Digital quantification of p16-positive foci in fibrotic interstitial lung disease is associated with a phenotype of idiopathic pulmonary fibrosis with reduced survival.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is associated with increased expression of cyclin-dependent kinase inhibitors such as p16 and p21, and subsequent induction of cell cycle arrest, cellular senescence, and pro-fibrotic gene expression. We sought to link p16-expression with a diagnosis of IPF or other fibrotic interstitial lung diseases (ILDs), radiographic pattern, senescent foci-specific gene expression, antifibrotic therapy response, and lung transplant (LTx)-free survival. METHODS: Eighty-six cases of fibrosing ILD were identified with surgical lung biopsy. Immunohistochemistry for p16 was performed on sections with the most active fibrosis. p16-positive foci (loose collection of p16-positive fibroblasts with overlying p16-positive epithelium) were identified on digital slides and quantified. Cases were scored as p16-low (≤ 2.1 foci per 100 mm2) or p16-high (> 2.1 foci per 100 mm2). Twenty-four areas including senescent foci, fibrotic and normal areas were characterized using in situ RNA expression analysis with digital spatial profiling (DSP) in selected cases. RESULTS: The presence of p16-positive foci was specific for the diagnosis of IPF, where 50% of cases expressed any level of p16 and 26% were p16-high. There was no relationship between radiographic pattern and p16 expression. However, there was increased expression of cyclin-dependent kinase inhibitors, collagens and matrix remodeling genes within p16-positive foci, and cases with high p16 expression had shorter LTx-free survival. On the other hand, antifibrotic therapy was significantly protective. DSP demonstrated that fibroblastic foci exhibit transcriptional features clearly distinct from that of normal-looking and even fibrotic areas. CONCLUSIONS: We demonstrated the potential clinical applicability of a standardized quantification of p16-positive fibroblastic foci. This method identifies an IPF phenotype associated with foci-specific upregulation of senescence-associated and matrix remodeling gene expression. While these patients have reduced LTx-free survival, good response to antifibrotic therapies was observed in those who were treated.

Osteopontin lung gene expression is a marker of disease severity in pulmonary arterial hypertension.

BACKGROUND AND OBJECTIVE: Osteopontin (OPN) is a pleiotropic cytokine involved in the proliferation of pulmonary artery smooth muscle cells (PA-SMC). OPN is upregulated in the lungs of patients with pulmonary hypertension (PH) associated with pulmonary fibrosis, suggesting that the lung is a source of OPN. We hypothesized that OPN lung expression is elevated in Group I pulmonary arterial hypertension (PAH) and is correlated to haemodynamics. METHODS: Microarray analysis (Affymetrix) was performed after RNA was extracted from explanted lungs in 15 patients with Group I PAH who underwent lung transplantation (LTx) and 11 normal controls. PA pressure levels were recorded intraoperatively, immediately before starting LTx. Serum OPN levels were measured in subjects with PAH, Group II PH and normal controls on the day of right heart catheterization. RESULTS: OPN was among the top five upregulated genes in PAH compared to normal controls, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR). OPN expression was similar and equally elevated in different subtypes of PAH. A strong significant correlation was observed between mean pulmonary arterial pressure and OPN gene expression. Ingenuity pathway analysis showed the involvement of OPN in functions and networks relevant to angiogenesis, cell death and proliferation of PA-SMC. OPN serum levels did not differ in subjects with Group I PAH and Group II PH. CONCLUSION: In the lungs of patients with severe PAH, OPN is highly expressed and the level of expression is significantly correlated to disease severity. OPN may play an important role in the vascular remodelling process of PAH.

Comprehensive gene expression profiling identifies distinct and overlapping transcriptional profiles in non-specific interstitial pneumonia and idiopathic pulmonary fibrosis.

BACKGROUND: The clinical-radiographic distinction between idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) is challenging. We sought to investigate the gene expression profiles of IPF and NSIP vs. normal controls. METHODS: Gene expression from explanted lungs of patients with IPF (n = 22), NSIP (n = 10) and from normal controls (n = 11) was assessed. Microarray analysis included Significance Analysis of Microarray (SAM), Ingenuity Pathway, Gene-Set Enrichment and unsupervised hierarchical clustering analyses. Immunohistochemistry and serology of proteins of interest were conducted. RESULTS: NSIP cases were significantly enriched for genes related to mechanisms of immune reaction, such as T-cell response and recruitment of leukocytes into the lung compartment. In IPF, in contrast, these involved senescence, epithelial-to-mesenchymal transition, myofibroblast differentiation and collagen deposition. Unlike the IPF group, NSIP cases exhibited a strikingly homogenous gene signature. Clustering analysis identified a subgroup of IPF patients with intermediate and ambiguous expression of SAM-selected genes, with the interesting upregulation of both NSIP-specific and senescence-related genes. Immunohistochemistry for p16, a senescence marker, on fibroblasts differentiated most IPF cases from NSIP. Serial serum levels of periostin, a senescence effector, predicted clinical progression in a cohort of patients with IPF. CONCLUSIONS: Comprehensive gene expression profiling in explanted lungs identifies distinct transcriptional profiles and differentially expressed genes in IPF and NSIP, supporting the notion of NSIP as a standalone condition. Potential gene and protein markers to discriminate IPF from NSIP were identified, with a prominent role of senescence in IPF. The finding of a subgroup of IPF patients with transcriptional features of both NSIP and senescence raises the hypothesis that "senescent" NSIP may represent a risk factor to develop superimposed IPF.

Successful Treatment of Fibrosing Organising Pneumonia Causing Respiratory Failure with Mycophenolic Acid.

Organising pneumonia (OP) is usually promptly responsive to corticosteroid treatment. We describe a series of 3 cases of severe, progressive, biopsy-proven fibrosing OP causing respiratory failure. All cases presented with peribronchial and subpleural consolidations, had a fibro-inflammatory infiltrative component in the alveolar septa, and only had a partial and unsatisfactory response to corticosteroids. However, they responded to mycophenolic acid (MPA) treatment with resolution of respiratory failure as well as clinical and functional improvement. MPA as an additional treatment option for aggressive forms of fibrosing OP and interstitial lung disease needs to be further explored.

A case of tinea incognito.

Tinea incognito is a dermatophyte infection of the skin that presents atypically because it has previously been treated with imunnosuppresive medication. Herein we present a case of a middle-aged man who was initially clinically diagnosed to have plaque-type psoriasis on his arms. Over the course of two months of topical hydrocortisone and calciptriol treatment as well as phototherapy, the rash worsened. At the time of presentation to hospital the patient had a pruritic, widespread, sloughing, erythematous rash with areas of eschar. A punch biopsy skin confirmed dermatophyte fungal infection of the skin. Fungal culture was positive for Trichophyton Rubrum and the eruption resolved with systemic anti-fungal therapy. Patient specific risk factors for atypical presentation included poor hygiene and hepatatic disease.

The effect of tidal volume on systemic inflammation in Acid-induced lung injury.

BACKGROUND: Overwhelming systemic inflammation has been implicated in the progression of acute lung injury (ALI) leading to multiple organ failure (MOF) and death. Previous studies suggest that mechanical ventilation (MV) may be a key mediator of MOF through an upregulation of the systemic inflammatory response. OBJECTIVES: It was the aim of this study to investigate mechanisms whereby mechanical stress induced by different tidal volumes may contribute to the development of systemic inflammation and maladaptive peripheral organ responses in the setting of ALI. METHODS: An acid aspiration model of ALI was employed in 129X1/SVJ mice through an intratracheal administration of hydrochloric acid followed by MV employing either a low (5 ml/kg) or high (12.5 ml/kg) tidal volume ventilation for 120 min. The isolated perfused mouse lung setup was used to assess the specific contribution of the lung to systemic inflammation during MV. Furthermore, lung perfusate collected over the course of MV was used to assess the effects of lung-derived mediators on activation (expression of a proadhesive phenotype) of liver endothelial cells. RESULTS: High tidal volume MV of acid-injured lungs resulted in greater physiologic and histological indices of lung injury compared to control groups. Additionally, there was an immediate and significant release of multiple inflammatory mediators from the lung into the systemic circulation which resulted in greater levels of mRNA adhesion molecule expression in liver endothelial cells in vitro. CONCLUSIONS: This study suggests that MV, specifically tidal volume strategy, influences the development of MOF through an upregulation of lung-derived systemic inflammation resulting in maladaptive cellular changes in peripheral organs.

A Rare Case of Inflammatory Myofibroblastic Tumor Causing Left Pulmonary Artery Stenosis and Successfully Treated with Localized Radiotherapy in a Patient with Perinuclear Antineutrophil Cytoplasmic Antibody Vasculitis.

Inflammatory myofibroblastic tumor (IMT) of the lung is a rare neoplasm that commonly behaves in an indolent fashion and is generally treated with complete surgical excision. The management of unresectable IMT presents a significant challenge, especially in cases with multiple comorbidities, and a consensus has yet to be reached on the most appropriate first-line modality. We present a case of unresectable IMT causing severe stenosis of the left pulmonary artery in a patient on immunosuppressive therapy for perinuclear antineutrophil cytoplasmic antibody vasculitis. The patient was successfully treated with localized radiotherapy to a total dose of 45 Gy in five weeks, and has been followed for more than seven years since treatment. In this case report, we review the pertinent literature and illustrate the difficulties in diagnosing and treating rare neoplasms in a patient with significant medical comorbidities.

Topical imiquimod therapy for lentigo maligna.

Lentigo maligna (LM) presents a challenge for complete surgical excision. Imiquimod is a topical immune-response modifier that acts on the immune system. We report our experience using imiquimod 5% cream as a surgical alternative for treatment of LM. Consecutive patients between December 2004 and February 2006 with LM were treated with topical imiquimod. Data on patient and lesion characteristics, side effects of therapy, posttreatment biopsy results, and follow-up was collected. Seven patients were treated with imiquimod 5 nights/wk for 12.4 weeks. Complete histologic and clinical resolution was seen in 86% (6 of 7 patients), at 19.1 months follow-up. Side effects included erythema (86%) and crusting (71%), resulting in dose alteration in 71% of patients. Topical imiquimod therapy demonstrates a high response rate for treatment of LM, with tolerable side effects. Further investigation into its efficacy in the treatment of LM in controlled clinical trials is warranted.

Endobronchial Carcinoid Tumour with Extensive Ossification: An Unusual Case Presentation.

Carcinoid tumour is a well-known primary endobronchial lung neoplasm. Although calcifications may be seen in up to 30% of pulmonary carcinoid tumours, near complete ossification of these tumours is an unusual finding. Such lesions can prove diagnostically challenging at the time of intraoperative frozen section as the latter technique requires thin sectioning of the lesion for microscopic assessment. We present an unusual case of endobronchial carcinoid tumour with extensive ossification in a 45-year-old male. Preliminary intraoperative diagnosis was achieved through the alternative use of cytology scrape smears. The final diagnosis was confirmed after decalcification of the tumour. The prognostic implications of heavily ossified carcinoid tumours remain elusive. Long-term clinical follow-up of these patients is recommended.

Performance of residents using digital images versus glass slides on certification examination in anatomical pathology: a mixed methods pilot study.

BACKGROUND: It is anticipated that many licensing examination centres for pathology will begin fully digitizing the certification examinations. The objective of our study was to test the feasibility of a fully digital examination and to assess the needs, concerns and expectations of pathology residents in moving from a glass slide-based examination to a fully digital examination. METHODS: We conducted a mixed methods study that compared, after randomization, the performance of senior residents (postgraduate years 4 and 5) in 7 accredited anatomical pathology training programs across Canada on a pathology examination using either glass slides or digital whole-slide scanned images of the slides. The pilot examination was followed by a post-test survey. In addition, pathology residents from all levels of training were invited to participate in an online survey. RESULTS: A total of 100 residents participated in the pilot examination; 49 were given glass slides instead of digital images. We found no significant difference in examination results between the 2 groups of residents (estimated marginal mean 8.23/12, 95% confidence interval [CI] 7.72-8.87, for glass slides; 7.84/12, 95% CI 7.28-8.41, for digital slides). In the post-test survey, most of the respondents expressed concerns with the digital examination, including slowly functioning software, blurring and poor detail of images, particularly nuclear features. All of the respondents of the general survey (n = 179) agreed that additional training was required if the examination were to become fully digital. INTERPRETATION: Although the performance of residents completing pathology examinations with glass slides was comparable to that of residents using digital images, our study showed that residents were not comfortable with the digital technology, especially given their current level of exposure to it. Additional training may be needed before implementing a fully digital examination, with consideration for a gradual transition.

Incidental Merkel cell carcinoma in a cutaneous horn: a case report.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine malignancy, which usually presents as an asymptomatic, rapidly growing, firm nodule on sun-damaged skin. We present a 93-year-old female who presented with a "cutaneous horn" on the face. On excision, histologic examination revealed a combined squamous cell carcinoma in situ with underlying MCC. Merkel cell polyomavirus immunohistochemistry was negative in this lesion. This case report highlights the significant association between MCC and squamous cell carcinoma and the uncommon clinical presentation of this combined tumor in the form of a cutaneous horn.

Effects of nebulized diethylenetetraamine-NONOate in a mouse model of acute Pseudomonas aeruginosa pneumonia.

OBJECTIVE: Endogenous and exogenous nitric oxide (NO) may have important antibacterial effects in patients with pneumonia. NO administration has been limited to the continuous inhalation of gas-phase NO (ie, inhaled NO [iNO]). Intermittent nebulization of NONOates, novel NO donors, may permit the continuous intrapulmonary delivery of NO. Thus, we assessed the effects of nebulized diethylenetetraamine-NONOate (DETA-NO) in a model of acute Pseudomonas aeruginosa pneumonia. DESIGN: Randomized, controlled study. SUBJECTS: Male C57Bl/6 mice. INTERVENTIONS: Pneumonia was induced by intratracheal instillation of P aeruginosa (3 x 10(7) CFU in 50 microL). Pneumonia and sham mice were randomized to receive no treatment, nebulized DETA-NO (12.5 or 125 micromol) at 4 h and 12 h, or continuous iNO for 24 h (10 or 40 ppm) until they were killed at 24 h. MAIN RESULTS: The nebulization of DETA-NO was associated with a marked increase in mean (+/- SEM) exhaled NO levels (after nebulization, 484 +/- 34 parts per billion [ppb]; baseline, 13.4 +/- 0.4 ppb; p < 0.01) and plasma levels of nitrites/nitrates (after nebulization, 73 +/- 28 microM; at baseline, 14 +/- 3 microM; p < 0.05). Nebulized DETA-NO decreased the pulmonary bacterial load in mice with pneumonia by 65 +/- 19% (p < 0.05 vs untreated mice) but had no effect on pulmonary leukocyte infiltration. Although the growth of P aeruginosa colonies in vitro was impaired on exposure to DETA-NO, growth was similarly impaired by exposure to DETA nucleophile/backbone alone. CONCLUSIONS: The nebulization of DETA-NO provides a method for the prolonged intrapulmonary delivery of NO. The antibacterial effect of DETA-NO in vivo and in vitro is due, in large part, to the DETA nucleophile moiety and is independent of NO, suggesting a limited therapeutic role for exogenous NO in pneumonia.

Current biopsy practices for suspected melanoma: A survey of family physicians in Southwestern Ontario.

BACKGROUND: Family physicians (FPs) have an opportunity to diagnose pigmented lesions early with a timely biopsy. OBJECTIVE: To assess current biopsy practices used by FPs in diagnosing melanoma. METHODS: A computer-generated random sample of 200 practicing FPs from large and small communities in Southwestern Ontario was identified from the College of Physicians and Surgeons of Ontario physician directory. Paper-based surveys exploring practice setting, basic melanoma knowledge, biopsy practices and referral wait times were mailed using a modified Dillman protocol. RESULTS: The response rate was 50% and respondents reflected the demographic characteristics of FPs nationwide as per the National Physician Survey. Knowledge testing revealed reasonable mean (± SD) scores (3.2±1.03 of 5). Twenty percent of respondents would always perform an excisional biopsy of skin lesions suspicious for melanoma. The remaining 80% would avoid an excisional biopsy in an aesthetically sensitive area and if there was risk of failure to close the defect primarily, among other reasons. If an excisional biopsy were not performed, one-half of respondents would perform an incisional biopsy (eg, punch biopsy). In large communities, 24% of patients were not seen by a surgeon within six months when referred without a tissue biopsy, leading to delayed diagnosis. DISCUSSION: Educating and supporting FPs to perform incisional biopsies in cases for which excisional biopsies are inappropriate should result in earlier diagnosis of melanoma. CONCLUSION: FPs appropriately recognize that excisional biopsies are ideal in melanoma management and one-half will move on to an incisional biopsy when excision is not appropriate.

HISTORIQUE: Les médecins de famille (M) peuvent diagnostiquer des lésions pigmentées rapidement grâce à une biopsie. OBJECTIF: Évaluer les pratiques de biopsie actuelles des MF pour diagnostiquer un mélanome. MÉTHODOLOGIE: Un échantillon aléatoire créé par ordinateur de 200 MF en exercice de grandes et petites collectivités du sud-ouest de l'Ontario a été extrait du répertoire de l'Ordre des médecins et chirurgiens de l'Ontario. On leur a posté des sondages papier au sujet de leur lieu de pratique, de leurs connaissances de base sur les mélanomes, de leurs pratiques de biopsie et des temps d'attente avant l'aiguillage, selon un protocole de Dillman modifié. RÉSULTATS: Le taux de réponse s'élevait à 50 % et les répondants reflétaient les caractéristiques démographiques des MF au pays, conformément au Sondage national des médecins. Les tests sur les connaissances ont révélé des indices moyens (± ÉT) raisonnables (3,2±1,03 sur 5). Vingt pour cent des répondants effectueraient toujours une biopsie-exérèse des lésions cutanées en cas de présomption de mélanome. Les 80 % restants éviteraient une biopsie-exérèse dans une zone esthétiquement fragile et en présence d'un risque de ne pas fermer l'anomalie primaire, entre autres. S'ils rejetaient la biopsie-exérèse, la moitié des répondants effectueraient une biopsie incisionnelle (p. ex., biopsie à l'emporte-pièce). Dans les grandes collectivités, 24 % des patients n'étaient pas vus par un chirurgien dans les six mois s'ils étaient aiguillés sans biopsie des tissus, ce qui s'associait à un retard de diagnostic. EXPOSÉ: L'enseignement et le soutien des MF à effectuer une biopsie incisionnelle lorsque la biopsie-exérèse ne convient pas devraient favoriser un diagnostic plus rapide des mélanomes. CONCLUSION: Le MF indiquait correctement que les biopsies-exérèses sont idéales pour prendre en charge les mélanomes et la moitié opterait pour une biopsie incisionnelle lorsque l'excision ne convient pas.

Obtaining high cure rates for challenging facial malignancies: A new method for producing rapid, accurate, high-quality frozen sections.

PURPOSE: The authors developed a new system to provide rapid, accurate, full-face frozen sections. OBJECTIVE: To evaluate the efficacy of the system when applied to the treatment of nonmelanoma cutaneous malignancies using Mohs micrographic surgery (MMS). METHODS: Patients undergoing MMS procedures between 2003 and 2007 for nonmelanoma head and neck cutaneous malignancies were prospectively collected. Specimens were prepared either in a traditional cryostat-based manner or using the new system. RESULTS: A total of 196 patients with 234 head and neck nonmelanoma cutaneous malignancies were included. The majority of tumours were basal cell carcinomas (89.5%). Of these, 38% demonstrated aggressive histologies (sclerosing or micronodular), and 30% were recurrent. On average, two levels (range one to six) and four blocks (range two to 23) were required to obtain clear margins. The mean defect size was 3.68 cm(2) (range 0.13 cm(2) to 37.68 cm(2)). Over the five-year study period, there were two recurrences in 234 cases (less than 1%), which compares favourably with other MMS series. The new system was associated with a shorter operative time than traditional specimen preparation (102 min versus 131 min; P=0.004). The new and traditional specimen preparation groups were similar in terms of the number of previous recurrences (29% versus 30%; P=1.00), defect size (3.7 cm(2) versus 4.0 cm(2); P=0.81) and the number of levels required (1.9 versus 1.5; P=0.05). CONCLUSIONS: The new system enables fast, accurate, full-face frozen section specimens that are ideal for MMS. The speed of specimen preparation is demonstrated by faster operative times, and a low recurrence rate attests the accuracy and quality of the sections.

The perimeter technique for lentigo maligna: an alternative to Mohs micrographic surgery.

BACKGROUND: Lentigo maligna (LM) presents a challenge for complete surgical excision because of its extensive subclinical spread and predilection for the face. OBJECTIVE: To report our experience using the staged perimeter technique as an alternative to Mohs micrographic surgery for treatment of LM. METHODS: The perimeter procedure was performed on 11 patients with LM between March 2003 and June 2004. Data on patient and lesion characteristics, number of stages required to obtain clear margins, and follow-up was obtained by chart review. RESULTS: A mean of 1.9 stages were required to achieve clear margins. A mean of 7 tissue specimens were sent to pathology per patient for evaluation. After a mean follow-up of 4.7 months, all patients were free of recurrence. CONCLUSIONS: The perimeter technique is a simple method of margin-controlled excision of LM. The main advantage is that all margins are examined with permanent sections. The main drawback is that multiple operative sessions are required to complete the procedure. This technique does not require specific Mohs training and is therefore applicable to non-Mohs surgeons.

Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis.

The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS-/-, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to - (iNOS+/+ donor bone marrow-transplanted into iNOS-/- recipient mice) and - to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS-/- mice and in - to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to - chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.