Cutaneous Specimens Research Articles

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ON THE BACK COVER: Immunofluorescence analysis of a histological section derived from a cutaneous squamous cell carcinoma specimen. S100A4 (green), primarily expressed by fibroblast cells, cytokeratin 14 (red), primarily expressed by basal epidermal cells, and DAPI (blue) to mark cell nuclei.Credit: Alexandra Pittar, Centre for Cancer Biology, an Alliance between SA Pathology and the University of South Australia, Adelaide, Australia. image

Differences in the pathological, transcriptomic, and prognostic implications of lymphoid structures between primary and metastatic cutaneous melanomas

BackgroundWhile the prognostic role of tertiary lymphoid structures (TLS) has been well studied in solid cancers, the prevalence and impact of immature precursor lymphoid structures known as lymphoid aggregates (LA)...

The classification of melanocytic gene signatures.

Gene expression profiling technologies have revolutionized cell biology, enabling researchers to identify gene signatures linked to various biological attributes of melanomas, such as pigmentation status, differentiation state, proliferative versus invasive capacity, and disease progression. Although the discovery of gene signatures has significantly enhanced our understanding of melanocytic phenotypes, reconciling the numerous signatures reported across independent studies and different profiling platforms remains a challenge. Current methods for classifying melanocytic gene signatures depend on exact gene overlap and comparison with unstandardized baseline transcriptomes. In this study, we aimed to categorize published gene signatures into clusters based on their similar patterns of expression across clinical cutaneous melanoma specimens. We analyzed nearly 800 melanoma samples from six gene expression repositories and developed a classification framework for gene signatures that is resilient against biases in gene identification across profiling platforms and inconsistencies in baseline standards. Using 39 frequently cited published gene signatures, our analysis revealed seven principal classes of gene signatures that correlate with previously identified phenotypes: Differentiated, Mitotic/MYC, AXL, Amelanotic, Neuro, Hypometabolic, and Invasive. Each class is consistent with the phenotypes that the constituent gene signatures represent, and our classification method does not rely on overlapping genes between signatures. To facilitate broader application, we created WIMMS (what is my melanocytic signature, available at https://wimms.tanlab.org/), a user-friendly web application. WIMMS allows users to categorize any gene signature, determining its relationship to predominantly cited signatures and its representation within the seven principal classes.

Study of MIC of silver and zinc oxide nanoparticles, strong and cost-effective antibacterial against biofilm-producing Acinetobacter baumannii in Shiraz, Southwest of Iran

BackgroundAcinetobacter baumannii resistant strains lead to increased mortality, treatment costs, and an increase in the length of hospitalization. Nowadays, nanoparticles are considered a substitute for antibiotics. This study aimed to determine the MIC of Silver (Ag) and Zinc Oxide (ZnO) Nanoparticles (NPs) on Biofilm-Producing Acinetobacter baumannii and determine the relationship between MIC and frequency of efflux pump genes in cutaneous specimens in Shiraz, Southwest Iran in 2021–2022.MethodsIn this study, specimens were collected from April 2021 to June 2022 at Namazi and Faqihi Hospitals in Shiraz. Investigation of biofilm production in multidrug resistance (MDR) isolates was done by the microtiter plate method. Synthesized nanoparticles were characterized by UV–vis spectrum, X-ray diffraction (XRD), and electron microscopy. The MIC of AgNPs and ZnONPs for isolates was done using the method described in the CLSI guideline (2018). The antibacterial effect of MIC of NPs on inanimate objects was done by colony counts. The prevalence of efflux pump genes (adeR, adeC, adeA, abeM, adeK, adeI) was also investigated by PCR technique.ResultsThe highest ceftriaxone resistance (68%) and lowest colistin resistance (7%) were identified. 57% of isolates were MDR. In addition, 71.9% could produce biofilm and 28.1% of isolates could not produce biofilm. The average size of AgNPs and ZnONPs in the present study is 48 and < 70 nm, respectively. The nanoparticles were spherical. The MIC and the MBC of the ZnONPs were in the range of 125 to 250 µg/mL respectively. Also, for AgNPs, the MIC and the MBC were in the range of 62.5 to 250 µg/ml, respectively. AbeM gene had the highest frequency and the AdeK gene had the lowest frequency. Statistical analysis showed that there is a relationship between the frequency of adeA, adeC, and adeM genes with the MIC of AgNPs and ZnONPs.ConclusionAccording to the results of the present study, inanimate objects such as scalpels in contact with AgNPs (6000 µg/ml for 240 min) or ZnONPs (5000 µg/ml for 120 min) can be free of biofilm producing Acinetobacter baumannii with efflux pump genes.

Exploring the correlation between high HRD status and immunotherapy response in melanoma.

e21541 Background: Homologous recombination deficiency (HRD) status demonstrates DNA instability in tumor cells and has been implicated as a response marker for immune checkpoint inhibitors (ICI). Here, we explore the correlation between high HRD status and ICI response biomarkers, homologous recombination repair (HRR) mutations, and the prognostic advantage of high HRD status following ICI in cutaneous melanoma patients. Methods: Melanoma samples (N = 2158) were profiled for genomic loss of heterozygosity (LOH) by NGS at Caris Life Sciences (Phoenix, AZ). As a surrogate for HRD, LOH-high was defined as LOH at ≥ 16% of the segments analyzed (up to 552). PD-L1 expression was assessed using IHC (SP142; positive (+): ≥ 1%). Mismatch repair deficiency/microsatellite instability-high (dMMR/MSI-H) was tested by IHC/NGS.Tumor mutational burden (TMB)-high was defined as ≥ 10 mutations/Mb. interferon gamma (IFN-g) and T-cell inflamed scores. Real-world overall survival (rw-OS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from the start of immunotherapy until the last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied where appropriate, with p-values adjusted for multiple comparisons. Results: Melanoma patient samples include - cutaneous: 1853; acral: 130; mucosal: 175, with LOH-high observed in 8.3% of cutaneous specimens, 13.1% of acral, and 30.9% of mucosal. Firstly, most biomarkers associated with response to ICI (PD-L1, TMB-high, total neoantigen load, IFN-g score, T-cell inflamed level, and dMMR/MSI-H) were not significantly different between LOH-high and LOH-low cohorts in all melanoma subtypes. However, TMB-high was significantly more prevalent in LOH-low cutaneous melanoma patients (54.9% vs 63.3%, p = 0.039). Prevalence of HRR gene mutations was not significantly different between high and low LOH groups in all cohorts, except for the ATRX gene which exhibited a significantly higher prevalence in the high LOH cutaneous (8.6% vs 1.3%, p &lt; 0.001) and mucosal (28.8% vs 2.5%, p &lt; 0.00001) melanoma specimens. Analysis of rw-OS showed no improvement in the treatment of LOH-high cutaneous melanoma patients with ICI compared to LOH-low (HR: 1.397 [95% CI: 0.759- 2.569], p = 0.281). Conclusions: This study demonstrates that HRDness reflected by the high-LOH status in melanoma did not correlate with mutations in HRR genes. In addition, melanoma specimens with high HRD status did not exhibit an overall higher prevalence of biomarkers associated with ICI response. Our study did not demonstrate improved survival outcomes in patients with high HRD status following anti-PD1 therapies. Future investigation is needed to assess other therapeutic approaches for these patients such as PARP inhibitors.

Performance of the Xpert™ Mpox PCR assay with oropharyngeal, anorectal, and cutaneous lesion swab specimens

Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

Usefulness of Xpert MTB/RIF Ultra for rapid diagnosis of extrapulmonary tuberculosis in Tunisia

Extrapulmonary tuberculosis (EPTB) remains a challenging diagnosis. The purpose of this study was to assess the accuracy of Xpert MTB/RIF Ultra (Cepheid, USA) for rapid diagnosis of EPTB in Tunisia. Eight hundred and forty-seven extrapulmonary samples collected from 2017 to 2021, were subjected to Xpert MTB/RIF Ultra. Microscopy and culture were performed for all the specimens. The accuracy of Xpert Ultra was evaluated in comparison to the culture. Xpert Ultra diagnosed EPTB with a global sensitivity of 80.66% (74.3–85.75) and specificity of 70.87% (67.31–74.20). The molecular test was most accurate when performed in cerebrospinal fluids, bones and joints and cutaneous specimens showing a sensitivity of 100% and a specificity ranging from 70.60 to 91.11%. In lymph node samples comprising aspirates and biopsies, the sensitivity of Xpert Ultra was high 87.50% (77.23–93.53), however, the specificity was 51.08% (44.67–57.46). For pleural samples, the Xpert Ultra sensitivity was 77.50% (68.34–84.68) ranging from 71.43 to 80% in pleural biopsies and fluids respectively. The specificity in all pleural specimens was 79.56% (74.40–83.91). Xpert Ultra showed promise in the diagnosis of EPTB. The performances varied according to the site of the disease. The test may be more valuable if used in combination with other diagnostic modalities.

Immune Infiltrate Profiling of Cutaneous Melanoma with Spatial Transcriptomics

Background and Objective:In 2023, an estimated 97,610 melanomas will be diagnosed in the U.S. Immunotherapy has revolutionized care for melanoma patients and highlighted the prognostic value of characterizing immune infiltrates in melanoma tissue. This project used spatial transcriptomics (ST) to analyze immune infiltrates in cutaneous melanoma specimens.&#x0D; Methods:Visium Spatial Gene Expression platform generated datasets from formalin fixed paraffin embedded cutaneous melanoma specimens were analyzed using Loupe Browser software v6.5.0 (10x Genomics Inc.). Unsupervised clustering and manual region selection identified areas enriched in immune cell specific differentially expressed genes (DEGs). The transcript for Collagen alpha-1(XIX) chain (COL19A1) was identified as a DEG co-expressed with B cell marker DEGs. Fluorescence activated cell sorting was used to isolate primary B and T cells from mouse secondary lymphoid organs and human peripheral blood mononuclear cells to measure COL19A1 with quantitative PCR (qPCR).&#x0D; Results:Transcripts for CD19 (B cell marker) were concentrated in an immune DEG- rich region adjacent to a melanoma micrometastasis in one specimen. There was overlap between the tertiary lymphoid structure [TLS] markers SELL, CXCR5, CCR7, and CXCL13 with CD19 and/or COL19A1. Though qPCR revealed minimal Col19a1 in mouse B and T cells, COL19A1 was abundant and specific for human B cells.&#x0D; Conclusions &amp; Potential Impact:This study demonstrates the feasibility and potential of using ST to characterize immune cell infiltrates in melanoma. Though COL19A1 expression is typically understood to be enriched in muscle and neuronal cells, the studies presented here suggest this transcript could be a novel human (but not mouse) B cell marker. Future studies are needed to determine if collagen XIX (protein) is expressed in B cells and if it is, what the biological functions of this protein are in B cells under physiologic and pathologic (e.g. melanoma) conditions.

Frequency and features of malignant tumors clinically mimicking cutaneous cysts: A retrospective chart review.

Many cutaneous lesions are clinically suspected as "cyst"; however, following histopathological examination, are found to be more significant lesions. Here, we examine the frequency and features of malignancies with cutaneous cysts in the clinical differential. A retrospective study of surgical pathology specimens at the James A. Haley Veterans' Hospital from January 2018 to December 2022 was conducted. Cutaneous specimens containing the clinical diagnosis of "cyst" were included. The clinicopathological features were summarized. Premalignant or malignant neoplasms accounted for 4.5% of all specimens submitted with cysts in the clinical differential. Most cyst-mimicking cancers were basal cell carcinoma (BCC) or squamous cell carcinoma (SCC); however, cancers with poorer prognoses, such as Merkel cell carcinoma and melanoma, also clinically masqueraded as cysts. The BCCs were predominately nodular, and the SCCs were largely well-differentiated and invasive. Many exhibited clinical signs and symptoms compatible with benign cysts, such as central punctum, pain, and rapid growth. Identified risk factors included history of prior non-melanoma skin cancer diagnosis, previous excision, and immunosuppression. Many lesions clinically concerning cutaneous cysts were found to be malignancies following histopathological review. Accordingly, following biopsy all cyst-like lesions should be examined microscopically, especially in certain clinical contexts in which the incidence of skin cancer is increased.

1022. Detection of Mpox virus with the Cepheid Xpert Mpox assay in oropharyngeal, anorectal and cutaneous lesion swab specimens

Abstract Background The global spread of Mpox in 2022 largely affected men who have sex with men and exposures via the oropharynx and anorectum are thought to contribute to sexual transmission. New molecular platforms including point-of-care assays for detection of Mpox DNA from cutaneous lesion samples have received emergency use authorization in the United States but these technologies are not validated for use with other sample types, including oropharyngeal or anorectal swabs. Methods Anorectal, oropharyngeal and cutaneous lesion swabs swabs were initially collected and frozen dry at -80C during clinical evaluation for suspected Mpox at a large HIV care center in Atlanta, GA. Swabs were thawed to room temperature and eluted in 3 mL of transport medium prior to testing on the Cepheid Xpert platform, which was performed according to the assay's instructions for use. DNA extracted from 140 μL of the same sample was used for non-variola orthopoxvirus (E9L-NVAR) PCR as a reference test and RNaseP PCR as a sample adequacy control. Results Positive agreement between the Xpert Mpox assay and in-house PCR was 100% for 9 cutaneous lesion swabs, 8 oropharyngeal swabs, and 5 anorectal swabs (see Figure). There were no false-negative results returned by the Xpert. Overall negative agreement was 86.7% (26/30). The Xpert assay was positive in 6 specimens where viral DNA was not detected by the NVOG assay including two specimens that also did not have detectable RNAseP. 4 of these 6 specimens with discordant results were from patients with viral DNA detected at other anatomic sites. Results of the Xpert Mpox assay, NVAR PCR, and sample adequacy controls for cutaneous lesion, oropharyngeal and anorectal specimens. The Xpert assay was positive for 100% of specimens when the non-variola orthopoxvirus (NVAR) target detected by our reference assay and there were no false negative results. The Xpert assay also detected viral targets in 6 specimens where no viral DNA was detected by the NVAR assay. However, four of these specimens were collected from patients with Mpox DNA detected at other anatomic sites. Conclusion The Xpert Mpox assay had perfect positive agreement with the NVAR PCR assay in anorectal, oropharyngeal and cutaneous lesion swab specimens suggesting it has high sensitivity for diagnosis of Mpox infection at the point-of-care, including with not-yet validated sample types. Specificity of the assay is difficult to estimate from our data, as a likely explanation of our results is that the Xpert Mpox assay has a lower limit of detection compared to our reference assay. This is evidenced by detection of Mpox DNA with high Ct values in samples from patients with viral DNA confirmed by both assays at other anatomic sites. The Xpert Mpox assay can be expected to perform well with oropharyngeal and anorectal swab specimens in clinical settings. Disclosures Jonathan Colasanti, MD, MSPH, DKB MED LLC: Honoraria|Prime Education LLC: Advisor/Consultant

The evaluation of IgG4 and IgG expression in cutaneous Rosai-Dorfman disease

ObjectiveThe authors investigated the expression of IgG4 and IgG in cutaneous Rosai-Dorfman Disease (CRDD) to further improve the understanding of this disease. MethodsThe authors retrospectively reviewed the clinicopathological features of 23 CRDD patients. The authors diagnosed CRDD by the presence of emperipolesis and immunohistochemical (IHC) staining of histiocytes consisting of S-100(+)/CD68(+)/CD1a(-) cells. The expressions of IgG and IgG4 in cutaneous specimens were assessed by IHC (EnVision) and quantitatively calculated by a medical image analysis system. ResultsAll 23 patients, including 14 males and 9 females, were confirmed to have CRDD. Their ages ranged from 17 to 68 years (mean 47.91 ± 14.16). The most frequently affected skin regions were the face, followed by the trunk, ears, neck, limbs, and genitals. In 16 of these cases, the disease presented as a single lesion. IHC staining of sections showed that IgG was positive (≥ 10 cells/High-Power Field [HPF]) in 22 cases, while IgG4 was positive (≥ 10 cells/HPF) in 18 cases. Moreover, the IgG4/IgG proportion ranged from 1.7% to 85.7% (mean 29.50 ± 24.67%, median 18.4%) in the 18 cases. Study limitationsIn the majority of studies, as well as in the current study, the design. RDD is a rare disease, so the sample size is small. In the next studies to come, the authors will expand the sample for multi-center verification and in-depth study. ConclusionThe positive rates of IgG4 and IgG and the IgG4/IgG ratio assessed through IHC staining may be important in understanding the pathogenesis of CRDD.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

Molecular and clinical correlates of HER3 expression highlights its potential role as a therapeutic target in melanoma

Overexpression of the epidermal growth factor receptor family member HER3 (erbB3) has been implicated in several types of cancer and recently drugs targeting HER3 have shown promising clinical activity. In melanoma, HER3 overexpression has been linked to both metastasis formation and resistance to drug therapy in cell culture models. Here, we sought to characterise the expression of HER3 in 187 melanoma biopsies (149 cutaneous, 38 mucosal) using immunohistochemistry, as well as to analyse the association between HER3 expression and molecular, clinical and pathological variables. A subset of the cutaneous melanoma specimens was taken prior to treatment with immune checkpoint blockade therapy (pre-ICB) (n=79). HER3 expression (≥1+) was observed in 136 of 187 samples (∼73%). HER3 expression was found to be markedly lower in the mucosal melanomas, with 17 of the 38 tumours (∼45%) demonstrating no HER3 expression. In cutaneous melanomas, there was a negative association between HER3 expression and mutational load, a positive association with NRAS mutational status, and a trend of negative association with PD-L1 expression. In the pre-ICB cohort, an association was found between high HER3 expression (≥2+) and overall survival after anti-PD-1-based immunotherapy. Overall, our results indicate that HER3 is a promising therapeutic avenue in cutaneous melanoma worthy of further clinical evaluation.

An epidemiological study of the predictors of multidrug resistance and methicillin resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory in Tennessee, USA.

Understanding drivers of multidrug resistance (MDR) and methicillin resistance, which have increased among canine staphylococcal isolates, is essential for guiding antimicrobial use practices. Therefore, the objective of this study was to identify predictors of MDR and methicillin resistance among Staphylococcus spp. commonly isolated from canine clinical specimens. This retrospective study used records of canine specimens submitted to the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory for bacterial culture and antimicrobial susceptibility testing between 2006 and 2017. Records from 7,805 specimens positive for the following Staphylococcus species were included for analysis: Staphylococcus pseudintermedius, Staphylococcus aureus, Staphylococcus coagulans (formerly Staphylococcus schleiferi subspecies coagulans), and Staphylococcus schleiferi (formerly S. schleiferi subsp. schleiferi). Generalized linear regression models were fit using generalized estimating equations (GEE) to identify predictors of MDR (defined as resistance to three or more antimicrobial classes) and methicillin resistance among these isolates. Multidrug resistance (42.1%) and methicillin resistance (31.8%) were relatively common. Isolates from skeletal (joint and bone) specimens had the highest levels of MDR (51.3%) and methicillin resistance (43.6%), followed by cutaneous specimens (45.8% multidrug-resistant, 37.1% methicillin resistant). Staphylococcus species, specimen site, and clinical setting were significant (p<0.01) predictors of both outcomes. Compared to S. pseudintermedius, S. schleiferi had higher odds of methicillin resistance, while S. coagulans and S. schleiferi had lower odds of MDR. The odds of both MDR and methicillin resistance for isolates from hospital patient specimens were significantly higher than those from referral patients for urine/bladder and otic specimens. Odds of MDR among isolates from skeletal specimens of hospital patients were also higher than those of referral patients. Staphylococcus isolates in this study had substantial levels of MDR and methicillin resistance. Differences in the odds of these outcomes between referral and hospital patient isolates did not persist for all specimen sites, which may reflect differences in diagnostic testing and antimicrobial use practices with respect to body site or system. Judicious antimicrobial use, informed by culture and susceptibility testing, is important to limit treatment failures and curb selection pressure.

SH2 domain-containing protein tyrosine phosphatase-2 isenriched in eyelid specimens of rosacea.

Rosacea is a cutaneous disease that may secondarily affect the ocular surface. Due to the vision threatening, cosmetic, psychological, and work productivity impact, the identification of cellular targets that govern rosacea would enhance our understanding of the biology of the disease and delineate targets for therapeutic manipulation. To characterize the involvement of SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) in the pathogenesis of rosacea. Specimens from elective ectropion surgery (n=20) were processed from patients with rosacea (n=10) and control patients (n=10). Immunohistochemistry (IHC) and quantitative western blotting (WB) were performed to identify and quantify the presence of SHP2 and 4G10 (a phosphotyrosine antibody) in rosacea compared to normal tissue. IHC samples were graded according to an intensity scale (0-4). Mann-Whitney statistical analyses were performed via a dedicated computerized software package. On WB, SHP2 was expressed in higher concentrations in rosacea specimens (p<0.05). On IHC, SHP2 was enriched in the epidermis in rosacea (p<0.05), although 4G10 levels were not statistically significantly different between the two groups (p>0.05). SHP2 is enriched in cutaneous specimens of rosacea, suggesting a critical role for this protein in the disease and indicating a modifiable therapeutic moiety.

P393 Clinical spectrum and molecular characterization of Rhytidhysteron spp. (now Gloniopsis) from India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjective Rhytidhysteron spp. are emerging dematiaceous fungi implicated in subcutaneous infections in immunocompromised as well as immunocompetent individuals. Diagnosis of Rhytidhysteron spp is delayed owing to non-sporulation and misidentification. The present study was conducted to characterize Rhytidhysteron spp. isolated from India by molecular techniques.MethodWe retrieved a total of eight isolates of Rhytidhysteron spp from our National Culture Collection for Pathogenic Fungi (NCCPF) that were received from various centers across India. Colony morphology on Sabouraud's dextrose agar (SDA) and potato dextrose agar (PDA) was recorded. Molecular identification was performed by sequencing internal transcribed spacer (ITS) and 28 s region of rDNA. Phylogenetic tree was constructed using the Neighbor-Joining method along with sequences of standard strains retrieved from the NCBI database.Results Rhytidhysteron spp was confirmed by sequencing from cutaneous specimens of eight cases. Six patients were immunocompetent while two were post-renal transplant recipients. Phenotypic features on SDA and PDA revealed growth of greyish-black mycelia on the obverse with black pigmentation on reverse after 7 days of incubation. On microscopic examination of lactophenol cotton blue mount, only dematiaceous septate hyphae without any spores were noted. On phylogenetic analysis of 28 s region, four of our isolates were closely clustered with Gloniopsis calami (Fig.1b), whereas two isolates with G. pneumonia and one each with G. percutanea, and Gloniopsis spp. (Fig.1b). On ITS phylogeny, the same four isolates closely clustered with G. calami (Fig.1a) as in 28 s region, one each isolate clustered with G. pneumonia, and G. percutanea while two isolates formed a separate clade with Rhytidhysteron rufulum (Fig.1a).ConclusionThis fungus is often difficult to identify due to lack of sporulation making morphological identification challenging. Therefore, molecular sequencing is a must for its identification. However, identification using the sequence of ITS and 28 s rDNA does not clearly correlate. Hence to confirm the identity additional genes needs to be sequenced and analyzed.

Histopathologic Evaluation of Cutaneous Squamous Cell Carcinoma Debulk Specimens Before Mohs Micrographic Surgery and Its Influence on Squamous Cell Carcinoma Staging.

Weinhammer, Annika MD*; Bennett, Daniel D. MD*; Eickhoff, Jens PhD†; Xu, Yaohui G. MD, PhD* Author Information

1201. Diphtheria in Veterans Health Administration (VHA), 2000-2021

BackgroundDiphtheria is caused by Corynebacterium diphtheriae and can cause respiratory or skin infections. Transmission occurs primarily person-to-person via respiratory tract and rarely from skin lesions or fomites. In the Veterans Health Administration (VHA), we perform surveillance for nationally notifiable diseases such as diphtheria. In early 2021, there were 4 alerts for C. diphtheriae. Therefore, we investigated diphtheria prevalence in VHA over the last 20 years.MethodsIsolates of C. diphtheriae were identified from VHA data sources from 1/1/2000-2/28/2021. Patient demographics, co-morbidities, microbiologic data, treatment, outcomes, and vaccination status were obtained via electronic medical record (EMR) review.Results33 C. diphtheriae isolates were identified representing 32 unique individuals. 17 isolates were identified from 2000-2015 and 16 were identified from 2016-2021. Isolates were from cutaneous (16), blood (10), urine (4), pulmonary (2), and throat (1) specimens. In 11 individuals, clinical significance was unclear (no antibiotics given, note mentioned that it was being considered a contaminant - i.e., isolate may have been incorrectly labeled as “C. diphtheriae” instead of “diphtheroid”). Only 3 isolates had toxin testing documented. One C. diphtheriae biovar gravis blood isolate was associated with sepsis without another source identified. The throat isolate was a nontoxigenic strain. No cutaneous isolates underwent susceptibility testing, but all 15 individuals received antibiotics (1 patient had 2 isolates). 11 had additional organisms identified in addition to C. diphtheriae. Table 1 describes demographics, co-morbidities, and vaccination status of cutaneous cases. Only 1 case (in 2021) had EMR documentation of local public health department reporting.Table 1. Characteristics of Unique Individuals with Cutaneous Diphtheria Isolates in VHA, 2000-2021 ConclusionNearly as many isolates have been identified in the last 5.5 years compared to the previous 15 years which may be related to more robust molecular identification methods available in VHA. Most C. diphtheriae isolated was from cutaneous sources that were acute in onset. About 33% were identified as C. diphtheriae but were not treated. EMR documentation of toxin production and public health department reporting was lacking.Disclosures All Authors: No reported disclosures

27660 Parasite infection in a patient with a CD30 clonal T-cell lymphocytic variant of hypereosinophilic syndrome: Cause or coincidence

Hypereosinophilic syndromes (HES) are defined by the presence of blood and/or tissue hypereosinophilia producing organ damage. HES can arise from a primary (neoplastic) growth of eosinophils or a reactive proliferation of eosinophils secondary to parasitic infestations, inflammatory diseases or neoplasms. A lymphocytic variant (L-HES) has been recently described in which a clonal T-cell population releasing Th2-related cytokines induce eosinophil proliferation. Cutaneous lesions in HES are frequent and nonspecific. A 65-year-old Dominican male presented with a progressive generalized rash and dyspnea for 2 years. A skin biopsy was performed, showing an unspecific spongiotic dermatitis. Blood tests revealed eosinophilia (6.4ˆ3/uL) and IgE 1.050 UI/mL. Strongyloides stercolaris, Schistosoma mansoni, and Hymenolepsis nana were detected in the stool. Antiparasitic treatment was carried out and stool parasites were no longer detectable. However, both the dyspnea and the skin rash progressed. Hyperesosinophilia persisted. A CT scan and a lung biopsy confirmed an eosinophilic pneumonia. A new cutaneous specimen showed a perivascular and interstistitial infiltrated composed of eosinophils and atypical CD30+ T cells. Bone marrow biopsy was normal. Clonality of T-cell receptor was detected in peripheral blood. Brentuximab and meplizumab were introduced with rash and dyspnea improvement. Herein, we present a case of HES in which a parasite infestation was first diagnosed and correctly treated, yet without resolution of the symptoms. The subsequent hematologic investigations and skin biopsy revealed the presence of a clonal, atypical T cell proliferation consistent with the diagnosis of a L-HES. To our knowledge, this coincidence has not previously described.

Loss of Lymphotoxin Alpha-Expressing Memory B Cells Correlates with Metastasis of Human Primary Melanoma.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.