A rapid genotyping panel for detection of primary central nervous system lymphoma.

Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N = 1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0 to 617 days. Permanent histopathology confirmed PCNSL in 142 of 152 biopsies (93.4%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6% sensitivity and 100% specificity (95% confidence interval [CI]: 44.1% to 70.4% and 87.2% to 100%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8% sensitivity and 100% specificity (95% CI: 56.2%-74.5% and 83.9%-100%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Synthesis and Characterization of a Magnetically Active 19F Molecular Beacon.

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine (19F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the 19F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the 19F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the 19F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

Developmental Deconvolution for Classification of Cancer Origin.

Cancer is partly a developmental disease, with malignancies named based on cell or tissue of origin. However, a systematic atlas of tumor origins is lacking. Here we map the single-cell organogenesis of 56 developmental trajectories to the transcriptomes of over 10,000 tumors across 33 cancer types. We deconvolute tumor transcriptomes into signals for individual developmental trajectories. Using these signals as inputs, we construct a developmental multilayer perceptron (D-MLP) classifier that outputs cancer origin. D-MLP (ROC-AUC: 0.974 for top prediction) outperforms benchmark classifiers. We analyze tumors from patients with cancer of unknown primary (CUP), selecting the most difficult cases in which extensive multimodal workup yielded no definitive tumor type. Interestingly, CUPs form groups distinguished by developmental trajectories, and classification reveals diagnosis for patient tumors. Our results provide an atlas of tumor developmental origins, provide a tool for diagnostic pathology, and suggest developmental classification may be a useful approach for patient tumors. SIGNIFICANCE: Here we map the developmental trajectories of tumors. We deconvolute tumor transcriptomes into signals for mammalian developmental programs and use this information to construct a deep learning classifier that outputs tumor type. We apply the classifier to CUP and reveal the developmental origins of patient tumors. See related commentary by Wang, p. 2498. This article is highlighted in the In This Issue feature, p. 2483.

Temporary Regulatory Deviations and the Coronavirus Disease 2019 (COVID-19) PCR Labeling Update Study Indicate What Laboratory-Developed Test Regulation by the US Food and Drug Administration (FDA) Could Look Like.

The coronavirus disease 2019 (COVID-19) response necessitated innovations and a series of regulatory deviations that also affected laboratory-developed tests (LDTs). To examine real-world consequences and specify regulatory paradigm shifts, legislative proposals were aligned on a common timeline with Emergency Use Authorization (EUA) of LDTs and the US Food and Drug Administration (FDA)-orchestrated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) labeling update study. The initial EUA adoption by LDT developers shows that the FDA can have oversight over LDTs. We used efficiency-corrected microcosting of our EUA PCR assay to estimate the national cost of the labeling update study to $0.3 to $1.4 million US dollars. Labeling update study performance data showed lower average detection limits in commercial in vitro diagnostic (IVD) assays versus LDTs (32,000 ± 75,000 versus 71,000 ± 147,000 nucleic acid amplification tests/mL; P = 0.04); however, comparison also shows that FDA review of IVD assays and LDTs did not prevent differences between initial and labeling update performance (IVD assay, P < 0.0001; LDT, P = 0.003). The regulatory shifts re-emphasized that both commercial tests and LDTs rely heavily on laboratory competence and procedures; however, lack of performance data on authorized tests, when clinically implemented, precludes assessment of the benefit related to regulatory review. Temporary regulatory deviations during the pandemic and regulatory science tools (ie, reference material) have generated valuable real-world evidence to inform pending legislation regarding LDT regulation.

Lymph node metastases develop through a wider evolutionary bottleneck than distant metastases.

Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms.

MET Alterations Are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer.

PURPOSE: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. EXPERIMENTAL DESIGN: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. RESULTS: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. CONCLUSIONS: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

Gene expression-based enrichment of live cells from adipose tissue produces subpopulations with improved osteogenic potential.

INTRODUCTION: Mesenchymal stem cells have been increasingly used for cell-based therapies. Adipose-derived stem/stromal cells (ASCs) from the stromal vascular fraction (SVF) of fat tissue are a particularly attractive option for cell based therapy given their accessibility and relative abundance. However, their application in both clinical and basic science investigations is complicated by the isolation of differentiable cells within the SVF. Current enrichment strategies, such as monolayer passaging and surface marker-based sorting, can be time-consuming or overly stringent. Ideally, a population of cells with great regenerative capacity could be isolated with high yields so that extensive in vitro manipulation is not necessary. The objective of this study was to determine whether SVF cells sorted based on expression of alkaline phosphatase liver/bone/kidney (ALPL) resulted in populations with increased osteogenic differentiation potential. METHODS: SVF samples were obtained from four, human donors and processed to isolate initial, heterogeneous cell populations. These SVF cells underwent a four day osteogenic priming period, after which they were treated with a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA. Cells were separated into positive and negative groups using fluorescence-activated cell sorting (FACS) then differentiated down the osteogenic lineage. Differentiation was assessed by measuring calcified matrix production in each sample. RESULTS: Cells positive for ALPL expression (ALPL+) represented approximately 34% of the gated population, while cells negative for ALPL expression (ALPL-) represented approximately 18%. ALPL+ cells produced 3.7-fold and 2.1-fold more calcified matrix than ALPL- and unsorted SVF cells, respectively, indicating a significant improvement in osteogenic differentiation. Further, ALPL+ cells showed increases in metabolite production for both adipogenesis and chondrogenesis, suggesting that the enrichment process yields an enhanced multipotent phenotype. Osteogenic differentiation response and cell yields for ALPL+ cells were markedly improved over surface marker-sorted samples. CONCLUSION: This study demonstrates a novel method to enrich heterogeneous SVF cells for increased osteogenic potential. The procedure requires less time and results in higher yields of therapeutically useful cells than other existing approaches. Gene expression-based sorting of MSCs is a potentially paradigm-shifting approach that could benefit applications spanning from basic science to clinical therapy.