A Genotype-Phenotype Study of High-Resolution FMR1 Nucleic Acid and Protein Analyses in Fragile X Patients with Neurobehavioral Assessments.

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying FMR1 silencing, CGG repeat expansion, is well characterized; however, delineation of the pathway from DNA to RNA to protein using biosamples from well characterized patients with FXS is limited. Since FXS is a common and prototypical genetic disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD), a comprehensive assessment of the FMR1 DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority. We applied nine sensitive and quantitative assays evaluating FMR1 DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of FMR1 expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different FMR1 expansions, with emphasis on those with FXS (N = 42 total, N = 31 with FMRP measurements). The group with FMRP data was also evaluated comprehensively in terms of its neurobehavioral profile, which allowed molecular-neurobehavioral correlations. FMR1 CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with findings of previous FMR1 genomic and protein studies. They also demonstrated a high level of agreement between blood and buccal specimens. These assays further corroborated previous reports of the relatively high prevalence of methylation mosaicism (slightly over 50% of the samples). Molecular-neurobehavioral correlations confirmed the inverse relationship between overall severity of the FXS phenotype and decrease in FMRP levels (N = 26 males, mean 4.2 ± 3.3 pg FMRP/ng genomic DNA). Other intriguing findings included a significant relationship between the diagnosis of FXS with ASD and two-fold lower levels of FMRP (mean 2.8 ± 1.3 pg FMRP/ng genomic DNA, p = 0.04), in particular observed in younger age- and IQ-adjusted males (mean age 6.9 ± 0.9 years with mean 3.2 ± 1.2 pg FMRP/ng genomic DNA, 57% with severe ASD), compared to FXS without ASD. Those with severe ID had even lower FMRP levels independent of ASD status in the male-only subset. The results underscore the link between FMR1 expansion, gene methylation, and FMRP deficit. The association between FMRP deficiency and overall severity of the neurobehavioral phenotype invites follow up studies in larger patient cohorts. They would be valuable to confirm and potentially extend our initial findings of the relationship between ASD and other neurobehavioral features and the magnitude of FMRP deficit. Molecular profiling of individuals with FXS may have important implications in research and clinical practice.

Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay.

OBJECTIVE: Expansion of the G4C2 repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. METHODS: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3' sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to ∼5.8 kb amplicons (∼950 G4C2 repeats). RESULTS: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3'-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. CONCLUSION: This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype-genotype correlations and therapeutic development in ALS/FTD.

Histological features associated with vemurafenib-induced skin toxicities: examination of 141 cutaneous lesions biopsied during therapy.

Dermatologic toxicities (DTs) associated with vemurafenib therapy include actinic keratosis (AK), verruca vulgaris (VV), keratoacanthoma (KA), and invasive squamous cell carcinoma (SCC), which may share histological features. The authors report the histological features to aid in distinguishing among these DTs. A 3-year retrospective examination of the authors' surgical pathology database was conducted and 141 cases of vemurafenib-associated DTs from 33 patients were identified. DTs were categorized into 3 groups: (1) cutaneous epithelial proliferations (CEP), (2) melanocytic lesions, and (3) inflammatory dermatoses. The authors compared the groups using analysis of variance, and P<0.05 was considered significant. CEP (n=120) accounted for 85% of all DTs biopsied. The most frequent diagnosis in the CEP category was VV (40%), followed by invasive SCC (24%) and AK (21%). KA was diagnosed in 3% of CEP. Histologically, AK, VV, KA, and invasive SCC may demonstrate similar morphological features in superficial sampled specimens. The mitotic rate was significantly higher in invasive SCC than other CEP (P<0.003). The median tumor thickness of SCC was 2.60 mm. Evaluating the base of the keratinized lesion will aid in distinguishing the histological type of CEP and the management of the DTs; thus, a deep shave or punch biopsy may be warranted for patients who received vemurafenib therapy.

Melanocytic differentiation is present in a significant proportion of nonpigmented diffuse neurofibromas: a potential diagnostic pitfall.

Whereas the pigmented (melanotic) variant of diffuse neurofibroma (DNF) with positivity for melanocytic markers is well recognized, expression of melanocytic markers in nonpigmented DNF has not been systematically studied. We analyzed 28 unselected consecutive DNFs for expression of melanocytic markers, including melan A, microphthalmia transcription factor (MITF), and HMB-45 antigen. For comparison, we also analyzed 40 localized skin neurofibromas and 7 intraneural neurofibromas. One case of nonpigmented DNF was analyzed by electron microscopy. Of the 28 DNFs studied by immunohistochemistry, 3 were pigmented and 25 nonpigmented. The 3 pigmented DNFs and 9 of 25 (36%) nonpigmented DNFs expressed melan A, MITF, and HMB-45 antigen. These markers were expressed either focally or more diffusely, typically in a minority of the lesional cells, and usually both in the dermal and subcutaneous portion of the DNF. Melan A was expressed in the largest number of the lesional cells (up to 50%), whereas only a small fraction of the melan A-positive cells (from 5% to 10% in most cases) also expressed HMB-45 antigen. None of the 47 non-DNFs expressed these markers. Ultrastructurally, melanosomes were present in some cells in nonpigmented DNF that expressed the melanocytic markers. Twenty-three of 28 (82%) DNFs, including 10 of 12 (83%) DNFs with melanocytic differentiation, were associated with neurofibromatosis type 1. Expression of melanocytic markers, including melan A, HMB-45 antigen, and MITF in DNF is a potential pitfall in differential diagnosis with melanocytic lesions that may clinically or histopathologically resemble DNF, in particular congenital melanocytic nevus with neurotization and neurofibroma-like melanoma.

Bi-clonal, multifocal primary cutaneous marginal zone B-cell lymphoma: report of a case and review of the literature.

Bi-clonality is a rare phenomenon seen in approximately 5% of chronic B-cell lymphoproliferative disorders. Both true bi-clonality and somatic hypermutation resulting in intraclonal evolution have been described. We present the case of a 37-year-old female who developed extranodal marginal zone B-cell lymphoma with immunohistochemical studies showing monotypic immunostaining of plasma cells for immunoglobulin lambda light chain on her right arm in 2008. Three years later, she developed a second focus of extranodal marginal zone B-cell lymphoma on her left arm, but immunohistochemical studies demonstrated monotypic immunostaining of plasma cells for immunoglobulin kappa light chain confirmed after repeat analysis. Evaluation for systemic lymphoma with laboratory and imaging studies was negative. Together, the findings were consistent with bi-clonal, multifocal extranodal primary cutaneous marginal zone B-cell lymphoma. We present this case to highlight a rare phenomenon within primary cutaneous marginal zone lymphomas.

Histopathologic and immunohistochemical characterization of rash to human epidermal growth factor receptor 1 (HER1) and HER1/2 inhibitors in cancer patients.

PURPOSE: Human epidermal growth factor receptor (HER) 1 and HER 1/2 inhibitors have shown benefit against a wide range of solid tumors. However, their use is associated with rash in 40% to 90% of patients, which impacts quality of life and interrupts antineoplastic therapy. The pathologic characteristics of affected skin remain unclear, precluding development of rational therapies. The aim of this study was to evaluate differences in histologic and immunohistochemical alterations in rash caused by lapatinib, a dual HER1/2 inhibitor (HER1/2i), and the single HER1 inhibitors (HER1i) cetuximab, erlotinib, and panitumumab. EXPERIMENTAL DESIGN: For each of the four drugs, skin biopsies were collected and analyzed from 8 patients with rash (n = 32). Blinded independent histologic analysis and automated measurement of 17 skin biomarkers involved in proliferation, differentiation, and inflammation were conducted. RESULTS: Increased expression of pAKT and decreased dermal K16 and p27 for HER1/2i when compared with each of the HER1i were observed. In addition, decreased epidermal atrophy and follicular neutrophilic infiltrate were evidenced in the skin of patients on HER1/2i when compared with HER1i. CONCLUSIONS: We found a lower inhibition of epidermal kinetics and decreased inflammation in HER1/2i-induced rash. These findings underscore differences in skin toxicity as related to specificity of HER blockade, concordant with clinical tolerability and decreased severity of skin toxicity seen with the HER1/2i lapatinib compared with the HER1 inhibitors cetuximab, erlotinib, and panitumumab.

An immunohistochemical analysis of pseudomelanocytic nests mimicking melanoma in situ: report of 2 cases.

Several case reports have discussed the difficulty in differentiating junctional pseudomelanocytic nests as a result of lichenoid inflammation from a true melanocytic neoplasm. Even immunohistochemistry can be misleading in these cases with both Melan-A and Mart-1 frequently resulting in false positivity as a result of nonspecific labeling of nonmelanocytic cells containing melanosomes. We present a series of 2 similar cases which were initially misdiagnosed as melanoma in situ likely as a result of Mart-1 positivity of the pseudomelanocytic nests. However, in our review, a significant lichenoid reaction was apparent at the dermal-epidermal junction. Staining with microphthalmia-associated transcription factor (MITF) showed a normal density of melanocytes along the dermal-epidermal junction and failed to uniformly label the Mart-1-positive pseudomelanocytic nests. In both patients, medications frequently resulting in fixed drug eruptions were identified, and a final diagnosis of fixed drug eruption was rendered in both cases. In light of these findings we suggest MITF is a more useful marker for evaluating lentiginous proliferations along the dermal-epidermal junction particularly when dealing with the differential diagnosis of lichenoid reaction with pseudomelanocytic nests versus melanoma in situ.

Combined IL-8 and TGF-beta blockade efficiently prevents neutrophil infiltrates into an A549-cell tumor.

Neutrophil infiltrates into tumors have been reported in certain circumstances to reduce tumor growth and in other circumstances to augment tumor growth, particularly by facilitating metastasis. Neutrophil chemotaxis can be facilitated by both interleukin-8 (IL-8) and transforming growth factor-beta (TGF-beta). However, the combined effects of these two cytokines on neutrophil tumor infiltrates is unknown, and we considered the possibility that studying the combined effects might resolve apparent contradictions with regard to neutrophil effects on tumor development. First, we determined that a simultaneous IL-8 and TGF-beta blockade is far more efficient at eliminating the neutrophil infiltrate from an A549 derived tumor than is blockade of either cytokine alone. Blockade of IL-8 alone, led to smaller tumors, consistent with the known inhibitory role of TGF-beta on A549 cell proliferation. Blockade of TGF-beta alone rescued the tumor growth but led to reduced metastasis volume. Surprisingly, blockade of both cytokines rescued both tumor volume and metastasis, underscoring the difficulty of understanding the effects of complete tumor cytokine elaboration profiles by isolating the effects of only one cytokine.

Local recurrence of ductal carcinoma in situ after skin-sparing mastectomy.

BACKGROUND: The incidence of local recurrence (LR) after conventional total mastectomy for ductal carcinoma in situ (DCIS) ranges from 1% to 3%. Skin-sparing mastectomy (SSM) preserves the native skin envelope to facilitate immediate breast reconstruction. Because DCIS is generally not clinically apparent, there is a potential for inadequate excision when SSM is performed. Risk factors for local recurrence after SSM for DCIS are examined. STUDY DESIGN: A retrospective review of 223 consecutive patients with DCIS treated by SSM and immediate reconstruction was performed. Age younger than 50 years, tumor size > 40 mm, high tumor grade, tumor necrosis, surgical margins < 1 mm, type of biopsy (excisional versus core), and SSM type were examined as risk factors for recurrence. RESULTS: Mean followup was 82.3 months (range 4.9 to 123.2 months). Recurrences developed in 11 patients (5.1%), including: local (n = 7; 3.3%), regional (n = 2; 0.9%), and distant (n = 2; 0.9%). All seven local recurrences were detected by physical examination. No patients received adjuvant radiation therapy. Two of 19 patients with surgical margins < 1 mm developed LR (10.5%). Univariate analysis showed high tumor grade (p = .019) to influence LR. CONCLUSIONS: The incidence of local recurrence of DCIS after SSM is similar to conventional total mastectomy. Reexcision of close margins should be performed if possible and adjuvant radiation therapy should be considered.

A pilot quality-of-life instrument for acne rosacea.

BACKGROUND: No rosacea-specific quality-of-life (QOL) instrument exists. OBJECTIVE: We sought to develop a validated, reliable rosacea-specific instrument. METHODS: From 6 in-depth interviews, we composed 21 rosacea-specific items. These items and Skindex-29 were administered in a validation cohort (n = 59). Internal consistency reliability and reproducibility were measured with Cronbach's coefficient alpha and intraclass correlation coefficient, respectively. Responsiveness was assessed comparing baseline with 4- to 6-month responses. Construct validity was assessed with principal axes factor analyses. Discriminant validity was examined with an additional 38 patients comparing differences in responsiveness between the rosacea-specific QOL instrument and Skindex. RESULTS: Reliability was high (Cronbach's alpha: 0.82-0.97, intraclass correlation coefficient: 0.70-0.95). The rosacea-specific QOL instrument showed preliminary responsiveness for patients with improved disease (P