Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Clinical outcomes and response of patients applying topical therapy for pyoderma gangrenosum: A prospective cohort study.

BACKGROUND: Pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. OBJECTIVE: We sought to estimate the effectiveness of topical therapies in the treatment of patients with PG. METHODS: This was a prospective cohort study of UK secondary care patients with a clinical diagnosis of PG that was suitable for topical treatment (recruited between July 2009 and June 2012). Participants received topical therapy after normal clinical practice (primarily topical corticosteroids [classes I-III] and tacrolimus 0.03% or 0.1%). The primary outcome was speed of healing at 6 weeks. Secondary outcomes included the following: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality of life; treatment failure; and recurrence. RESULTS: Sixty-six patients (22-85 years of age) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28 of 66 (43.8%) ulcers healed by 6 months. The median time to healing was 145 days (95% confidence interval, 96 days to ∞). Initial ulcer size was a significant predictor of time to healing (hazard ratio, 0.94 [95% confidence interval, 0.88-1.00); P = .043). Four patients (15%) had a recurrence. LIMITATIONS: Our study did not include a randomized comparator. CONCLUSION: Topical therapy is potentially an effective first-line treatment for PG that avoids the possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone.

Is speed of healing a good predictor of eventual healing of pyoderma gangrenosum?

BACKGROUND: Pyoderma gangrenosum is a rare inflammatory skin condition. Two prospective studies have evaluated treatments for pyoderma gangrenosum using a primary outcome of healing speed at 6 weeks. OBJECTIVE: Using data from both studies we assessed the predictive value of 3 early predictors for healing at 6 months: speed of healing, Investigator Global Assessment (IGA), and resolution of inflammation, recorded at 2 and 6 weeks. METHODS: Logistic regression models were applied and the effectiveness of the 3 measures was assessed through estimating the positive and negative predictive values and the area under the receiver operating characteristic curve. RESULTS: The positive and negative predictive value at 6 weeks were, respectively, 63.5% (95% confidence interval [CI] 52.4%-73.7%) and 74.6% (95% CI 62.5%-84.5%) for speed of healing; 80% (95% CI 68.7%-88.6%) and 74.2% (95% CI 64.1%-82.7%) for IGA; and 72.1% (95% CI 59.9%-82.3%) and 68.1% (95% CI 57.7%-77.3%) for resolution of inflammation. IGA had the best combined positive predictive value, negative predictive value, and area under the receiver operating characteristic curve at 2 and 6 weeks. LIMITATIONS: We were limited by data available from existing datasets. CONCLUSION: Speed of healing, IGA, and resolution of inflammation were all shown to be good predictors of eventual healing of pyoderma gangrenosum.

Fatal Case of Primary Cutaneous Aggressive T-Cell Lymphoma Switching From a CD4+ to a CD8+ Phenotype: Progressive Disease With Bexarotene and Romidepsin Treatment.

A 77-year-old white male presented to the clinic with two isolated cutaneous tumors on his forehead. A cutaneous biopsy showed a focally folliculotropic CD4 cutaneous lymphoma. The tumors were irradiated with a complete response, and he was started on oral bexarotene. He experienced localized cutaneous relapse 3 months into treatment. These new tumors now revealed a surprisingly CD8 cytotoxic phenotype, but with the same clone. A systemic workup was negative. His regimen was switched to romidepsin, and he was treated with local radiation again. Another 3.5 months passed in remission until he developed widespread cutaneous tumors. Positron emission tomography/computed tomography revealed multifocal systemic disease involving his diaphragm, liver, distal duodenum, proximal jejunum, anterior chest wall including pectoral muscles, and lungs without significant adenopathy. He died a few days later. Given his full clinical and pathological course, he was given the diagnosis of an aggressive primary cutaneous T-cell lymphoma, unspecified.

Prematurity reduces the severity and need for treatment of neonatal abstinence syndrome.

AIM: This study determined the influence of prematurity on the manifestation and treatment of neonatal abstinence syndrome (NAS). METHODS: This was a medical record review of Australian infants exposed to opiates in 2004 and 2007. Finnegan scores were obtained for 215 of 361 (59%) preterm infants under 37-week gestation and 694 of 1178 (59%) term infants. RESULTS: The mean and standard deviation (SD) gestational ages were 34 (3) and 38 (3) weeks for preterm and term infants, respectively. Maternal daily methadone doses were similar for the preterm and term infants with a mean (SD) of 79 mg (39) versus 72 mg (38) (p = 0.06). Maximum Finnegan scores were significantly lower in preterm infants (10 versus 11, p = 0.01), scores were positively correlated with gestation and fewer preterm infants were medicated for NAS (40% versus 48% p = 0.05). Maximum median daily and interquartile range morphine doses were lower for preterm than term infants (0.5 mg/kg/day (0.3-0.6) versus 0.5 mg/kg/day (0.4-0.7), p = 0.02). CONCLUSION: Preterm infants were just as likely to be monitored for withdrawal as term infants, but their Finnegan scores were lower and fewer preterm infants were treated for NAS. Whether this indicates decreased NAS severity or physiological immaturity is uncertain. Other means of evaluating NAS in preterm infants are warranted, especially long-term outcomes.

Evaluation of Novel Targets, Including CC-Chemokine Receptor 4, in Adult T-Cell Acute Lymphoblastic Leukemia/Lymphoma: A Mayo Clinic Clinical and Pathologic Study.

CONTEXT.­: Unlike B-cell acute lymphoblastic leukemia/lymphoma (ALL/LBL), there have been few therapeutic advances in T-cell ALL (T-ALL)/LBL, an aggressive ALL/LBL subtype. OBJECTIVE.­: To perform a focused tissue array study to elucidate tumor markers of therapeutic potential in T-ALL/LBL. DESIGN.­: Using immunohistochemistry, we evaluated expression of leukemic antigens of interest, specifically CC-chemokine receptor 4 (CCR4), among others, on available remnant diagnostic material, including tumor tissue slides obtained from formalin-fixed, paraffin-embedded preserved tissues. RESULTS.­: Our analysis identified, for the first time, expression of CCR4 in T-ALL/LBL in 11 of 27 cases (40.7%) and confirmed common expression of BCL2, CD38, and CD47, as reported previously. We also identified the expression of CD123 in 4 of 26 cases (15.4%), whereas BCL6 and PDL1 were expressed in a small number of T-ALL/LBL cases. The potential novel target CCR4 was significantly more common in the Pre/Pro-T immunophenotypic subtype, 6 of 9 (66.7%, P = .01). No additional differences in clinical and epidemiologic variables were noted among positive or negative CCR4 cases. CONCLUSIONS.­: These findings support preclinical and clinical testing of therapies targeting CCR4, CD47, BCL2, CD38, and CD123 in T-ALL/LBL, and may help guide the development of targeted clinical trials in T-ALL/LBL, a rare disease in urgent need of novel therapies.

Profile of infants born to drug-using mothers: a state-wide audit.

AIMS: To ascertain the characteristics and short-term outcomes of infants born to illicit drug-using mothers in public hospitals in the state of New South Wales and the Australian Capital Territory during 2004. METHODS: Patients were identified retrospectively by hospital records searches using ICD-10 morbidity codes and records of local Drug and Alcohol Services. Records were reviewed on site. All public hospitals (n= 101) with obstetric services were included. RESULTS: A total of 879 (1.4%, 95% confidence interval: 1.3-1.5%) drug-using mothers were identified from 62,682 confinements. Opiates (46.8%), amphetamines (23.0%) and polydrug (16.4%) exposure were most common. There were eight stillbirths. Among these 871 infants, prematurity (23.6%) and low birthweight (27.1%) were common and 51.1% were admitted to nurseries for further care. Two infants died. Major congenital anomalies were detected in 15 infants. Pharmacological treatment for withdrawal was required for 202 (23.2%), and 143 (70.8%) infants were discharged home on medication. Infants who completed inpatient pharmacological treatment were hospitalised longer (median 26.0 vs. 12.0 days) and were more likely to be premature (37.3 vs. 14.0%). Child-at-risk notifications affected 40.6% of the infants, and 7.6% were fostered prior to discharge. A total of 333 (38.2%) infants were breastfed at discharge. CONCLUSIONS: Our regional study highlights a substantial prevalence of drug use in pregnancy with considerable adverse perinatal and hospital outcomes in infants born to these mothers. Coordinated health care and resources are needed to support these mother-infant pairs because of their social, medical and mental-health issues.

Rapid Expansion of a Teledermatology Web Application for Digital Dermatology Assessment Necessitated by the COVID-19 Pandemic: Retrospective Evaluation.

BACKGROUND: The COVID-19 pandemic necessitated a change in the provision of outpatient care in dermatology. OBJECTIVE: A novel, asynchronous, digital consultation platform was codeveloped with 2 National Health Service dermatology teams to improve access and enhance choice in outpatient care. METHODS: The rollout of the platform was accelerated during the initial COVID-19 lockdown, and its wider use across 2 Scottish health boards was retrospectively evaluated. Integrated with the hospital booking system and electronic patient record, the platform provides an alternative to face-to-face consultations, using information and images submitted by the patients. RESULTS: In total, 297 new patient consultations and 108 return patient consultations were assessed, and 80% (324/405) of the images submitted were of satisfactory quality. The consultations were, on average, 3 minutes shorter than equivalent face-to-face interactions, and a total of 5758 km of patient travel was avoided. Outcomes included web-based reviews (66/405, 16.3%), face-to-face reviews (190/405, 46.9%), biopsies (46/405, 11.4%), discharge (89/405, 22%), and other treatments or investigations (14/405, 3.5%). High levels of patient satisfaction (92/112, 82.1%) were reported. CONCLUSIONS: Digital dermatology assessments are now included in the choices for consultation types that are available to patients, helping to augment service capacity during pandemic recovery.

Membranous nephropathy: a rare renal manifestation of IgG4-related systemic disease.

This is a case of IgG4-related systemic disease (ISD) and its rare renal manifestation as membranous nephropathy (MGN). The patient presented with peripheral edema, hematuria and proteinuria. 24-h urine revealed proteinuria of up to 15 g/d. Renal biopsy revealed MGN and many IgG4-positive plasma cells. Serum IgG4 levels were elevated at 750 mg/dl (9 - 89 mg/d). Biopsies of multiple tissues revealed many IgG4-positive plasma cells. Prednisone was initiated after making the diagnosis of ISD. However, the patient progressed into renal failure and eventually needed dialysis despite rituximab therapy. The renal manifestation of ISD typically shows tubulointerstitial nephritis, but, rarely, it may include glomerular abnormalities such as MGN or membranoproliferative glomerulonephritis. Castleman disease (CD) and ISD share similarities in pathology, particularly in the lymph nodes. Rituximab has shown promising results in some patients with ISD, CD, and MGN, and its use should be considered in steroid-resistant cases. Clinicians need to be made aware of ISD and its varied presentations. Timely initiation of steroid therapy can induce remission, and delay in therapy can cause permanent organ damage. Therefore, clinicians must have a high index of suspicion to make an early diagnosis of ISD in order to initiate appropriate treatment.

Rare Multisystem Histiocytic Sarcoma on 18F-FDG PET/CT.

Histiocytic sarcoma (HS) is a rare malignancy with morphologic and immunophenotypic features indicating histiocytic differentiation. We present a case of HS with multisystem involvement, including an obstructing mass in the pancreatic head. 18F-FDG PET/CT is a valuable tool in staging this rare entity and in assessing the response to therapy. Knowing the diverse metastatic pattern of HS will help avoid imaging pitfalls on clinical 18F-FDG PET/CT scans.

The Role and Pitfall of F18-FDG PET/CT in Surveillance of High Grade Pulmonary Lymphomatoid Granulomatosis.

Lymphomatoid granulomatosis (LYG) is an uncommon angiocentric and angiodestructive T-cell rich, Epstein-Barr virus (EBV) positive B-cell multisystem lymphoproliferative disorder, predominately affecting the lungs. Since both clinical presentation and radiographic imaging findings, including X-ray and computed tomographic (CT), are nonspecific, the ultimate diagnosis of LYG relies on lung tissue sample diagnosis with its WHO grading based on the degree of cytologic atypia, necrosis and density of EBV positive B-cells. In addition, its histopathologic grading is correlated with clinical manifestation with high grade LYG mimicking aggressive B-cell lymphoma. Discordant grading between pulmonary and cutaneous LYG lesion has have been observed and might be a potential diagnostic and prognostic pitfall. F18-FDG PET/CT has been used to monitor disease progression and treatment response. In this study, we reviewed and summarized the clinical role of F18-FDG PET/CT in the surveillance of high grade pulmonary LYG, and examined its limitations in grading multisystem LYG.

Utility of TRBC1 Expression in the Diagnosis of Peripheral Blood Involvement by Cutaneous T-Cell Lymphoma.

Peripheral blood involvement by cutaneous T-cell lymphoma is typically assessed by flow cytometry and plays a critical role in diagnosis, classification, and prognosis. Simplified strategies to detect tumor cells (Sezary cells) fail to exclude reactive subsets, whereas tumor-specific abnormalities are subtle and inconsistently present. We implemented a flow cytometric strategy to detect clonal Sezary cells based on the monotypic expression of one of two mutually exclusive TCR constant ß chains, TRBC1 and TRBC2. Analysis of CD4+ T-cell subsets and TCR variable ß classes from healthy donors showed polytypic TRBC1 staining. Clonal Sezary cells were identified by TRBC1 staining in 56 of 111 (50%) samples from patients with cutaneous T-cell lymphoma, accounting for 7-18,155 cells/µl and including 13 cases (23%) lacking tumor-specific immunophenotypic abnormalities. CD4+ T-cell subsets from 86 patients without T-cell lymphoma showed polytypic TRBC1 staining, except for five patients (6%) with minute T-cell clones of uncertain significance accounting for 53-136 cells/µl. Assessment of TRBC1 expression within a comprehensive single-tube flow cytometry assay effectively overcomes interpretative uncertainties in the identification of Sezary cells without the need for a separate T-cell clonality assay.

Flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: International guidelines for assay characteristics.

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported.

International guidelines for the flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: Assay development/optimization, validation, and ongoing quality monitors.

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency.

Mastocytosis.

OBJECTIVES: The 2019 Workshop of the Society for Hematopathology/European Association for Haematopathology received and reviewed cases covering the spectrum of mastocytosis and related diseases, including morphologic mimics, focusing on recent updates and relevant findings for pathologists. METHODS: The workshop panel reviewed 99 cases of cutaneous and systemic mastocytosis (SM) and SM and associated hematologic neoplasms (SM-AHN). RESULTS: Despite a common theme of KIT mutation (particularly D816V), mastocytosis is a heterogeneous neoplasm with a wide variety of presentations. This spectrum, including rare subtypes and extramedullary organ involvement, is discussed and illustrated by representative cases. CONCLUSIONS: In the age of targeted treatment aimed at KIT, the accurate diagnosis and classification of mastocytosis has major implications for therapy and further interventions. Understanding the clinical, pathologic, and genetic findings of mastocytosis is crucial for selecting the proper tests to perform and subsequent arrival at a correct diagnosis in this rare disease.

Reactive Eosinophil Proliferations in Tissue and the Lymphocytic Variant of Hypereosinophilic Syndrome.

OBJECTIVES: The 2019 Society for Hematopathology and European Association for Haematopathology Workshop reviewed the spectrum of neoplastic, nonneoplastic, and borderline entities associated with reactive eosinophilia in tissue. METHODS: The workshop panel reviewed 46 cases covered in 2 workshop sessions. RESULTS: The 46 cases were presented with their consensus diagnoses during the workshop. Reactive eosinophilia in lymph nodes and other tissues may be accompanied by or be distinct from peripheral blood eosinophilia. Reactive etiologies included inflammatory disorders such as Kimura disease and IgG4-related disease, which may show overlapping pathologic features and reactions to infectious agents and hypersensitivity (covered in a separate review). Hodgkin, T-cell, and B-cell lymphomas and histiocytic neoplasms can result in reactive eosinophilia. The spectrum of these diseases is discussed and illustrated through representative cases. CONCLUSIONS: Reactive eosinophilia in lymph nodes and tissues may be related to both nonneoplastic and neoplastic lymphoid proliferations and histiocytic and nonhematolymphoid processes. Understanding the differential diagnosis of reactive eosinophilia and the potential for overlapping clinical and pathologic findings is critical in reaching the correct diagnosis so that patients can be treated appropriately.

Myeloid/Lymphoid Neoplasms Associated With Eosinophilia and Rearrangements of PDGFRA, PDGFRB, or FGFR1 or With PCM1-JAK2.

OBJECTIVES: To summarize cases submitted to the 2019 Society for Hematopathology/European Association for Haematopathology Workshop under the category of myeloid/lymphoid neoplasms with eosinophilia and PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2 rearrangements, focusing on recent updates and relevant practice findings. METHODS: The cases were summarized according to their respective gene rearrangement to illustrate the spectrum of clinical, laboratory, and histopathology manifestations and to explore the appropriate molecular genetic tests. RESULTS: Disease presentations were heterogeneous, including myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs), MDS/MPN, acute myeloid leukemia, acute B- or T-lymphoblastic lymphoma/acute lymphoblastic lymphoma (ALL/LBL), or mixed-lineage neoplasms. Frequent extramedullary involvement occurred. Eosinophilia was common but not invariably present. With the advancement of RNA sequencing, cryptic rearrangements were recognized in genes other than PDGFRA. Additional somatic mutations were more frequent in the FGFR1-rearranged cases. Cases with B-ALL presentations differed from Philadelphia-like B-ALL by the presence of an underlying MPN. Cases with FLT3 and ABL1 rearrangements could be potential candidates for future inclusion in this category. CONCLUSIONS: Accurate diagnosis and classification of this category of myeloid/lymphoid neoplasms has important therapeutic implications. With the large number of submitted cases, we expand our understanding of these rare neoplasms and improve our ability to diagnose these genetically defined disorders.