No abstract

Fungal infections are a rising threat to our immunocompromised patient population as well as other non-immunocompromised patients with various medical conditions. However, little progress has been made in the past decade to improve fungal diagnostics. To jointly address this diagnostic challenge, the Fungal Diagnostics Laboratory Consortium (FDLC) was recently created. The FDLC consists of 26 laboratories from the United States and Canada that routinely provide fungal diagnostic services for patient care. A survey of fungal diagnostic capacity among the 26 members of the FDLC was recently completed, identifying the following diagnostic gaps: lack of molecular detection of mucormycosis; lack of an optimal diagnostic algorithm incorporating fungal biomarkers and molecular tools for early and accurate diagnosis of Pneumocystis pneumonia, aspergillosis, candidemia, and endemic mycoses; lack of a standardized molecular approach to identify fungal pathogens directly in formalin-fixed paraffin-embedded tissues; lack of robust databases to enhance mold identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; suboptimal diagnostic approaches for mold blood cultures, tissue culture processing for Mucorales, and fungal respiratory cultures for cystic fibrosis patients; inadequate capacity for fungal point-of-care testing, to detect and identify new, emerging or under-recognized, rare or uncommon fungal pathogens, and performance of antifungal susceptibility testing. In this commentary, the FDLC delineates the most pressing unmet diagnostic needs and provides expert opinion on how to fulfill them. Most importantly, the FDLC provides a robust laboratory network to tackle these diagnostic gaps and ultimately to improve and enhance the clinical laboratory’s capability to rapidly and accurately diagnose fungal infections.

Invasive fungal infections are increasing in prevalence because of an expanding population of immunocompromised individuals. To reduce morbidity and mortality, it is critical to accurately identify fungal pathogens to guide treatment. Current methods rely on histopathology, fungal culture, and serology, which are often insufficient for diagnosis. Herein, we describe the use of a laboratorydeveloped internal transcribed spaceretargeted amplicon-based next-generation sequencing (NGS) assay for the identification of fungal etiology in fungal stainepositive formalin-fixed, paraffinembedded tissues by using Illumina MiSeq. A total of 44 specimens from 35 patients were included in this study, with varying degrees of fungal burden from multiple anatomic sites. NGS identified 20 unique species across the 54 total organisms detected, including 40 molds, 10 yeasts, and 4 dimorphic fungi. The histopathologic morphology and the organisms suspected by surgical pathologist were compared with the organisms identified by NGS, with 100% (44/44) and 93.2% (41/44) concordance, respectively. In contrast, fungal culture only provided an identification in 27.3% (12/44) of specimens. We demonstrated that NGS is a powerful method for accurate and unbiased fungal identification in formalin-fixed, paraffin-embedded tissues. A retrospective evaluation of the clinical utility of the NGS results also suggests this technology can potentially improve both the speed and the accuracy of diagnosis for invasive fungal infections.

Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+ NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100 nM EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (n=8) vs. age- and sex-matched normal healthy donors (n=8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NKLGLL patients.

Large granular lymphocyte (LGL) leukemia is a rare hematological disorder with expansion of the T‐cell or natural killer (NK) cell lineage. Signal transducer and activator of transcription 3 (STAT3) exhibits somatic activating mutations in 30%‐40% of LGL leukemia cases. Transcriptional targets of STAT3 include inflammatory cytokines, thus previous studies have measured cytokine levels of LGL leukemia patients compared to normal donors. Sphingolipid metabolism is a growing area of cancer research, with efforts focused on drug discovery. To date, no studies have examined serum sphingolipids in LGL leukemia patients, and only one study compared a subset of cytokines between the T‐LGL and NK‐LGL subtypes. Therefore, here, we included both LGL leukemia subtypes with the goals of (a) measuring serum sphingolipids for the first time, (b) measuring cytokines to find distinctions between the subtypes, and (c) establishing relationships with STAT3 mutations and clinical data. The serum analyses identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) significantly different in the LGL leukemia group compared to normal donors. In a mixed STAT3 mutation group, D661Y samples exhibited the highest mean corpuscular volume (MCV) values. We explored this further by expanding the cohort to include larger groups of single STAT3 mutations. Male D661Y STAT3 samples had lower Hgb and higher MCV compared to wild type (WT) or Y640F counterparts. This is the first report examining large groups of individual STAT3 mutations. Overall, our results revealed novel serum biomarkers and evidence that D661Y mutation may show different clinical manifestation compared to WT or Y640F STAT3.