The Daily Sign-Out

DNA Methylation and Gene Expression in Idiopathic Pulmonary Fibrosis

In case you may have missed this one, Ivana Yang and colleagues from the University of Colorado have published a fascinating article in the December 1, 2014 issue of American Journal of Respiratory and Critical Care Medicine, "Relationship of DNA Methylation and Gene Expression in Idiopathic Pulmonary Fibrosis," that explores possible epigenetic mechanisms through DNA methylation that may be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF).

Many of these studies are being done in various cancers, but this one suggests one or more programs that unfold with IPF.

 

Multifocal Micronodular Pneumocyte Hyperplasia Mimicking Miliary TB

Saturday morning interesting case report!

Download Multifocal pneumocyte hyperplasia mimicking miliary TB case report Lee 2014

 

Primary Ciliary Dyskinesia: Update and Review

 A very thorough and up-to-date review of primary ciliary dyskinesia, which is a heterogenous group of related disorders.  Most of us learned about the diagnostic ultrastructural abnormalities described in these patients, but one point of interest for me is that up to 30% of patients have normal cilia by EM and/or nondiagnostic ciliary waveform changes.  Thus, some of the "pearls" of this paper for me was information on a new diagnostic test measuring nasal nitric oxide and annotation of PCD-causing genetic mutations.  Finally, check out the links to these sites which provide a wealth of additional information on these disorders.  Great reference paper for anyone interested in pulmonary medicine and pathology.

 

Genetic Disorders of Mucociliary Clearance Consortium

Primary Ciliary Disease Foundation

 

Michael R. Knowles, Leigh Anne Daniels, Stephanie D. Davis, Maimoona A. Zariwala, and Margaret W. Leigh "Primary Ciliary Dyskinesia. Recent Advances in Diagnostics, Genetics, and Characterization of Clinical Disease", American Journal of Respiratory and Critical Care Medicine, Vol. 188, No. 8 (2013), pp. 913-922.

doi: 10.1164/rccm.201301-0059C 

Abstract

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia that leads to oto-sino-pulmonary diseases and organ laterality defects in approximately 50% of cases. The estimated incidence of PCD is approximately 1 per 15,000 births, but the prevalence of PCD is difficult to determine, primarily because of limitations in diagnostic methods that focus on testing ciliary ultrastructure and function. Diagnostic capabilities have recently benefitted from (1) documentation of low nasal nitric oxide production in PCD and (2) discovery of biallelic mutations in multiple PCD-causing genes. The use of these complementary diagnostic approaches shows that at least 30% of patients with PCD have normal ciliary ultrastructure. More accurate identification of patients with PCD has also allowed definition of a strong clinical phenotype, which includes neonatal respiratory distress in >80% of cases, daily nasal congestion and wet cough starting soon after birth, and early development of recurrent/chronic middle-ear and sinus disease. Recent studies, using advanced imaging and pulmonary physiologic assessments, clearly demonstrate early onset of lung disease in PCD, with abnormal air flow mechanics by age 6–8 years that is similar to cystic fibrosis, and age-dependent onset of bronchiectasis. The treatment of PCD is not standardized, and there are no validated PCD-specific therapies. Most patients with PCD receive suboptimal management, which should include airway clearance, regular surveillance of pulmonary function and respiratory microbiology, and use of antibiotics targeted to pathogens. The PCD Foundation is developing a network of clinical centers, which should improve diagnosis and management of PCD.

 

Chronic Bronchitis and Chronic Obstructive Lung Disease Review

Although commonly thought of synonymous, or, at least, forming a spectrum, chronic bronchitis is distinct from COPD.  This review focuses on the pathophysiology of chronic bronchitis and goblet cell hyperplasia and the association between small airways disease and worse outcomes in patients with COPD.  A state-of-the-art review.

Victor Kim and Gerard J. Criner "Chronic Bronchitis and Chronic Obstructive Pulmonary Disease", American Journal of Respiratory and Critical Care Medicine, Vol. 187, No. 3 (2013), pp. 228-237.

doi: 10.1164/rccm.201210-1843CI

 

Abstract

Chronic bronchitis (CB) is a common but variable phenomenon in chronic obstructive pulmonary disease (COPD). It has numerous clinical consequences, including an accelerated decline in lung function, greater risk of the development of airflow obstruction in smokers, a predisposition to lower respiratory tract infection, higher exacerbation frequency, and worse overall mortality. CB is caused by overproduction and hypersecretion of mucus by goblet cells, which leads to worsening airflow obstruction by luminal obstruction of small airways, epithelial remodeling, and alteration of airway surface tension predisposing to collapse. Despite its clinical sequelae, little is known about the pathophysiology of CB and goblet cell hyperplasia in COPD, and treatment options are limited. In addition, it is becoming increasingly apparent that in the classic COPD spectrum, with emphysema on one end and CB on the other, most patients lie somewhere in the middle. It is known now that many patients with severe emphysema can develop CB, and small airway pathology has been linked to worse clinical outcomes, such as increased mortality and lesser improvement in lung function after lung volume reduction surgery. However, in recent years, a greater understanding of the importance of CB as a phenotype to identify patients with a beneficial response to therapy has been described. Herein we review the epidemiology of CB, the evidence behind its clinical consequences, the current understanding of the pathophysiology of goblet cell hyperplasia in COPD, and current therapies for CB.

 

All the RAGE in Asthma

My lab's CAP inspection is finally over!!! Now I can get back to more regular blogging (without guilt).

This month's American Journal of Pathology features an intriguing article that proposes a central role for receptor for advanced glycation end products (RAGE) in the pathogenesis of asthma (abstract).  

First, some background.  RAGE is a multiligand receptor for ligands including S100 proteins, HMGB1, amyloid beta, and heparin.  There are two forms of RAGE: membrane RAGE (mRAGE) which is thought to be proinflammatory, and soluble (i.e., cytoplasmic) RAGE (sRAGE) considered to be anti-inflammatory.  RAGE transcripts and proteins are preferentially expressed in lung.

This study uses a house dust mite (HDM) antigen sensitization/challenge model in RAGE knockout (RAGE-ko) mice to directly assess the role of RAGE in asthma/allergic airway disease (A3D).  The authors confirm these results using a different model, ovalbumin, to confirm that the same effects of RAGE are independent of the antigen challenge.

The authors measured cell counts in BALF obtained from Diff-Quik stained sections; they measured BALF for levels of eotaxin, eotaxin-2, IL-4, IL-5, and IL-13 by ELISA.  The prepared RNA from whole lung and performed qRT-PCR for RAGE, IL-5, and IL-13.  FFPE lung was examined for RAGE by immunofluorescence and tissue eosinophilia. Finally (if you can picture this), they measured respiratory mechanics in both wt and RAGE-ko mice.

Most of the measures of A3D are not seen in RAGE-knockout mice after HDM challenge compared to wt mice.  Following HDM challenge, RAGE-knockout mice showed no significant changes in pulmonary function testing; no increased eosinophilic infiltrates, goblet cell hyperplasia, or increased mucin expression; no increases in IL-5, IL-13, eotaxin, or eotaxin-2.  The latter suggests that canonical mediators of eosinophil recruitment and airway remodeling are abrogated in the absence of RAGE and that RAGE likely acts as a proximal mediator in one or more proinflammatory pathways that lead to the functional and pathologic changes seen in A3D.  However, IL-4, IgE, and HDM-specific IgG1 are the same between wt and RAGE-knockout mice--indicating that humoral immunity remains intact in the absence of RAGE and that IgE alone does not appear to play a significant role in this model.  The ovalbumin model showed similar data indicating that the role of RAGE in murine A3D is independent of the particular epitopes involved in antigen sensitization and challenge.

This is a very interesting paper that challenges some longstanding ideas about the understanding of the pathogenesis of asthma.  Certainly worth noting as follow-up studies come out.

 

Hypersensitivity Pneumonitis: recent updates

There have been two recent papers reviewing hypersensitivity pneumonitis (HP) that I had to summarize for myself and am sharing with you. While idiopathic pulmonary fibrosis/usual interstitial pneumonitis (IPF/UIP) may be the prototype for interstitial lung disease, I think that hypersensitivity pneumonitis is more frequently encountered or strongly considered in the differential diagnosis as a non-neoplastic lung biopsy specimen in “the daily sign-out,” at least in community-based general surgical pathology practice, and especially since the majority of IPF cases are now diagnosed by high-resolution CT scans without biopsy. Thus, being a practical sort, I suggest that a solid appreciation for the clinical, radiographic and pathological features of HP would help resolve many cases you might encounter in practice.

The paper by Selman, Pardo and King in the August 15, 2012 Am J Respir Crit Care Med (abstract) is more clinically oriented, while the paper by Herbst and Myers in the August 2012 Arch Pathol Lab Med (abstract) focuses on the role of surgical biopsy in the diagnosis of HP. I refer (defer?) to the Dail and Hammar Pulmonary Pathology textbook for the definitive description of the pathological findings in HP. (I also suggest that curling that textbook--volume 1--with 10 reps for each arm will give you beastly biceps in a few weeks’ time (hey--Kindle version anytime soon?).

One of the interesting aspects of the AJRCCM paper is the discussion on pathogenesis. The authors review the “2-hit” hypothesis: that there is a pre-existing genetic susceptibility and/or environmental factors, followed by exposure to one of the many antigens associated with HP. The antigens can be roughly divided into 3 groups: 1) thermophilic actinomycetes (Saccharopolyspora rectivirgula) as well as a variety of other fungi (Aspergillus, Penicillium spp); 2) a complex mixture of high- and low-molecular weight proteins from avian serum, feces, and feathers; 3) water-borne non-tuberculous mycobacteria. Importantly, no genetic factors have been consistently associated with HP. But certain polymorphisms have been identified that are associated with increased risk, including HLA-DR and -DQ polymorphisms, polymorphisms in the genes coding for TAP transport peptides MHC molecules for presentation to cytotoxic T cells, and polymorphisms in β type 8 (PSMB8)--an immunoproteasome catalytic subunit that participates in degradation of ubiquinated proteins to generate peptides for antigen presentation by class I MHC molecules. Finally, the authors discuss the role of cigarette smoking in HP. It is somewhat curious but known clinically that HP is less frequent in smokers compared to non-smokers. It is hypothesized that nicotine blunts the response to antigen exposure by its effects on decreasing macrophage activation, decreasing lymphocyte proliferation, and impairing T-cell function. However, when HP does occur in smokers, they tend to develop a chronic clinical course characterized by more recurrent episodes of disease and have a poorer survival rate compared with HP in non-smokers.

Classically (“”), HP is divided into acute, subacute, and chronic forms. However, there are no accepted clinical criteria that distinguish the various forms. Moreover, there is little information regarding the latency period between antigen exposure and disease onset. Indeed, it is uncertain that these divisions truly represent different stages of the disease.

Recently, cluster analysis studies suggest a two-cluster model for HP that correlates with observed clinical profile and the type of antigen exposure. Cluster 1 corresponds to the clinically “acute” form of HP. It is associated with an influenza-like syndrome occurring a few hours after a substantial exposure, more recurrent systemic symptoms, and a normal chest xray. Cluster 1 tends to occurs in individuals exposed to thermophilic actinomyces or fungi (≈”farmer’s lung”). Cluster 2 corresponds to the clinically “chronic” form of HP. It is associated with more features of severe, chronic disease (e.g. digital clubbing, hypoxemia, restrictive pattern by PFTs, fibrosis on HRCT scans) as well as slowly progressive chronic respiratory disease. This cluster tends to occur in individuals with bird antigen exposure. As pointed out by Herbst and Myers, this is the form one would be more likely to encounter in a biopsy situation as it mimics IPF/UIP or fibrotic NSIP. Of note, HRCT features that would favor HP over UIP/NSIP include lobular areas with decreased attenuation and air trapping, centrilobular nodules, and lack of lower zone predominance. The cluster analysis by 

Pathology of HP

As one may have surmised already, “acute” HP is just not biopsied very often so it is not very well-characterized. When specimens have been examined in which patients were diagnosed clinically with acute HP, the histologic findings resemble acute fibrinous organizing pneumonia. The “chronic” form of HP is much better studied and characterized as consists of a histologic triad of chronic interstitial inflammation, nonnecrotizing granulomas, and cellular bronchiolitis.  While this triad is seen in around 80% of patients with the clinical diagnosis of HP, it is not specific for HP.

The chronic interstitial infiltrate consists of a patchy and variably intense lymphoplasmacytic interstitial infiltrate that is frequently most intense around small bronchi and bronchioles. A salient point by Herbst and Myers (that I have noticed myself as well) is that this infiltrate may be accompanied by alveolar macrophages in adjacent alveoli with occasional aggregates of foamy macrophages suggesting lipid pneumonia. They also note that large numbers of foamy macrophages are frequently seen in pigeon-breeder’s disease. Importantly, necrosis and vasculitis are not seen. Nonnecrotizing granulomas may be found around respiratory bronchi or bronchioles as well as in the interstitium or in alveoli (so basically anywhere). These are “poorly-formed” (cf. sarcoidal granulomas) and composed of epithelioid histiocytes and Langhans’ giant cells. One helpful tip is the use of cathepsin K by IHC to help identify microgranulomas, particularly in “subacute” cases, but I think it would probably be helpful when granulomas don’t pop out at you. Another notable point is that the giant cells frequently contain cholesterol clefts, Schaumann bodies, and/or birefringent oxalate crystals. The cellular bronchiolitis is somewhat self-explanatory btu is primarily lymphocytic and extends into adjacent alveolar interstitium.

Herbst and Myers also point out some other significant “soft” findings that can be valuable in the diagnosis of HP. Small foci of organizing pneumonia pattern are found in up to 60% of patients diagnosed with HP. Small airways involvement may be found (more frequently in farmer’s lung than pigeon breeder’s disease) and adds an obstructive component. Mast cells are a constant finding and are frequently found in areas of interstitial fibrosis. And last but not least--interstitial fibrosis. Significant intersitial fibrosis is frequently found in patients who are biopsied months to years after the onset of symptoms as well as in those who have repetitive or persistent exposure to the putative antigen. Histologically, the fibrosis may have a UIP-like pattern identical to IPF.

As a personal note, I have found that the diagnosis of HP is typically made by carefully correlating the biopsy findings with the patient’s clinical history and radiographic findings in collaboration with the pulmonologist and radiologist. The paper by Selman, Pardo and King highlights important clinical findings of which we should be aware in consideration of the histological findings and the paper by Herbst and Myers emphasizes those histological features that might lead one to consider HP perhaps with less than adequate or preliminary clinical information. Both papers are timely and highly recommended but I hope I’ve been able to summarize them adequately for you.

Emergent respiratory viral pathogens

Jartii et al provide an excellent review new viral respiratory infections in the May 2012 Current Opinion in Pulmonary Medicine in you are looking for a concise update on this topic.  Here are some key points:

1. Human metapneumovirus (MPV) causes upper and lower respiratory tract infections in all ages but particularly in young children where the prevalence in respiratory infections is about 5% (cf. 2-5% in adults).  Overall, MPV infections are less or similar in severity to RSV or influenza virus infections. The most severe infections include bronchiolitis, exacerbations of asthma, and pneumonia. Although adults usually have only minor symptoms, MPV may cause serious morbidity and mortality in the elderly in long-term care facilities. MPV is a single-stranded envelped RNA virus in the family Paramyxoviridae and is most closely related to RSV genetically and clinically. Diagnosis is based on PCR but IF assays are now available.
2. New coronaviruses NL63 and HKU1 have been identified in a small percentage of individuals of all ages but have primarily been associated with mild upper respiratory tract infections. Chronic disease, immunosuppression, institutionalization and old age increase the risk of severe infection. CoVs are large plus-strand enveloped RNA viruses; currently, diagnosis of CoV-NL63 and -HKU1 is made only with RT-PCR.
3. Human bocaviruses (HBoV) include 4 species and belong to the Parvoviridae family. HBoVs are minute non-enveloped linear single-strand DNA viruses. HBoV1 shows a predilection for children and has been associated with upper and lower respiratory tract infections, acute asthma and pneumonia in children. Diagnosis is based on serology or PCR in serum due to persistence of HBoV DNA in nasophayngeal secretions.
4. Human rhinovirus (HRV) is the most common respiratory pathogen in all ages but PCR techniques have facilitated the discovery of two new groups, C and D, and possibly over 50 new HRV strains have been discovered in addition to the two former major genetic groups, A and B, and 99 HRV serotypes. HRV-C appears to be highly clinically significant. HRVs are small nonenveloped minus- sensed RNA viruses belonging to Picornaviridae. Diagnosis is based on PCR.
5. KI and WU polyomaviruses are newly discovered polyomaviruses that may be a cause of respiratory infections in children. Torque Teno Viruses are another newly discovered group of highly diverse viruses. High TTV loads in nasal secretions or plasma have positively correlated with markers of eosinophilic inflammation, bronchiectasis and idiopathic pulmonary fibrosis, congenital mannose-binding lectin deficiencies, and autoimmune rheumatoid disease.
6. All of these "new" respiratory viruses can be detected with PCR-based methodologies. But, since there is neither a specific treatment or vaccine for these viral infections, there really isn't a clinical need for diagnostic testing. Testing could be practical, however, in isolating infected patients in hospitals or LTC settings and in decreasing unnecessary antibiotic therapy. Quantitative PCR techiniques may be useful in establishing viral detection as the cause of respiratory illness.

Biomarkers in COPD

Chronic obstructive lung disease (COPD) is defined functionally as airflow limitation that is not fully reversible.  It is a MAJOR cause of morbidity and mortality in the U.S. and worldwide.  COPD has two distinct major, but often coexisting, forms that represent different manifestations of COPD: emphysema and chronic bronchitis.  Emphysema can be defined morphologically, either by radiography or pathology, while chronic bronchitis is defined clinically based upon the duration of productive cough and exclusion of other causes.  Treatment is focused on preventing progression of disease and the development of acute exacerbations, which are a major cause of hospitalizations, morbidity and mortality.  Common to both major forms is small airways inflammation and an abnormal inflammatory response either in large airways (chronic bronchitis) or in alveoli (emphysema).

COPD is also associated with low-grade systemic inflammation and biomarkers that could predict increased risk for mortality from progressive disease or acute exacerbations might be greatly beneficial in providing more intesive surveillance and treatment of this group.  A team of researchers led by Tufts University School of Medicine screened blood samples from people in the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) to see if inflammatory biomarkers could predict disease outcome (abstract).  The study included 1843 patients who were followed for 3 years.  168 of the 1843 patients (9.1%) died.  Serum or plasma levels of fibrinogen, chemokine ligand 18, surfactant protein D, C-reative protein, Clara cell secretory protein-16, interleukin-6, IL-8, and tumor necrosis factor-alpha were measured at recruitment and at subsequent visits.

The addtion of a panel of biomarkers significantly improved predictive power and was able to better predict outcomes compared with current clinical variables, including age, BODE index, and hospitalization history. The addition of IL-6 provided the most improvement of discriminatory power, but this was improved further with the addition of all biomarkers measured.

Incidentalomas revisited

Dr. Bruce Friedman posted a thoughtful blog on Lab Soft News recently concerning "incidentalomas" ("How to Avoid the Risks of a CT Incidentaloma").  This post happened to coincide with a new working committee at my hospital commissioned with developing a virtual clinic for following patients who have incidental lung nodules discovered.

Perhaps unlike other sites, there are widely disseminated radiographic recommendations for following lung nodules (Fleishner criteria).  Of interest, a recent article found that despite widespread awareness of these criteria, actual incorporation of these criteria into practice has been found wanting--only around 50% of radiology practices actually used these recommendations in reports.  One issue that we will be challenged with is how to track these patients across multiple different providers for appropriate follow-up.  One model we are considering is already in place: using a breast "navigator" to follow-up patients with abnormal mammograms.  This issue arose recently because of a series of patients presented at our Tumor Board who had incidental lung nodules identified but were not followed appropriately and then were re-discovered with inoperable advanced stage disease months later.

This idea also came up just today in a conversation I was having with a urologist colleague regarding renal incidentalomas treated with cryoablation.  I recently diagnosed an incidentally discovered 2.5-cm mass in an 80-year-old woman on CT-guided needle biopsy as an muscle-predominant angiomyolipoma (mini-case report on its way!).  For better or worse, the tumor was ablated probable before it was even grossed!  But, to return to Dr. Friedman's post, what if the patient was simply observed with serial imaging?  The urologist was unaware of any recommendations similar to the Fleishner criteria for lung nodules.  If you, dear reader, know of any please post them in a comment.

Finally, today's email brought a "Published Ahead of Print" article from Journal of Thoracic Oncology on-- mediastinal incidentalomas.  This is a prospective study of 88 patients in which mediastinal lymphadenopathy was found as an incidental finding on CT scans for indications other than staging.  The title is a bit deceptive but the scenario is common.  The finding of incidental mediastinal lymph nodes on CT were characterized by multiple Naruke stations involved, relative small sizes of nodes (median ranging from 6 to 14 mm for all nodes), and coexistence with hilar adenopathy.  In this study, sarcoidosis was diagnosed by EBUS-FNA in 22% of patients and 2 patients developed lung cancer 2 years after initial finding.

While I concur with the basic premise of the post (i.e. that one shouldn't jump into a full-blown diagnostic work-up of an incidental finding), I think we should also insist on developing evidence-based criteria for managing these types of findings such as, Fleishner criteria for lung nodules.

Lymphomatoid granulomatosis: new insights

A great deal has been learned over the past few years about lymphomatous granulomatosis (LG), a rare pulmonary lymphoproliferative disease with a high mortality rate.  The eminent pulmonary pathologist, Anna-Louise Katzenstein, and co-authors, Erika Doxtader and Sonia Narendra, have published an excellent (free!) review e-article in American Journal of Surgical Pathology that I recommend highly.

The pathologic features of LG and post-transplant lymphoproliferative disorder (PTLD) are essentially identical, so the diagnosis of LG should not be made in organ transplant recipients.  Similarly, the pathologic features of LG are so similar to iatrogenic immunodeficiency-associated lymphoproliferative disorder that the latter diagnosis should be given consideration if the patient has a history of receiving methotrexate or other immunosuppresive drugs.