Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.

PD-L1 expression in inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumor is a rare mesenchymal tumor occurring at many anatomic sites, with a predilection for children and young adults. Often indolent, they can be locally aggressive and can metastasize, resulting in significant morbidity and mortality. Therapeutic options are often limited. The identification of underlying kinase mutations has allowed the use of targeted therapy in a subset of patients. Unfortunately, not all tumors harbor mutations and resistance to tyrosine kinase inhibitor therapy is a potential problem. We hypothesized that these tumors may be amenable to PD-L1 therapy given the immune nature of the tumor. PD-L1 expression in inflammatory myofibroblastic tumors has not yet been defined. The purpose of this study was to explore PD-L1 expression in inflammatory myofibroblastic tumors, as adaptive PD-L1 expression is known to enrich for response to anti-PD-1/PD-L1 therapies. Expression of PD-L1 (clone SP142) was assessed in 35 specimens from 28 patients. Positivity was defined as membranous expression in ≥5% of cells and evaluated separately in tumor and immune cells. Adaptive vs. constitutive patterns of tumor cell PD-L1 expression were assessed. PD-L1 status was correlated with clinicopathologic features. CD8+ T cell infiltrates were quantified by digital image analysis. ALK status was assessed by immunohistochemistry and/or FISH. Twenty-four (69%) tumors had PD-L1(+) tumor cells and 28 (80%) showed PD-L1(+) immune cells. Most recurrent and metastatic tumors (80%) and ALK(-) tumors (88%) were PD-L1(+). Adaptive PD-L1 expression was present in 23 (96%) of PD-L1(+) tumors, which also showed a three-four fold increase in CD8+ T cell infiltration relative to PD-L1(-) tumors. Constitutive PD-L1 expression was associated with larger tumor size (p = 0.002). Inflammatory myofibroblastic tumors show frequent constitutive and adaptive PD-L1 expression, the latter of which is thought to be predictive of response to anti-PD-1. These data support further investigation into PD-1/PD-L1 blockade in this tumor type.

A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction.

The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence 1417EENKVR1422 and the terminal 1478TRL1480 (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

Occupational Asthma Due to Inhalation of Aerosolized Lipophilic Coating Materials.

We present a case of onset of severe asthma in a 59-year-old patient who worked in an aerospace plant. He was noted to have wheezing on exam and obstruction on PFTs. Review of his occupational history revealed exposure to lipophilic industrial compounds. We outline the radiographic and histologic findings that were found in the patient, and discuss occupational asthma due to inhalation of lipophilic compounds.

senX3-independent contribution of regX3 to Mycobacterium tuberculosis virulence.

BACKGROUND: Mycobacterium tuberculosis (Mtb) must adapt to various stress conditions during host infection. The two-component regulatory system (2CRS) SenX3-RegX3 is required for Mtb virulence. We showed recently that the senX3-regX3 intergenic region contains promoter activity, driving senX3-independent regX3 expression. In the current study, we tested the hypothesis that RegX3 has a SenX3-independent role in Mtb virulence. The gene expression patterns, growth, and survival of mutants containing transposon insertions in senX3 (senX3::Tn) and regX3 (regX3::Tn) were compared to those of their respective complemented strains and the isogenic wild-type parent strain during axenic growth in nutrient-rich broth, phosphate depletion, nutrient starvation, and in the lungs of BALB/c mice. RESULTS: regX3 expression was reduced in senX3::Tn during phosphate depletion and nutrient starvation, and expression of the phosphate-specific transport gene pstC2 was reduced similarly in senX3::Tn and regX3::Tn during phosphate depletion. Although senX3 and regX3 were each dispensable for Mtb growth in nutrient-rich broth, disruption of senX3 or regX3 caused a similar growth defect during phosphate depletion. Interestingly, senX3::Tn, in which monocistronic regX3 expression is preserved, showed significantly higher survival relative to regX3::Tn after 7 days of nutrient starvation (p <0.01), and in mouse lungs at Day 31 (p < 0.01), Day 62 (p < 0.01), and Day 124 (p = 0.05) after aerosol infection. CONCLUSION: Our data demonstrate the specificity of the senX3-regX3 2CRS for sensing and responding to low ambient phosphate, but also raise the possibility that RegX3 may function independently of its cognate sensor histidine kinase.

Potent rifamycin-sparing regimen cures guinea pig tuberculosis as rapidly as the standard regimen.

Strategies involving new drug combinations, as well as new uses of existing drugs, are urgently needed to reduce the time required to cure patients with drug-sensitive or multidrug-resistant (MDR) tuberculosis (TB). We compared the sterilizing activity of the standard first-line antitubercular regimen, rifampin-isoniazid-pyrazinamide (RHZ), with that of the novel regimen PA-824-moxifloxacin-pyrazinamide (PaMZ), which is currently being studied in clinical trials (NCT01498419), in the guinea pig model of chronic TB infection, in which animals develop necrotic granulomas histologically resembling their human counterparts. Guinea pigs were aerosol infected with ~2 log10 bacilli of wild-type Mycobacterium tuberculosis H37Rv, and antibiotic treatment was initiated 6 weeks after infection. Separate groups of animals received RHZ, PaMZ, or single or two-drug components of the latter regimen administered at human-equivalent doses 5 days/week for a total of 8 weeks. Relapse rates were assessed 3 months after discontinuation of treatment to determine the sterilizing activity of each combination regimen. PaMZ given at human-equivalent doses was safe and well tolerated for the entire treatment period and rendered guinea pig lungs culture negative more rapidly than RHZ did. After 1 month of treatment, 80% and 50% of animals in the RHZ and PaMZ groups, respectively, had lung culture-positive relapse. Both combination regimens prevented microbiological relapse when administered for a total of 2 months. Our data support the use of PaMZ as a novel isoniazid- and rifamycin-sparing regimen suitable for treatment of both drug-sensitive TB and MDR-TB.

Recent Advances in Genitourinary Tumors Updates From the 5th Edition of the World Health Organization Blue Book Series.

CONTEXT.­: Urinary and Male Genital Tumours is the 8th volume of the World Health Organization Classification of Tumours series, 5th edition. Released in hard copy in September 2022, it presents an update to the classification of male genital and urinary tumors in the molecular age. Building upon previous volumes in this series, significant effort has been made to harmonize terminology across organ systems for biologically similar tumors (eg, neuroendocrine tumors). Genomic terminology has been standardized and genetic syndromes covered more comprehensively. This review presents a concise summary of this volume highlighting new entities, notable modifications relative to the 4th edition, and elements of relevance to routine clinical practice. OBJECTIVE.­: To provide a comprehensive update on the World Health Organization classification of urinary and male genital tumors, highlighting updated diagnostic criteria and terminology. DATA SOURCES.­: The 4th and 5th editions of the World Health Organization Classification of Tumours: Urinary and Male Genital Tumours. CONCLUSIONS.­: The World Health Organization has made several changes in the 5th edition of the update on urinary and male genital tumors that pathologists need to be aware of for up-to-date clinical practice.

DICER1-associated metastatic abdominopelvic primitive neuroectodermal tumor with an EWSR1 rearrangement in a 16-yr-old female.

We report a case of a DICER1-associated EWSR1-rearranged malignant primitive neuroectodermal tumor (PNET) arising in a patient with DICER1 tumor predisposition syndrome. A 16-yr-old female with a history of multinodular goiter presented with a widely metastatic abdominal small round blue cell tumor with neuroectodermal differentiation. EWSR1 gene rearrangement was identified in the tumor by fluorescence in situ hybridization (FISH). Genetic analysis revealed biallelic pathogenic DICER1 variation. The patient was treated with an aggressive course of chemotherapy, surgery, and radiation with complete pathologic response. We believe this case to represent a new expression of the DICER1 tumor predisposition syndrome, an entity caused by deleterious germline mutations in the DICER1 gene, encoding a ribonuclease active in the processing of miRNA. Patients with germline mutations in DICER1 develop a diverse group of benign and malignant tumors. Some of these tumors have been noted to have immature neuroepithelium as a component, including the ciliary body medulloepithelioma and the recently described DICER1-associated presacral malignant teratoid neoplasm. To our knowledge, abdominal sarcomas that resemble PNET histology with an EWSR1 rearrangement have not previously been described as a classical expression of the DICER1 syndrome phenotype.

IDH1 and IDH2 mutations in lung adenocarcinomas: Evidences of subclonal evolution.

BACKGROUND: Selective IDH1 and IDH2 inhibitors have been approved for targeted therapy of acute myeloid leukemia. Clinical trials for solid tumors with IDH1 and IDH2 (IDH1/2) mutations are ongoing. Reports of IDH1/2-mutated non-small cell lung cancers (NSCLCs), however, are limited. METHODS: We evaluated IDH1/2 mutations in 1,924 NSCLC specimens (92% adenocarcinoma) using a next-generation sequencing assay. RESULTS: Retrospective quality assessments identified false detection of IDH1 c.395G>A (p.R132H) resulting from cytosine deamination (C:G→T:A) artifact in one specimen. IDH1/2 mutations were detected in 9 (0.5%) adenocarcinomas taken by fine-needle aspiration (n = 3), thoracentesis (n = 2) or core biopsy (n = 4). All nine adenocarcinomas showed high-grade features. Extensive clear cell change, however, was not observed. High expression (50% or greater) of PD-L1 was observed in two of five specimens examined. IDH1/2 mutations were associated with old age, smoking history, and coexisting KRAS mutation. Lower than expected variant allele frequency of IDH1/2 mutants and coexistence of IDH1/2 mutations with known trunk drivers in the BRAF, EGFR, and KRAS genes suggest they could be branching drivers leading to subclonal evolution in lung adenocarcinomas. Multiregional analysis of an adenocarcinoma harboring two IDH2 mutations revealed parallel evolution originating from a KRAS-mutated lineage, further supporting subclonal evolution promoted by IDH1/2 mutations. CONCLUSIONS: IDH1/2 mutations in NSCLCs are uncommon. They occur in adenocarcinomas with high-grade features and may be branching drivers leading to subclonal evolution. Accumulation of more IDH1/2-mutated NSCLCs is needed to clarify their clinicopathological characteristics and implications for targeted therapy.

Clonal Origin Evaluated by Trunk and Branching Drivers and Prevalence of Mutations in Multiple Lung Tumor Nodules.

INTRODUCTION: Differentiation between intrapulmonary metastasis (IPM) and multiple primary lung cancers (MPLC) in patients with synchronous or metachronous lung tumor nodules is critical but challenging. OBJECTIVE: We proposed an algorithm to evaluate clonal origin based on trunk (initiating) versus branching drivers and the prevalence of mutations in lung adenocarcinomas. METHODS: Driver mutations were examined using next-generation sequencing in five trunk driver genes (BRAF, EGFR, ERBB2, KRAS, and NRAS) and three branching driver genes (ATK1, PIK3CA, and TP53). RESULTS: Mutational profiling supported same clonality and likely same clonality, respectively, in 39 and 14 of 66 pairs of specimens with known identical clonal origin. Discordance of TP53 mutations (branching drivers) was observed in three pairs. Subsequent analyses of 30 pairs of synchronous or metachronous lung tumor nodules revealed different clonality and likely different clonality in 17 and 2 pairs, respectively, including three pairs with similar histomorphology; same clonality and likely same clonality in three and five pairs, respectively, including two pairs with different histomorphology; and inconclusive or noninformative results in three pairs. CONCLUSION: While discordance of trunk drivers indicated MPLC in patients with synchronous or metachronous lung tumor nodules, discordance of branching drivers did not exclude IPM. Concordance of uncommon drivers supported IPM, whereas concordance of common drivers did not exclude MPLC. Additional recommendations from official organizations are needed to guide applications of molecular markers in defining clonality of multiple lung tumor nodules.

The Utility of NKX2.2 and TLE1 Immunohistochemistry in the Differentiation of Ewing Sarcoma and Synovial Sarcoma.

Although molecular testing can definitively distinguish Ewing sarcoma (EWS) from synovial sarcoma (SS) it is frequently desirable to provide a confident preliminary diagnosis before such analysis can be completed. Recently, the nuclear markers NKX2.2 and TLE1 have been shown to have good sensitivity but imperfect specificity, respectively, for EWS and SS. However, the performance of these markers has not been extensively evaluated within this specific differential diagnosis. This study performed NKX2.2, TLE1, and CD99 immunohistochemistry in a group of EWS and SSs confirmed by reverse transcription-polymerase chain reaction to evaluate the utility of these novel markers in this context. NKX2.2 staining was overall 75% sensitive and 91.7% specific for EWS and was never seen in SS. Although the specificity of TLE1 staining was impacted by antibody used, it was at best only 75% specific for SS. However, a lack of reactivity had a 100% negative predictive value against a SS diagnosis. Overall, immunohistochemistry for NKX2.2 and TLE1 can provide a useful first step in helping to distinguish EWS and SS.

Clinical Validation of Coexisting Activating Mutations Within EGFR, Mitogen-Activated Protein Kinase, and Phosphatidylinositol 3-Kinase Pathways in Lung Cancers.

CONTEXT.­: Mutations within the same signature transduction pathway are redundant and, therefore, most are mutually exclusive. Laboratory errors, however, may introduce unexpected coexisting mutations. OBJECTIVE.­: To validate coexisting mutations within epidermal growth factor receptor (EGFR), mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. DESIGN.­: In this retrospective study for quality assessment of next-generation sequencing in a clinical diagnostics setting, coexisting mutations within EGFR, KRAS, NRAS, BRAF, AKT1, and PIK3CA genes were examined in 1208 non-small cell lung cancers. RESULTS.­: EGFR mutations did not coexist with BRAF mutations, neither kinase-activated nor kinase-impaired mutations. There was a low but similar incidence (3.3%-5.1%) of PIK3CA mutations in BRAF-, EGFR-, and KRAS-mutated lung cancers and a rare incidence of coexisting KRAS and EGFR mutations detected in 1 of 1208 lung cancers (0.08%) or 1 of 226 EGFR-mutated lung cancers (0.4%). Coexisting BRAF p.V600E mutation was observed in 3 of 4 AKT1 p.E17K-mutated lung cancers. Mutational profiling of DNA reisolated from subareas with the same or different histomorphology, using an alternative assay, confirmed that coexisting mutations might present within the same (whole or subclonal) population or different populations and clarified that the so-called coexisting activating KRAS and BRAF mutations originally reported in a specimen were indeed present in separate lung nodules submitted in the same block. CONCLUSIONS.­: The results supported that EGFR and BRAF mutations are early driver mutations in lung cancers. Guidelines from official organizations to establish standard operating procedures are warranted to validate unexpected coexisting mutations and, if clinically indicated, to determine their presence in the same or different tumor populations.

Clinical Validation of Discordant Trunk Driver Mutations in Paired Primary and Metastatic Lung Cancer Specimens.

OBJECTIVES: To propose an operating procedure for validation of discordant trunk driver mutations. METHODS: Concordance of trunk drivers was examined by next-generation sequencing in 15 patients with two to three metastatic lung cancers and 32 paired primary and metastatic lung cancers. RESULTS: Tissue identity was confirmed by genotyping 17 single-nucleotide polymorphisms within the panel. All except three pairs showed concordant trunk drivers. Quality assessment conducted in three primary and metastatic pairs with discordant trunk drivers indicates metastasis from a synchronous or remote lung primary in two patients. Review of literature revealed high discordant rates of EGFR and KRAS mutations, especially when Sanger sequencing was applied to examine primary and lymph node metastatic tumors. CONCLUSIONS: Trunk driver mutations are highly concordant in primary and metastatic tumors. Discordance of trunk drivers, once confirmed, may suggest a second primary cancer. Guidelines are recommended to establish standard operating procedures for validation of discordant trunk drivers.

Clinicopathologic study of calcifying fibrous tumor of the gastrointestinal tract: a case series.

Calcifying fibrous tumor (CFT) is a rare benign mesenchymal lesion known to arise at multiple body sites that may clinically mimic other more aggressive lesions in the gastrointestinal (GI) tract. In this study we describe the clinicopathologic findings of 28 GI tract CFTs. Tumors predominantly arose in middle-aged adults with a slight female predominance. The most commonly involved sites were small bowel and colon, followed by stomach and appendix. Tumors ranged from 0.3 to 9.3 cm (median 1.4 cm), and submucosa was the most commonly involved layer. All tumors were well circumscribed and unencapsulated. Microscopically, tumors were hypocellular and composed of spindle cells with abundant, haphazardly arranged hyalinized collagen. No necrosis and less than one mitosis per 10 HPF were identified in all cases. Calcification was present in most (81%) of the cases. All cases had lymphoplasmacytic inflammatory infiltrates either scattered throughout the lesion with occasional perivascular conglomeration or in the form of lymphoid aggregates. A lymphoplasmacytic cuff was usually present (81%). Immunostains showed variable CD34 immunoreactivity and variable numbers of IgG4-positive plasma cells. The lesional cells were negative for DOG-1, ALK-1, S100, C-kit, Sox10, Melan A, HMB45, desmin, CK7, and CK20, and showed cytoplasmic staining for ß-catenin. Follow-up information was available in 5 cases with no recurrences reported to date (mean follow-up, 3 years). CFT is a rare benign tumor that can occur in part of the GI tract and should be distinguished from other mesenchymal tumors due to its low risk of recurrence.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing.

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2-5% in 33 (4.3%) mutations and 2-10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations.

Mutational spectrum of intraepithelial neoplasia in pancreatic heterotopia.

Heterotopic pancreatic parenchyma recapitulates the normal pancreas in extrapancreatic locations and, on rare occasions, can even give rise to pancreatic adenocarcinoma. The genetic signatures of pancreatic adenocarcinoma and its precursor lesions are well characterized. We explored the genetic alterations in precursor lesions (intraductal papillary mucinous neoplasms [IPMN], pancreatic intraepithelial neoplasia [PanIN]) in patients with pancreatic heterotopias but without concomitant pancreatic ductal adenocarcinomas. This allowed us to determine whether the stereotypical dysplasia--infiltrating carcinoma sequence also occurs in these extrapancreatic foci. Seven cases of heterotopic pancreas with ductal precursor lesions were identified. These included 2 IPMNs with focal high-grade dysplasia and 5 PanINs with low- to moderate-grade dysplasia (PanIN grades 1-2). Neoplastic epithelium was microdissected and genomic DNA was extracted. Sequencing of commonly mutated hotspots (KRAS, TP53, CDKN2A, SMAD4, BRAF, and GNAS) in pancreatic ductal adenocarcinoma and its precursor lesions was performed. Both IPMNs were found to have KRAS codon 12 mutations. The identification of KRAS mutations suggests a genetic pathway shared with IPMN of the pancreas. No mutations were identified in our heterotopic PanINs. One of the possible mechanisms for the development of dysplasia in these lesions is field effect. At the time of these resections, there was no clinical or pathologic evidence of a prior or concomitant pancreatic lesion. However, a clinically undetectable lesion is theoretically possible. Therefore, although a field effect cannot be excluded, there was no evidence for it in this study.

Heterogeneity of resistance mutations detectable by nextgeneration sequencing in TKI-treated lung adenocarcinoma.

EGFR-mutated lung adenocarcinomas routinely develop resistance to tyrosine kinase inhibitors (TKI). To better characterize the relative frequencies of the resistance mechanisms, we analyzed 48 EGFR-mutated TKI-resistant specimens from 41 patients. Next-generation sequencing of post-treatment specimens detected EGFR p.T790M in 31 (79%) of 39 patients, PIK3CA mutations in 10 (26%), EGFR p.S768_V769delinsIL in one, and KRAS p.G12C in one. Five PIK3CA mutations were outside of codons 542, 545, and 1047. Three of four pre-treatment specimens did not carry the PIK3CA mutation found in the post-treatment sample. Small cell carcinoma transformation was identified in four patients; none had p.T790M, including two where p.T790M was identified in the co-existing adenocarcinoma. In p.T790M-mutated specimens, the allele frequency was less than 5% in 24% of cases. p.T790M allele frequency was usually lower than that of the sensitizing mutation indicating that the resistance mutation was present either in a subset of cells or, if the sensitizing mutation was amplified, in a subset of the sensitizing alleles of a dominant clone. Eight patients had multiple resistance mutations, suggesting either multiple separate resistant clones or a single clone harboring multiple resistance mechanisms. PIK3CA mutations appear to be a more significant resistance mechanism than previously recognized.

Adenocarcinoma arising in an extralobar sequestration: a case report and review of the literature.

Extralobar sequestration is a type of bronchopulmonary foregut malformation defined as an isolated portion of lung tissue with a systemic arterial supply, its own pleural investment, and no bronchial communication. While it may be recognized in utero or in the neonatal period, depending on its location and associated anomalies, it can also go unrecognized until later in life when it may present as a mass. We report the first case of adenocarcinoma arising in an extralobar sequestration. The patient was a 70-year old man with a 55 pack year smoking history who presented with chest discomfort and was found to have a 6.5 cm right lower lobe mass. Percutaneous biopsy of the mass was positive for adenocarcinoma. At surgery, the mass was noted to have a separate arterial connection, no bronchial communication, and its own pleural investment, consistent with an extralobar sequestration. Malignancy arising in pulmonary sequestrations is rare and the few reported cases have been in intralobar types. Carcinoma arising in this setting adds to the dilemma of whether or not these developmental anomalies should be excised or followed. Our tumor, while small, did have vascular invasion.

Pleural Fluid Cytology of the Polymorphous Variant of EBV-Positive Diffuse Large B-Cell Lymphoma: First Report and Distinction from a Reactive Process.

EBV-positive diffuse large B-cell lymphoma of the elderly is a newly described aggressive lymphoma predominantly affecting patients >50 years of age. Patients may present with nodal and/or extranodal involvement. The lung is one of the more common extranodal sites. The incidence of pleural fluid involvement is less well described. In one study by Oyama et al., pleural effusions were noted in nine percent of cases. Identification of pleural fluid involvement could be important as it may carry prognostic importance in staging (it typically occurs more often in cases with widespread disease), and it could be a relatively easy means of establishing a diagnosis in newly presenting cases. We report the first description of the pleural fluid cytology in a case of EBV-positive diffuse large B-cell lymphoma of the elderly.