitivity of 8G7G3/1 at high cutoff values and a lower specificity of SPT24 at low cutoff values. However, when the optimal cutoff value was used for each clone (>5% for 8G7G3/1 and >50% strong staining for SPT24), no significant difference in sensitivity (0.79 versus 0.82) or specificity (0.98 versus 0.98) was detected.

Conclusions: Both TTF-1 antibody clones tested are equally useful for reliably diagnosing lung ADC, but only when different cutoff values are used.

Synchronous Stage 3A EGFR Mutation–Positive Adenocarcinoma and Stage 1 ALK Mutation–Positive Adenocarcinoma in an Asian Female Never-Smoker

A. Takano1 (; A. Jain2; D. Lim2; E. H. Tan2; T. Lim1; D. Tan.2 1Department of Pathology, Singapore General Hospital (SGH), Singapore; 2National Cancer Centre of Singapore (NCCS), Singapore.

Context: EGFR and ALK mutations in NSCLC are considered to be mutually exclusive, and reports of concomitant mutations are rare. We describe a patient who harbored 2 synchronous lung adenocarcinomas of different histologic subtypes and mutations.

Design: This was the index case for our center's lung cancer sectioning protocol, to study lung cancer heterogeneity. This patient was initially staged as stage 3A EGFR mutation–positive lung adenocarcinoma with an exon 19 deletion, T3 (right upper lobe primary and a separate subcentimeter tumor in the same lobe) N2 (ipsilateral subcarinal and mediastinal lymph node involvement) M0 disease. The patient was treated with a neoadjuvant chemotherapy combination of gefitinib and gemcitabine/cisplatin, followed by right upper lobectomy and lymph node sampling.

Results: Histologic and molecular studies of the right upper lobectomy specimen revealed a synchronous EGFR mutant adenocarcinoma of acinar subtype (pT1 N2) with pathologic evidence of response to neoadjuvant systemic treatment, and a separate ALK mutation–positive adenocarcinoma of mucinous subtype (T1), which showed no response to treatment.

Conclusions: We describe, to our knowledge, a first case of a female Asian nonsmoker with synchronous EGFR and ALK mutation lung adenocarcinomas who was accurately restaged after lobectomy with histologic study and molecular profiling. The authors aim to highlight the possibility of coexisting synchronous EGFR and ALK mutation–positive tumors in patients, especially in cases of uneven response of separate tumors to targeted therapy. We also emphasize the unusual presence of ALK mutation in a mucinous adenocarcinoma subtype, which is known to harbor KRAS mutations in the white population.

Pathologic Acute Lung Injury: A Possible Subtype of Acute or Subacute Interstitial Pneumonia

T. Tanaka1,2; T. Hayashi1; T. Nakayama2; K. Tabata1,2; Y. Kashima1; S. Nunomura1,2; K. Kataoka3; Y. Kondoh3; T. Johkoh4; H. Taniguchi3; J. Fukuoka1,2 ( 1Department of Pathology, Nagasaki University Hospital, Nagasaki, Japan; 2Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; 3Department of Respiratory Medicine and Allergy, Tosei General Hospital, Seto, Japan; 4Department of Radiology, Kinki Central Hospital of Mutual Aid Association of Public School Teachers, Nagasaki, Japan.

Context: The aim of this study was to elucidate the pathologic features of patients presenting with acute or subacute interstitial pneumonia that did not fit with current histologic patterns, such as NSIP, OP, AFOP, or DAD, and were considered here as having pathologic acute lung injury (ALI).

Design: Patients with ALI were identified if all of the following criteria were satisfied: (1) development of dyspnea within 3 months after undergoing a surgical lung biopsy; (2) interstitial pneumonia histology did not fit with NSIP, OP, AFOP, or DAD; (3) lack of chronic fibrotic interstitial pneumonia; and (4) lack of known other named interstitial lung disease. Among the 227 consecutive patients, 19 patients were identified as having ALI and were examined semiquantitatively for chronic fibrosis (CF), fibroblastic focus (FF), hyaline membrane (HM), airspace edema (AE), airspace fibrin (AF), organizing pneumonia (OP), incorporated organization (IO), enlarged alveolar duct (EAD), pleural thickening (PT), cellular IP (CIP), and other specific findings.

Results: Mean age was 63.0 years (8 male and 11 female patients). A total of 8 of 19 patients had a smoking history. A total of 14 patients presented as having idiopathic disease, and 5 patients had connective tissue disease as a background. The common histologic features were IO (19 of 19), EAD (18 of 19), PT (18 of 19), and AE (17 of 19). AF was found in 8 of 19 patients. None showed CF, FF, or HM.

Conclusions: ALI pattern may be a distinct pathologic entity that shows IO, EAD, PT, and AE.

COLVα2: A Biomarker of Lung Fibrosis in Systemic Sclerosis?

W. Teodoro1 (; J. Morais1; P. Andrade1; A. Velosa1; P. Martin1; S. Carrasco1; E. R. Parra2; V. L. Capelozzi.2 1Rheumatology Division and 2Department of Pathology, Medical School, University of Sao Paulo, Sao Paulo, Brazil.

Context: The type V collagen (COLV) mutations are involved in collagen vascular diseases, such as systemic sclerosis (SSc), for which an unusual accumulation of this collagen has been demonstrated. Immunogenic properties, histoarchitecture differentiation, and changes in COLV mRNA expression may suggest that such modifications assigned to this protein may be correlated with SSc pathogenesis. Our aim in this study was to evaluate COLVα1 and COLVα2 expression in lung from SSc patients.

Design: Lung biopsies of 10 patients and 5 control participants were obtained from SSc patients according to protocols by the American College of Rheumatology and the ethics committee (CAPPesq 0960/08). COLVα1 and COLVα2 expression was evaluated by immunofluorescence and histomorphometry. COLVα1 and COLVα2 gene expression analysis was done using real-time qRT-PCR.

Results: COLVα2 immunostaining showed distorted and strongly thickened fibers in lung, with irregular bundles of COLVα2 distributed in parallel and perpendicular arrangements. These distributions result in a dense network around the vessels in SSc patients compared with a thin fiber pattern from the healthy controls. In contrast, COLVα1 expression was absent in lung interstitial compartments compared with respective controls. Histomorphometric analysis of SSc lung demonstrated increased COLVα2 expression in the bronchovascular interstitium compared with controls (P < .01), and the molecular profile demonstrated an increase of COLVα2 gene expression in SSc lung (P = .001).

Conclusions: The overexpression of COLVα2 fibers and COLVα2 gene expression suggest an interference with the fibrillogenesis process in lung fibrosis from SSc patients, reinforcing the participation of this protein collagen in the pathogenesis of SSc.

Decrease in Topoisomerase-1 Protein Synthesis and Pulmonary Collagen Remodeling Induced by Collagen V Nasal Tolerance in Systemic Sclerosis Model

A. Velosa1 (; W. Teodoro1; A. Santos Filho1; S. Fernezlian2; E. Parra2; V. Capelozzi.2 1Rheumatology Division and 2Department of Pathology, Medical School, University of Sao Paulo, Sao Paulo, Brazil.

Context: Autoantibodies against topoisomerase-1 (anti–Scl-70) are found to be associated with increased mortality and correlate with the extent of pulmonary fibrosis in systemic sclerosis (SSc). Our goal was to evaluate anti–Scl-70 antibodies and topoisomerase-1 expression in lung and to correlate these with pulmonary collagen remodeling in experimental SSc after collagen V (COL V)–induced nasal tolerance.

Design: Female New Zealand rabbits (N = 12) were immunized with 1 mg/mL COL V in Freund adjuvant (IM). After 150 days, 6 immunized animals were tolerated by nasal administration of type COL V (25 mg/d; IM-TOL) daily during 60 days. Anti–Scl-70 antibodies were evaluated by ELISA. Immunohistochemistry, histomorphometry, and RT-PCR evaluated expression of pulmonary topoisomerase-1; collagen types I, III, and V; and TGF-β in pulmonary parenchyma.

Results: A significant decrease in topoisomerase-1 expression by pulmonary endothelial cells was found when comparing IM-TOL versus IM (P = .02). No difference was found for the anti–Scl-70 frequency after tolerance. Total collagen content around the small vessels (P < .001) and bronchioles (P < .001), beyond mRNA expression for types I (P = .002) and V (P = .009) collagen, decreased in IM-TOL compared with IM. TGF-β expression decreased in endothelial (P < .001) and smooth muscle (P < .001) cells from pulmonary vessels, epithelial cells (P < .001), and interstitial fibroblasts (P < .001) in IM-TOL compared with IM.

Conclusions: The results showed that a direct link between nasal type V collagen tolerance and a decline in topoisomerase-1 expression may reduce pulmonary fibrosis, suggesting that strategies aimed at preventing the increase of type V collagen synthesis may have a greater impact in SSc.

Molecular Diagnostics in Lung Cancer Using Small Biopsy Specimens

Y. Yatabe ( Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, Nagoya, Japan.

Context: Assessment of molecular status for treatment of lung cancer patients in clinical practice today is essential, with candidates for molecular targeted therapy determined based on individual mutation status.

Design: We present recommendations and tips for processing tissues for both histologic diagnosis and molecular testing.

Results: A cytology imprint prepared before fixation can be a good source for chimeric transcription in RT-PCR. Longer fixation prevents PCR, whereas immunohistochemistry frequently fails because of shorter fixation. In addition, for successful molecular testing it is important to understand the pros and cons of individual techniques. For detecting an EGFR mutation, direct sequencing is easy and versatile, although its sensitivity is low. Thus, most phase 3 trials have used highly sensitive methods that can detect as few as 1% tumor cells in normal tissues. Mutation-specific antibodies for EGFR have also been reported, although they have some limitations. For detecting ALK fusion, immunohistochemistry is rapid and easy, but it lacks a standard protocol, like other companion diagnostic kits. At least it is known that specific antibody clones and sensitive detection methods are keys to successful detection. FISH is now a standard assay, although recent results have revealed variations in signal interpretation.

Conclusions: Today, only small biopsy samples are generally available for molecular testing because the treatments target advanced unresectable disease. Pathologists must play a pivotal role in tissue handling, being positioned between clinicians and molecular biologists.

Absence of Napsin A Expression in Invasive Mucinous Adenocarcinoma (IMC) and Mucinous Adenocarcinoma In Situ (AIS) by Immunohistochemistry

K. Zhang ( Geisinger Medical Laboratory, Geisinger Health System, Danville, Pennsylvania.

Context: A new lung adenocarcinoma classification by IASLC/ATS/ERS has defined two new entities of IMC and mucinous AIS (AIS). Expression of Napsin A on the tumors has not been well documented, and it is investigated in this study by IHC.

Design: We evaluated the expression of Napsin A, CK7, CK20, TTF-1, and CDX2 on 13 cases of IMC, 4 AIS cases, and 54 cases of other types of lung adenocarcinoma on routine sections. The intensity was graded as weak or strong. The distribution was recorded as negative (<5% of tumor cells stained), 1+ (5%–25%), 2+ (26%–50%), 3+ (51%–75%), or 4+ (>75%).

Results: A total of 92% (12 of 13) of IMC cases and 75% (3 of 4) of AIS cases lacked expression of Napsin A. Two positive IMC and AIS cases revealed only 1+, with weak positivity. All IMC and AIS cases showed CK7+; 64% of IMC and 73% of AIS cases were CK20+, with most cases being 1+ or 2+ positivity. A total of 92% of IMC cases and 100% of AIS cases were TTF-1. The TTF-1+ IMC case showed only 1+, with weak nuclear positivity. CDX2+ was observed in 30% of IMC cases and 25% of AIS cases with 1+ weak positivity. A total of 93% of other types of adenocarcinoma showed Napsin A+ with 4+; 100% showed CK7+; 89% showed TTF-1+; and 0% showed CDX2+.

Conclusions: (1) Expression of Napsin A on IMC and mucinous AIS is uncommon, which may serve as a useful adjunctive marker for defining and diagnosing the tumors. (2) Lepidic pattern of the tumors can be well illustrated by Napsin A+ preexisting benign alveolar pneumocytes beneath overlying Napsin A mucinous columnar cells.