The blood proteomic signature of prurigo nodularis reveals distinct inflammatory and neuropathic endotypes: A cluster analysis.

BACKGROUND: Prurigo nodularis (PN) is an extremely pruritic, chronic inflammatory skin disease. Little is known about systemic inflammation in PN. OBJECTIVE: To characterize plasma inflammatory biomarkers in patients with PN and investigate the presence of disease endotypes. METHODS: In this cross-sectional study, Olink proteomic analysis was performed on plasma samples from patients with PN (n = 29) and healthy controls (n = 18). RESULTS: Patients with PN had increased levels of 8 circulating biomarkers compared to controls, including tumor necrosis factor, C-X-C Motif Chemokine Ligand 9, interleukin-12B, and tumor necrosis factor receptor superfamily member 9 (P < .05). Two PN clusters were identified in cluster 1 (n = 13) and cluster 2 (n = 16). Cluster 2 had higher levels of 25 inflammatory markers than cluster 1. Cluster 1 had a greater percentage of patients with a history of myelopathy and spinal disc disease compared with cluster 2 (69% vs 25%, P = .03). Patients in cluster 2 were more likely to have a history of atopy (38% in cluster 2 vs 8% in cluster 1, P = .09). LIMITATIONS: Small sample size precludes robust subgroup analyses. CONCLUSION: This study provides evidence of neuroimmune-biased endotypes in PN and can aid clinicians in managing patients with PN that are nonresponsive to traditional therapies.

TrkA overexpression in non-tumorigenic human breast cell lines confers oncogenic and metastatic properties.

BACKGROUND/PURPOSE: TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS: To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS: TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS: Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.

Undetectable Tumor Cell-Free DNA in a Patient With Metastatic Breast Cancer With Complete Response and Long-Term Remission.

The ability to serially monitor tumor-derived cell-free DNA (cfDNA) brings with it the potential to measure response to anticancer therapies and detect minimal residual disease (MRD). This report describes a patient with HER2-positive metastatic breast cancer with an exceptional response to trastuzumab and nab-paclitaxel who remains in complete remission several years after cessation of therapy. Next-generation sequencing of the patient's primary tumor tissue showed several mutations, including an oncogenic hotspot PIK3CA mutation. A sample of cfDNA was collected 6 years after her last therapy and then analyzed for mutant PIK3CA using digital PCR. No detectable mutations associated with the primary tumor were found despite assaying >10,000 genome equivalents, suggesting that the patient had achieved a molecular remission. Results of this case study suggest that serial monitoring of MRD using liquid biopsies could provide a useful method for individualizing treatment plans for patients with metastatic disease with extreme responses to therapy. However, large-scale clinical studies are needed to validate and implement these techniques for patient care.

The estrogen receptor-alpha S118P variant does not affect breast cancer incidence or response to endocrine therapies.

PURPOSE: Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. METHODS: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. RESULTS: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. CONCLUSIONS: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability.

The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1.

HER2 missense mutations have distinct effects on oncogenic signaling and migration.

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.

PIK3CA mutations and TP53 alterations cooperate to increase cancerous phenotypes and tumor heterogeneity.

BACKGROUND/PURPOSE: The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. EXPERIMENTAL DESIGN/METHODS: We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. RESULTS: Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. CONCLUSIONS: This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

Single-Nucleotide Polymorphism Leading to False Allelic Fraction by Droplet Digital PCR.

BACKGROUND: Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS: Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA. RESULTS: Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele. CONCLUSIONS: These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers.

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

A Polycythemia Vera JAK2 Mutation Masquerading as a Duodenal Cancer Mutation.

Next-generation sequencing (NGS) is increasingly being used in cancer care to identify both somatic tumor driver mutations that can be targeted for therapy, and heritable mutations in the germline associated with increased cancer risk. This report presents a case of a JAK2 V617F mutation falsely identified as a duodenal cancer mutation via NGS. The patient was found to have a history of polycythemia vera, a disorder with a high incidence of JAK2 somatic mutations. Buccal cell DNA showed heterozygosity for the mutation, suggesting that it was potentially germline. However, subsequent resequencing of tumor, adjacent normal tissue, and fingernail DNA confirmed the mutation was somatic, and its presence in tumor and buccal cells resulted from contaminating blood cells. This report highlights important nuances of NGS that can lead to misinterpretation of results with potential clinical implications.

A phosphoproteomic screen demonstrates differential dependence on HER3 for MAP kinase pathway activation by distinct PIK3CA mutations.

The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways.

Single-Cell RNA Sequencing Reveals Dysregulated POSTN+WNT5A+ Fibroblast Subclusters in Prurigo Nodularis.

Prurigo nodularis (PN) is an intensely pruritic, inflammatory skin disease with a poorly understood pathogenesis. We performed single-cell transcriptomic profiling of 28,695 lesional and nonlesional PN cells. Lesional PN has increased dysregulated fibroblasts (FBs) and myofibroblasts. FBs in lesional PN were shifted toward a cancer-associated FB-like phenotype, with POSTN+WNT5A+ cancer-associated FBs increased in PN and similarly so in squamous cell carcinoma. A multicenter cohort study revealed an increased risk of squamous cell carcinoma and cancer-associated FB-associated malignancies (breast and colorectal) in patients with PN. Systemic fibroproliferative diseases (renal sclerosis and idiopathic pulmonary fibrosis) were upregulated in patients with PN. Ligand-receptor analyses demonstrated an FB neuronal axis with FB-derived WNT5A and periostin interactions with neuronal receptors melanoma cell adhesion molecule and ITGAV. These findings identify a pathogenic and targetable POSTN+WNT5A+ FB subpopulation that may predispose cancer-associated FB-associated malignancies in patients with PN.

Single-cell RNA sequencing reveals dysregulated fibroblast subclusters in prurigo nodularis.

Prurigo nodularis (PN) is an intensely pruritic, chronic inflammatory skin disease that disproportionately affects black patients. However, the pathogenesis of PN is poorly understood. We performed single-cell transcriptomic profiling, ligand receptor analysis and cell trajectory analysis of 28,695 lesional and non-lesional PN skin cells to uncover disease-identifying cell compositions and genetic characteristics. We uncovered a dysregulated role for fibroblasts (FBs) and myofibroblasts as a key pathogenic element in PN, which were significantly increased in PN lesional skin. We defined seven unique subclusters of FBs in PN skin and observed a shift of PN lesional FBs towards a cancer-associated fibroblast (CAF)-like phenotype, with WNT5A+ CAFs increased in the skin of PN patients and similarly so in squamous cell carcinoma (SCC). A multicenter PN cohort study subsequently revealed an increased risk of SCC as well as additional CAF-associated malignancies in PN patients, including breast and colorectal cancers. Systemic fibroproliferative diseases were also upregulated in PN patients, including renal sclerosis and idiopathic pulmonary fibrosis. Ligand receptor analyses demonstrated increased FB1-derived WNT5A and periostin interactions with neuronal receptors MCAM and ITGAV, suggesting a fibroblast-neuronal axis in PN. Type I IFN responses in immune cells and increased angiogenesis/permeability in endothelial cells were also observed. As compared to atopic dermatitis (AD) and psoriasis (PSO) patients, increased mesenchymal dysregulation is unique to PN with an intermediate Th2/Th17 phenotype between atopic dermatitis and psoriasis. These findings identify a pathogenic role for CAFs in PN, including a novel targetable WNT5A+ fibroblast subpopulation and CAF-associated malignancies in PN patients.

Multisystem organ failure secondary to acute generalised exanthematous pustulosis (AGEP) with atypical presentation resembling septic shock.

A woman was admitted for sepsis secondary to cellulitis. After clinical improvement of sepsis, non-follicular small pustules were observed on the trunk, limbs and face while vesicles/bullae and skin exfoliation were noted on upper extremities. Larger systemic manifestations included fever, hypertension and tachycardia. Laboratory results revealed neutrophilic leukocytosis, eosinophilia, mild transaminitis and acute renal failure. Despite treatment for potential sepsis and discontinuation of offending agents, her condition worsened leading to haemodynamic instability and renal failure requiring vasopressor support, intubation and continuous veno-venous haemodialysis. Skin biopsy revealed a diagnosis of acute generalised exanthematous pustulosis (AGEP), a rare condition usually caused by antibiotic treatment. The suspected offending drug was clindamycin, with possible combined effects by metronidazole and/or vancomycin. Improvement of skin manifestations were seen within 48 hours of starting systemic steroids. Here, we present an uncharacteristic case of AGEP clinically presenting with atypical skin lesions, severe systemic involvement mimicking septic shock, which culminated in multisystem organ failure.

Circulating plasma IL-13 and periostin are dysregulated type 2 inflammatory biomarkers in prurigo nodularis: A cluster analysis.

Importance: Prurigo nodularis (PN) is a chronic heterogeneous inflammatory skin disease. Objective: To elucidate which components of type 2 inflammation are dysregulated systemically in PN. Design: Whole blood was obtained from PN patients with uncontrolled disease and control patients without pruritus. Plasma was assayed for IL-4, IL-5, IL-13, IgE, and periostin. ANOVA was utilized to compare PN and control patients and multiple-hypothesis adjusted p-value was calculated with the significance threshold at 0.05. Clustering was performed using K-means clustering. Participants: PN patients (n = 29) and controls (n = 18) from Johns Hopkins Dermatology had similar age sex, and race distributions. Results: Single-plex assays of the biomarkers demonstrated elevated circulating plasma IL-13 (0.13 vs. 0.006 pg/mL, p = 0.0008) and periostin (80.3 vs. 60.2 ng/mL, p = 0.012) in PN compared to controls. IL-4 (0.11 vs. 0.02 pg/mL, p = 0.30) and IL-5 (0.75 vs. 0.40 pg/mL, p = 0.10) were not significantly elevated, while IgE approached significance (1202.0 vs. 432.7 ng/mL, p = 0.08). Clustering of PN and control patients together revealed two clusters. Cluster 1 (n = 36) consisted of 18 PN patients and 18 controls. Cluster 2 (n = 11) consisted entirely of PN patients (p < 0.01). Cluster 2 had higher levels of IL-13 (0.33 vs. 0.008 pg/mL, p = 0.0001) and IL-5 (1.22 vs. 0.43 pg/mL, p = 0.03) compared to cluster 1. Conclusion and relevance: This study demonstrates elevation of IL-13 and periostin in the blood of PN patients, with distinct clusters with varying degrees of type 2 inflammation. Given this heterogeneity, future precision medicine approaches should be explored in the management of PN.

NOTCH1 PEST domain variants are responsive to standard of care treatments despite distinct transformative properties in a breast cancer model.

Activating variants in the PEST region of NOTCH1 have been associated with aggressive phenotypes in human cancers, including triple-negative breast cancer (TNBC). Previous studies suggested that PEST domain variants in TNBC patients resulted in increased cell proliferation, invasiveness, and decreased overall survival. In this study, we assess the phenotypic transformation of activating NOTCH1 variants and their response to standard of care therapies. AAV-mediated gene targeting was used to isogenically incorporate 3 NOTCH1 variants, including a novel TNBC frameshift variant, in two non-tumorigenic breast epithelial cell lines, MCF10A and hTERT-IMEC. Two different variants at the NOTCH1 A2241 site (A2441fs and A2441T) both demonstrated increased transformative properties when compared to a non-transformative PEST domain variant (S2523L). These phenotypic changes include proliferation, migration, anchorage-independent growth, and MAPK pathway activation. In contrast to previous studies, activating NOTCH1 variants did not display sensitivity to a gamma secretase inhibitor (GSI) or resistance to chemotherapies. This study demonstrates distinct transformative phenotypes are specific to a given variant within NOTCH1 and these phenotypes do not correlate with sensitivities or resistance to chemotherapies or GSIs. Although previous studies have suggested NOTCH1 variants may be prognostic for TNBC, our study does not demonstrate prognostic ability of these variants and suggests further characterization would be required for clinical applications.

Hierarchical tumor heterogeneity mediated by cell contact between distinct genetic subclones.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact-mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor-positive and -negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.

Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity.

BACKGROUND: Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. DESIGN AND METHODS: Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. RESULTS: Recovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. CONCLUSIONS: We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.