Harman S. Sekhon

Mouse Nkrp1-Clr Gene Cluster Sequence and Expression Analyses Reveal Conservation of Tissue-Specific MHC-Independent Immunosurveillance

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed (“functional”), or unlicensed (“hypofunctional”). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m -/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab’)2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

Myopericytoma: A Pleural-Based Spindle Cell Neoplasm Off the Beaten Path

Myopericytoma is a recently described hemangiopericytoma-like neoplasm with myoid differentiation. These tumors are typically located in the subcutaneous and soft tissues of the extremities. The authors report a rare pleural-based pulmonary myopericytoma in a 58-year-old woman. The lesion was grossly homogeneous and well circumscribed. Microscopically, it was composed of densely packed spindle cells organized as whorls and short interlacing fascicles with a concentric perivascular distribution. Immunohistochemical reactions were positive for vimentin, smooth muscle actin (SMA), muscle-specific actin, and Bcl-2 and negative for desmin, h-caldesmon, cytokeratin, and CD34. Atypically, increased mitotic activity was noted, but no other malignant features were identified. The differential diagnoses are discussed with specific emphasis on solitary fibrous tumor of the pleura, which is the most common benign pleural-based spindle cell neoplasm and may be a diagnostic pitfall with potentially harmful consequences.

Spontaneous Pneumothorax and Lung Carcinoma: Should One Consider Synchronous Malignant Pleural Mesothelioma?

Abstract: We describe the clinical and pathologic findings of a 68-year-old smoker with previous asbestos exposure who presented with spontaneous hydropneumothorax and was diagnosed with synchronous undifferentiated lung carcinoma and incidental malignant pleural mesothelioma. The synchronous occurrence of these two neoplasms is an extremely rare event with fewer than 20 reported cases in the English literature. The accurate diagnosis of synchronous tumors can be extremely challenging and the identification of a concomitant mesothelioma in our case was not made until an extensive immunohistochemical analysis was done on the resection specimen. Spontaneous pneumothorax occurs much more commonly in patients with malignant mesothelioma than with primary lung carcinomas. Consequently, although synchronous pleural mesotheliomas and lung carcinomas are infrequent, this diagnosis should be considered when a patient with a lung mass and a history of asbestos exposure presents with spontaneous pneumothorax and pleural thickening on imaging. Identification of synchronous tumors is of critical importance for determining the patient’s stage and management and can have significant medicolegal implications should the patient seek compensation.

Vulvar squamous cell carcinoma (VSCC) as two diseases: HPV status identifies distinct mutational profiles including oncogenic fibroblast growth factor receptor 3

Purpose: Patients with advanced or recurrent invasive vulvar squamous cell carcinoma (VSCC) have limited treatment options and a grave prognosis. Understanding the genomic landscape may facilitate the identification of new therapies and improve clinical outcomes. Experimental Design: A retrospective chart review and molecular analysis of patients with VSCC from 2000 to 2016 was performed at the Ottawa Hospital Research Institute. The presence of oncogenic human papillomavirus (HPV) was determined by nested PCR and amplified DNA was sequenced using the Ion AmpliSeq Cancer Hotspot v2 Panel. The patients were divided into two groups according to HPV status (HPV-positive versus HPV-negative) and clinical outcome correlated with mutation status using descriptive statistics. Results: In 43 VSCC patients, there was a high mutation rate in both HPV-positive (73%) and HPV-negative (90%) disease with the two subgroups expressing distinct genetic profiles. HPV-positive tumors were characterized by oncogenic mutations in PIK3CA (27%), FGFR3 (14%), and PTEN (9%), whereas HPV-negative tumors were found to have mutations in TP53 (57%), HRAS (24%), PI3KCA (19%), and CDKN2A (14%). Mutation S249C in FGFR3 occurred in 14% of HPV-positive tumors. While there were notable differences in the occurrence of TP53, HRAS, PTEN, and FGFR3 mutations according to HPV status, only the rate of TP53 mutations was statistically significant (P = 0.0004). No significant difference in prognosis was found between patients with HPV-positive and HPV-negative VSCC. Conclusions: HPV-positive VSCC is characterized by oncogenic FGFR3 mutations that helps classify this subtype as a separate disease. Inhibitors of FGFR3 merit consideration as a therapeutic strategy in this neglected cancer in women. Clin Cancer Res; 23(15); 4501–10. ©2017 AACR.

Three‐dimensional cell groups with disordered nuclei and cellular discohesion (3DDD) are associated with high sensitivity and specificity for cystoscopic urine cytopathological diagnosis of low‐grade urothelial neoplasia

Cystoscopic urine obtained before the resection of low‐grade urothelial carcinoma (LGUC), with adequate cytological sampling of the tumor, frequently revealed the presence of three‐dimensional cell groups with disordered nuclei and cellular discohesion (3DDD). 936 cystoscopic urine specimens were categorized into five groups: Group 1 (80 specimens) with biopsy‐proven LGUC within 6 months of cytologic examination, Group 2 (23 specimens) with biopsy proven LGUC within 6 to 36 months of cytologic examination, Group 3 (527 specimens) with a history of LGUC but no tumor for a period of greater than 3 years, Group 4 (300 specimens) with no association with LGUC, and Group 5 (6 specimens) with urinary lithiasis. Specimens with scant cellularity accounted for 20% of those in Group 1. For 3DDD in detecting LGUC in adequate cystoscopic urine, the sensitivity was 70%, specificity was 94%. Two‐ or three‐dimensional cell groups with ordered nuclei and/or cellular non‐discohesion were often seen in specimens from Groups 4 or 5. The 3DDD was present in a significant number of cases with concurrent negative cystoscopic findings but also positive LGUC in ensuing follow‐up. In these cases, 3DDD with or without tumor identified at concurrent cystoscopy were found to be morphologically similar. Furthermore, the presence of 3DDD in 8% of Group 3 likely represents urothelial dysplasia that is not cystoscopically detectable. The high specificity and sensitivity of 3DDD is demonstrated. These findings are consistent with the decreased cell adhesion and disordered nuclear arrangement of low grade urothelial neoplasia. Diagn. Cytopathol. 2014;42:555–563.

Factors influencing a specific pathologic diagnosis of non-small-cell lung carcinoma.

INTRODUCTION Historically, a non-small-cell lung carcinoma diagnosis, without pathologic subclassification, provided sufficient information to guide therapy. Evidence now demonstrates that pathologic subtype classification is central in selecting optimal treatment. This review aimed to identify factors associated with a specific pathologic diagnosis. METHODS All nonoperative cases of non-small-cell lung carcinoma (NSCLC) referred to the medical oncology divisions of the Ottawa Hospital Cancer Centre (2008) and Princess Margaret Hospital, Toronto (2007-2010) were identified. The charts were reviewed for demographics, diagnostic methods, and final diagnosis. Logistic regression was performed to identify variables associated with a specific diagnosis. RESULTS Of 739 patient records analyzed, 377 (51%) were men, 299 (40%) were aged over 70 years, and 510 (69%) had an Eastern Cooperative Oncology Group performance status of 0-2. Three hundred and eighty five (52%) of patients were diagnosed in a tertiary academic center. The lung primary was sampled in 503 (68%) of patients. Computed tomography-guided biopsy (n = 370, 50%) and bronchoscopy (n = 179, 24%) were the most common techniques. Four hundred and seventy seven (65%) of biopsies were cytologic specimens alone, and immunohistochemistry was performed in 337 (46%) of cases. The most common diagnoses were adenocarcinoma (n = 338, 46%), NSCLC not otherwise specified (n = 254, 34%), and squamous cell carcinoma (n = 115, 16%). Overall, 456 (62%) of patients received a specific pathologic diagnosis. Factors significantly associated with attaining a specific pathologic diagnosis were diagnosis outside an academic center (adjusted odds ratios [OR] 2.1 [95% CI, 1.41-3.14]; P = .0003), histologic laboratory samples (adjusted OR 1.58 [95% CI, 1.003-2.49]; P = .049), and immunohistochemical testing (adjusted OR 1.82 [95% CI, 1.25-2.70], P = .0021). CONCLUSIONS A significant minority of patients with NSCLC do not receive a specific pathologic diagnosis. In an era of individualized medicine, this may potentially impact optimal clinical management.

Cyclooxygenase-2 (COX-2) inhibition for prostate cancer chemoprevention: double-blind randomised study of pre-prostatectomy celecoxib or placebo.

To evaluate the biological effects of selective cyclooxygenase‐2 inhibition on prostate tissue in patients undergoing radical prostatectomy (RP).

Correction: Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion

[This corrects the article DOI: 10.1371/journal.ppat.1005446.].

Translation of Knowledge to Practice—Improving Awareness in NSCLC Molecular Testing

Background: Molecular testing in advanced lung cancer is standard in guiding treatment selection. However, population‐wide implementation of testing remains a challenge. We developed a knowledge translation intervention to improve understanding among diagnostic specialists about molecular testing and appropriate diagnostic sampling in lung cancer. Methods: Specialty‐specific education programs were developed from existing literature and input from Canadian leaders in lung pathology, respirology, interventional radiology, thoracic surgery, radiation oncology, and medical oncology. The programs, including key messages, review of current data, existing guidelines, group discussion, and participant feedback, were administered at provincial and national specialty meetings. Participant knowledge was assessed before and after the intervention by using anonymous questionnaires. Molecular (EGFR) testing rates in Ontario were also evaluated before and after the intervention period. Results: Ten programs were administered to diagnostic specialists, including respirologists, pathologists, thoracic surgeons, radiologists, radiation oncologists, and medical oncologists, with completion of 255 preintervention and 219 postintervention surveys. At baseline, 30% were unsure of tissue handling methods for molecular testing, 20% chose an incorrect technique, and half were unfamiliar with how to initiate testing. After intervention, specialist knowledge improved regarding tissue handling and appropriate fixation techniques and uncertainty decreased from 30% to 2% (p < 0.001). A 12% increase (relative increase 57%) in molecular (EGFR) testing requests in Ontario was observed over the intervention period (p = 0.0032). Conclusions: Significant knowledge gaps exist among diagnostic specialists regarding molecular testing and targeted therapy in lung cancer. This initiative significantly improved understanding of the importance and methods of successful molecular testing and correlated with increased testing rates.