Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Tinea versicolor in a premature infant.

Tinea versicolor (TV) is a fungal skin infection that classically affects adolescents and young adults. Occasionally, it may be seen on the face of infants. We report an unusual case of widespread cutaneous TV in a premature infant.

A prospective, comprehensive registry that integrates the molecular analysis of pediatric and adolescent melanocytic lesions.

BACKGROUND: Childhood melanocytic tumors represent a diagnostic and therapeutic challenge, and additional research is needed to better define the natural history of these tumors. METHODS: The authors developed a comprehensive, prospective registry called Molecular Analysis of Childhood Melanocytic Tumors for children and adolescents with an atypical Spitz tumor/Spitz melanoma (AST/SM), conventional or adult-type melanoma (CM), melanoma arising in a giant congenital nevus (MCM), or atypical melanocytic proliferation of other types (OT) to better define the clinical behavior of these lesions by incorporating an integrated clinicopathologic and molecular analysis using centralized pathology review and various platforms, including fluorescence in situ hybridization; array comparative genomic hybridization; and whole genome, exome, and capture targeted panels. RESULTS: From May 2016 to November 2019, 70 children were enrolled with a median age at diagnosis of 9.1 years. Thirty-seven had AST/SM, 17 had CM, 4 had MCM, and 12 had OT. Patients with AST/SM were younger (median age, 7 years), and their tumor most commonly arose in the extremities and trunk. The most common gene rearrangements included MAP3K8 and ALK. None of the 33 patients who underwent a TERT promoter mutation analysis had a mutation, and all patients were alive. Among the CM patients, the median age was 13 years; 11 had a BRAFV600E mutation, and 7 had a TERT promoter mutation. Three patients died of their disease. All 4 patients with MCM harbored an NRASQ61 mutation and died of their disease. The OT group was heterogenous, and all patients survived. CONCLUSIONS: The incorporation of an integrated clinicopathologic and genomic analysis identifies distinct subgroups of pediatric melanocytic lesions that have different clinical behaviors. The integration of this combined diagnostic modality can help to individualize diagnoses and treatments for these patients.

Acute onset traction-associated ulceration and alopecia.

Traction alopecia is a protracted form of hair loss resulting from persistent tension on the hair follicle . We report a case of a 9-year-old African American girl who presented with extensive scalp ulceration and alopecia 11 days after placement of a synthetic hair braid. Over a period of months, with consistent wound care and low-tension hairstyling, most of the hair regrew. We propose that the ulceration and hair loss in this case represents an acute variant of traction alopecia due to pressure-induced ischemia and necrosis.

Workshop Proceedings: Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?

A workshop entitled "Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?" was held at the 36th Annual Meeting of the American College of Toxicology in Summerlin, Nevada. The workshop was sponsored by Shire and Ultragenyx and was designed to present the nonclinical considerations for the development of various products for rare diseases. A panel of experts from industry and government highlighted the nonclinical considerations in developing toxicology programs supporting rare disease therapeutics, challenges in preclinical safety assessment, reviewed the current guidance, and presented the progress that has been made to date. The main learning from the workshop was that nonclinical testing of therapeutics targeting rare disease warrants special considerations, and early collaboration between sponsors and health authorities may help optimize the scope and timing of the supportive studies. Specific examples for nonclinical development programs for enzyme replacement therapy (ERT) were presented. Although the symposium focused on ERTs, the concepts are broadly applicable.

Primary cervical cancer screening with human papillomavirus: end of study results from the ATHENA study using HPV as the first-line screening test.

OBJECTIVES: ATHENA evaluated the cobas HPV Test as the primary screen for cervical cancer in women ≥25years. This reports the 3-year end-of-study results comparing the performance of HPV primary screening to different screening and triage combinations. METHODS: 42,209 women ≥25years were enrolled and had cytology and hrHPV testing. Women with abnormal cytology (≥atypical squamous cells of undetermined significance) and those HPV positive were referred to colposcopy. Women not reaching the study endpoint of CIN2+ entered the 3-year follow-up phase. RESULTS: 3-year CIR of CIN3+ in cytology-negative women was 0.8% (95% CI; 0.5-1.1%), 0.3% (95% CI 0.1-0.7%) in HPV-negative women, and 0.3% (95% CI; 0.1-0.6%) in cytology and HPV negative women. The sensitivity for CIN3+ of cytology was 47.8% (95% CI; 41.6-54.1%) compared to 61.7% (95% CI; 56.0-67.5%) for the hybrid strategy (cytology if 25-29years and cotesting with cytology and HPV if ≥30years) and 76.1% (95% CI; 70.3-81.8%) for HPV primary. The specificity for CIN3+ was 97.1% (95% CI; 96.9-97.2%), 94.6% (95% CI; 94.4-94.8%), and 93.5% (95% CI; 93.3-93.8%) for cytology, hybrid strategy, and HPV primary, respectively. Although HPV primary detects significantly more cases of CIN3+ in women ≥25years than either cytology or hybrid strategy, it requires significantly more colposcopies. However, the number of colposcopies required to detect a single CIN3+ is the same as for the hybrid strategy. CONCLUSIONS: HPV primary screening in women ≥25years is as effective as a hybrid screening strategy that uses cytology if 25-29years and cotesting if ≥30years. However, HPV primary screening requires less screening tests.

Proof of concept for a banding scheme to support risk assessments related to multi-product biologics manufacturing.

A banding scheme theory has been proposed to assess the potency/toxicity of biologics and assist with decisions regarding the introduction of new biologic products into existing manufacturing facilities. The current work was conducted to provide a practical example of how this scheme could be applied. Information was identified for representatives from the following four proposed bands: Band A (lethal toxins); Band B (toxins and apoptosis signals); Band C (cytokines and growth factors); and Band D (antibodies, antibody fragments, scaffold molecules, and insulins). The potency/toxicity of the representative substances was confirmed as follows: Band A, low nanogram quantities exert lethal effects; Band B, repeated administration of microgram quantities is tolerated in humans; Band C, endogenous substances and recombinant versions administered to patients in low (interferons), intermediate (growth factors), and high (interleukins) microgram doses, often on a chronic basis; and Band D, endogenous substances present or produced in the body in milligram quantities per day (insulin, collagen) or protein therapeutics administered in milligram quantities per dose (mAbs). This work confirms that substances in Bands A, B, C, and D represent very high, high, medium, and low concern with regard to risk of cross-contamination in manufacturing facilities, thus supporting the proposed banding scheme.

Differentiating skin-limited and multisystem Langerhans cell histiocytosis.

OBJECTIVE: To identify features associated with multisystem involvement and therapeutic failure in patients with skin Langerhans cell histiocytosis (LCH). STUDY DESIGN: We reviewed medical records of 71 consecutive patients with LCH with skin involvement evaluated at Texas Children's Hospital and analyzed clinical features, laboratory results, and the presence of circulating cells with the BRAF-V600E mutation with respect to initial staging and clinical outcomes. RESULTS: Skin disease in patients older than 18 months of age at diagnosis was associated with the presence of multisystem disease (OR, 9.65; 95% CI, 1.17-79.4). Forty percent of patients referred for presumed skin-limited LCH had underlying multisystem involvement, one-half of these with risk-organ involvement. Patients with skin-limited LCH had a 3-year progression-free survival of 89% after initial therapy, and none developed multisystem disease. Patients with skin/multisystem involvement had a 3-year progression-free survival of 44% with vinblastine/prednisone therapy, and risk-organ involvement did not correlate with failure to achieve nonactive disease. Circulating cells with BRAF-V600E were detected at higher frequency in patients with multisystem involvement (8 of 11 skin/multisystem vs 1 of 13 skin-limited; P = .002). CONCLUSION: Skin-limited LCH necessitates infrequent therapeutic intervention and has a lower risk of progression relative to skin plus multisystem LCH. The less-aggressive clinical course and lack of circulating cells with the BRAF-V600E mutation in skin-limited LCH suggest a different mechanism of disease origin compared with multisystem or risk-organ disease.

The role of IL-1ß and TNF-α signaling in the genesis of cancer treatment related symptoms (CTRS): a study using cytokine receptor-deficient mice.

Cytotoxic chemotherapeutic agents often induce a cluster of cancer treatment related symptoms (CTRS). The purpose of this study was to develop a mouse model of CTRS to examine the role of IL-1ß and TNF-α signaling in the genesis of these symptoms. CTRS (change in wheel running activity, food intake, and body weight from baseline) were examined in wild type (WT) mice or mice lacking the TNF-α p55 (type 1) receptor (TNFR1-/-) and/or IL-1ß type 1 receptor (IL-1R1-/-) injected with four doses of cyclophosphamide/Adriamycin/5-fluorouracil (CAF) at 20-day intervals. Inflammatory cytokines in blood and tissues were measured using multiplex immunoassays and quantitative RT-PCR. ANOVA was used to examine differences between genotype and/or treatment group. Kaplan-Meier analysis was used to estimate survival rate. CAF rapidly increased IL-1ß and TNF-α signaling in WT mice. CAF induced acute CTRS immediately following drug injection which returned to baseline prior to the next CAF dose. Persistent CTRS were evident 3weeks after the 4th CAF dose. Acute but not persistent CTRS were associated with increased levels of IL-7, IL-9, KC, MCP-1, GCSF, and IP-10. This CAF induced inflammatory response was blunted in IL-1R1 deficient mice and absent in IL-1R1/TNFR1-deficient mice. IL-1R1-/- mice showed an identical pattern of CTRS to their WT counterparts. The assessment of CTRS in IL-1R1/TNF-R1-deficient mice was precluded by severe toxicity. Our data suggest that an important function of the IL-1ß and TNF-α driven inflammatory cascade is to promote recovery following exposure to cytotoxic agents.

Idiopathic eruptive macular pigmentation involving the distal extremities: a case report.

Idiopathic eruptive macular pigmentation is a disorder of pigmentation found in children and adolescents. It typically manifests as nonconfluent tan to brown macules involving the neck, trunk, and proximal extremities. We report the case of a 6-year-old girl with an atypical presentation involving the distal extremities. Histopathology confirmed the subtle increase in pigmentation of the basal layer of the epidermis. Treatments are largely ineffective. The condition is generally self-limited over months to years, thus treatment is unnecessary.

Concurrent hidradenitis suppurativa, inflammatory acne, dissecting cellulitis of the scalp, and pyoderma gangrenosum in a 16-year-old boy.

We report the case of a 16-year-old boy who presented to our clinic with concomitant hidradenitis suppurativa, inflammatory acne, dissecting cellulitis of the scalp, and pyoderma gangrenosum. Recent reports describe the co-occurrence of pyoderma gangrenosum, acne, and hidradenitis suppurativa. This case further expands the spectrum of concomitant pyoderma gangrenosum and dissecting cellulitis of the scalp.

Periungual Bowen's disease in a 4-year-old girl.

Bowen's disease (BD), or cutaneous squamous cell carcinoma (SCC) in situ, is rare in children. BD usually occurs in Caucasian adults on sun-exposed areas and may progress to invasive cutaneous SCC. Most cases of periungual BD have been linked to human papillomavirus infection. We report an immunocompetent child with periungual BD.

The ATHENA human papillomavirus study: design, methods, and baseline results.

OBJECTIVE: The objective of the study was to describe baseline data from Addressing the Need for Advanced HPV Diagnostics, a prospective, multicenter US cervical cancer screening trial. STUDY DESIGN: A total of 47,208 women aged 21 years or older undergoing routine screening were enrolled; liquid-based cytology and human papillomavirus (HPV) testing were performed. Women with abnormal cytology underwent colposcopy, as did high-risk HPV (hrHPV)-positive women and a random subset of women negative by both tests aged 25 years or older. Verification bias adjustment was applied; 95% confidence intervals were computed by the bootstrap method. RESULTS: The prevalence of cytologic abnormalities was 7.1%. hrHPV, HPV 16, and HPV 18 were detected using the cobas HPV Test in 12.6%, 2.8%, and 1.0% of women, respectively. Both cytologic abnormalities and hrHPV positivity declined with increasing age. The adjusted prevalence of cervical intraepithelial neoplasia grade 2 (CIN2) or greater in women aged 25-34 years was 2.3%, decreasing to 1.5% among older women. CONCLUSION: The Addressing the Need for Advanced HPV Diagnostics study provides important estimates of the prevalence of cytologic abnormalities, hrHPV positivity, and CIN2 or greater in a US screening population.

Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study.

BACKGROUND: The ATHENA study was designed to assess the performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping compared with liquid-based cytology for cervical cancer screening in a large US population aged 21 years and older. We did a subanalysis of this population to compare the screening performance of the cobas HPV test versus liquid-based cytology in women aged 25 years and older, and assess management strategies for HPV-positive women. METHODS: Women aged 25 years or older who were attending routine cervical screening were enrolled from 61 clinical centres in 23 US states. Cervical specimens were obtained for liquid-based cytology and HPV DNA testing with two first-generation assays (Amplicor HPV test and Linear Array HPV genotyping test) and the second-generation cobas HPV test (with individual HPV16 and HPV18 detection). Colposcopy and diagnostic biopsies were done on women with atypical squamous cells of undetermined significance (ASC-US) or worse cytology, those who tested positive with either first-generation HPV test, and a random sample of women who tested negative for HPV and cytology. All women not selected for colposcopy received their results and exited the study. Participants and colposcopists were masked to cytology and HPV test results until the colposcopy visit was completed. The primary endpoint for this substudy was histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3) or worse. This study is registered with ClinicalTrials.gov, number NCT00709891; the study is in the follow-up phase, which is due to be completed in December, 2012. FINDINGS: From May 27, 2008, to Aug 27, 2009, 47,208 women were enrolled, of whom 41,955 met our eligibility criteria. Valid cobas HPV and liquid-based cytology test results were available for 40,901 women (97%), who were included in this analysis. Of these, 4275 women (10%) tested cobas HPV positive and 2617 (6%) had abnormal cytology. 431 women were diagnosed with CIN2 or worse and 274 with CIN3 or worse. In women who had colposcopy, the cobas HPV test was more sensitive than liquid-based cytology for detection of CIN3 or worse (252/274 [92·0%, 95% CI 88·1-94·6] vs 146/274 [53·3%, 95% CI 47·4-59·1]; difference 38·7%, 95% CI 31·9-45·5; p<0·0001). Addition of liquid-based cytology to HPV testing increased sensitivity for CIN3 or worse to 96·7% (265/274, 95% CI 93·9-98·3), but increased the number of screen positives by 35·2% (5783/40,901 vs 4275/40,901) compared with HPV testing alone. As a triage test to identify CIN3 or worse in HPV-positive women, detection of HPV16, HPV18, or both alone was equivalent to detection of ASC-US or worse alone in terms of sensitivity (150/252 [59·5%] vs 133/252 [52·8%]; p=0·11) and positive predictive value (PPV) (150/966 [15·5%] vs 133/940 [14·1%]; p=0·20). Among HPV-positive women, detection of HPV16, HPV18, or both or low-grade squamous intraepithelial lesion or worse cytology had better sensitivity (182/252 [72·2%]; p<0·0001) and similar PPV (182/1314 [13·9%]; p=0·70) for detection of CIN3 or worse than ASC-US or worse cytology alone. Furthermore, detection of HPV16, HPV18, or both or high-grade squamous intraepithelial lesion or worse cytology had higher sensitivity (165/252 [65·5%]; p=0·0011) and PPV (165/1013 [16·3%]; p=0·031) for detection of CIN3 or worse than ASC-US or worse cytology alone. INTERPRETATION: HPV testing with separate HPV16 and HPV18 detection could provide an alternative, more sensitive, and efficient strategy for cervical cancer screening than do methods based solely on cytology. FUNDING: Roche Molecular Systems.

Cutaneous manifestations of malignancy.

PURPOSE OF REVIEW: Many types of cancer can cause lesions to appear on the skin. Cutaneous manifestations may be primary or secondary to an underlying malignancy. Skin lesions may appear prior to the onset of systemic malignancy or they may occur concurrently or following an established diagnosis. The purpose of this review is to educate medical providers about several types of pediatric malignancies that can present with cutaneous findings. The primary focus of this review will be cutaneous features of leukemias, lymphomas, neuroblastoma, some sarcomas, and Langerhans cell histiocytosis. RECENT FINDINGS: A wide range of cutaneous manifestations have been reported in systemic malignancies of children. In addition to providing a description of more typical features of skin lesions associated with these conditions, this review will summarize a few recent case reports of pediatric malignancies with unusual cutaneous presentations. SUMMARY: Systemic malignancy can present with a variety of cutaneous manifestations. Given that accurate, prompt diagnosis of malignancy can have tremendous prognostic significance, it is imperative that clinicians are familiar with features of skin lesions that may be seen in this setting.

Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys.

Recombinant human idursulfase, an intravenous enzyme replacement therapy indicated for treatment of somatic symptoms of mucopolysaccharidosis II (Hunter syndrome), is anticipated to have minimal benefit for the cognitive impairment associated with the severe phenotype. Because intrathecal (IT) administration of enzyme replacement therapy for other lysosomal enzyme disorders has shown efficacy in animal models, an IT formulation of idursulfase (idursulfase-IT) and a drug-delivery device (subcutaneous port connected to a lumbar IT catheter) were developed for treating central nervous system (CNS) involvement. In this chronic safety study, cynomolgus monkeys were dosed weekly with IV idursulfase (0.5 mg/kg) and every four weeks with idursulfase-IT (3, 30, and 100 mg) for six months, with device and vehicle controls treated similarly (n = 6, all groups). Necropsies were performed twenty-four hours post-final IT dose or after a recovery period (four weeks post-final dose in vehicle-control, 3 mg, and 100 mg IT groups: n = 6). No clinical signs or gross central nervous system lesions were observed. Compared to controls, more pronounced cellular infiltrates in brain and spinal cord meninges were noted, which largely resolved after the recovery period. Central nervous sytem levels of idursulfase-IT were dose dependent, as determined by enzyme activity and immunohistochemistry. The no-observed-adverse-effect level of idursulfase-IT was 100 mg.