Isaac Brownell

Hedgehog Signaling in Neonatal and Adult Lung

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Merkel Cell Carcinoma Therapeutic Update

Opinion statementMerkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine tumor of the skin. Early-stage disease can be cured with surgical resection and radiotherapy (RT). Sentinel lymph node biopsy (SLNB) is an important staging tool, as a microscopic MCC is frequently identified. Adjuvant RT to the primary excision site and regional lymph node bed may improve locoregional control. However, newer studies confirm that patients with biopsy-negative sentinel lymph nodes may not benefit from regional RT. Advanced MCC currently lacks a highly effective treatment as responses to chemotherapy are not durable. Recent work suggests that immunotherapy targeting the programmed cell death receptor 1/programmed cell death ligand 1 (PD-1/PD-L1) checkpoint holds great promise in treating advanced MCC and may provide durable responses in a portion of patients. At the same time, high-throughput sequencing studies have demonstrated significant differences in the mutational profiles of tumors with and without the Merkel cell polyomavirus (MCV). An important secondary endpoint in the ongoing immunotherapy trials for MCC will be determining if there is a response difference between the virus-positive MCC tumors that typically lack a large mutational burden and the virus-negative tumors that have a large number of somatic mutations and predicted tumor neoantigens. Interestingly, sequencing studies have failed to identify a highly recurrent activated driver pathway in the majority of MCC tumors. This may explain why targeted therapies can demonstrate exceptional responses in case reports but fail when treating all comers with MCC. Ultimately, a precision medicine approach may be more appropriate for treating MCC, where identified driver mutations are used to direct targeted therapies. At a minimum, stratifying patients in future clinical trials based on tumor viral status should be considered as virus-negative tumors are more likely to harbor activating driver mutations.

Cutaneous squamous and neuroendocrine carcinoma: genetically and immunohistochemically different from Merkel cell carcinoma

Cutaneous neuroendocrine (Merkel cell) carcinoma most often arises de novo in the background of a clonally integrated virus, the Merkel cell polyomavirus, and is notable for positive expression of retinoblastoma 1 (RB1) protein and low expression of p53 compared with the rare Merkel cell polyomavirus-negative Merkel cell carcinomas. Combined squamous and Merkel cell tumors are consistently negative for Merkel cell polyomavirus. Little is known about their immunophenotypic or molecular profile. Herein, we studied 10 combined cutaneous squamous cell and neuroendocrine carcinomas for immunohistochemical expression of p53, retinoblastoma 1 protein, neurofilament, p63, and cytokeratin 20 (CK20). We compared mutation profiles of five combined Merkel cell carcinomas and seven ‘pure’ Merkel cell carcinomas using targeted next-generation sequencing. Combined tumors were from the head, trunk, and leg of Caucasian males and one female aged 52–89. All cases were highly p53- and p63-positive and neurofilament-negative in the squamous component, whereas RB1-negative in both components. Eight out of 10 were p53-positive, 3/10 p63-positive, and 3/10 focally neurofilament-positive in the neuroendocrine component. Six out of 10 were CK20-positive in any part. By next-generation sequencing, combined tumors were highly mutated, with an average of 48 mutations per megabase compared with pure tumors, which showed 1.25 mutations per megabase. RB1 and p53 mutations were identified in all five combined tumors. Combined tumors represent an immunophenotypically and genetically distinct variant of primary cutaneous neuroendocrine carcinomas, notable for a highly mutated genetic profile, significant p53 expression and/or mutation, absent RB1 expression in the context of increased RB1 mutation, and minimal neurofilament expression.

Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium.

Significance The role of nerves in regulating stem cells is largely unknown. Here, we use the touch dome epithelium in skin as a model to study neural regulation of adult stem cells. We find that sensory nerves trophically maintain the touch dome epithelium by signaling with Sonic hedgehog (Shh) to lineage-specific stem cells. This novel aspect of touch dome innervation demonstrates retrograde paracrine signaling to sensory epithelium progenitors by afferent sensory neurons. Indeed, neural Shh is a key regulatory factor in the perineural niche required for long-term renewal of touch dome stem cells. We further demonstrate that Hedgehog upregulation alone is not sufficient to drive malignant expansion of mouse Merkel cells, despite reports of active Hedgehog signaling in Merkel cell carcinoma. The touch dome is a highly patterned mechanosensory structure in the epidermis composed of specialized keratinocytes in juxtaposition with innervated Merkel cells. The touch dome epithelium is maintained by tissue-specific stem cells, but the signals that regulate the touch dome are not known. We identify touch dome stem cells that are unique among epidermal cells in their activated Hedgehog signaling and ability to maintain the touch dome as a distinct lineage compartment. Skin denervation reveals that renewal of touch dome stem cells requires a perineural microenvironment, and deleting Sonic hedgehog (Shh) in neurons or Smoothened in the epidermis demonstrates that Shh is an essential niche factor that maintains touch dome stem cells. Up-regulation of Hedgehog signaling results in neoplastic expansion of touch dome keratinocytes but no Merkel cell neoplasia. These findings demonstrate that nerve-derived Shh is a critical regulator of lineage-specific stem cells that maintain specialized sensory compartments in the epidermis.

Selective radiotherapy for the treatment of head and neck Merkel cell carcinoma.

The role of radiotherapy (RT) in the management of Merkel cell carcinoma (MCC) is controversial. The authors of this report evaluated the rates and patterns of failure in a selected group of patients who underwent RT for MCC of the head and neck (HN).

Perivascular Hair Follicle Stem Cells Associate with a Venule Annulus

The perivascular microenvironment helps maintain stem cells in many tissues. We sought to determine if there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.5 (E14.5), when nascent hair placodes had blood vessels approaching them. By birth, a vascular annulus stereotypically surrounded the Keratin 15 negative (K15−) stem cells in the upper bulge, and remained associated with the K15− upper bulge throughout the hair cycle. The angiogenic factor Egfl6 was expressed by the K15− bulge and localized adjacent to the vascular annulus, which was comprised of post-capillary venules. Although denervation altered the phenotype of upper bulge stem cells, the vascular annulus persisted in surgically denervated mouse skin. The importance of the perivascular niche was further suggested by the fact that vascular annuli formed around the upper bulge of de novo reconstituted hair follicles prior to their innervation. Together, these findings demonstrate that the upper bulge is associated with a perivascular niche during the establishment and maintenance of this specialized region of hair follicle stem cells.

Evaluating blood levels of neuron specific enolase, chromogranin A, and circulating tumor cells as Merkel cell carcinoma biomarkers

Background Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Although used to monitor MCC patients, the clinical utility of neuron-specific enolase (NSE) and chromogranin A (ChrA) blood levels is untested. EpCAM-positive circulating tumor cells (CTC) reflect disease status in several epithelial tumors. Here we investigate the use of NSE and ChrA blood levels and CTC counts as biomarkers for MCC disease behavior. Methods NSE and ChrA blood levels from 60 patients with MCC were retrospectively analyzed; 30 patients were additionally screened for CTC. Biomarker values were correlated to clinical parameters. Results Despite routine use by some physicians, NSE and ChrA blood levels did not correlate with progression free survival, disease specific survival, or MCC recurrence. We found CTC in 97% of tested MCC patients. CTC counts were elevated in patients with active disease, suggesting their potential use in monitoring MCC. Conclusion NSE and ChrA levels were not effective in predicting outcomes or detecting recurrences of MCC. In contrast, CTC counts have potential utility as a biomarker for MCC disease behavior.

A Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development

The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.

Assessment of Cancer Cell Line Representativeness Using Microarrays for Merkel Cell Carcinoma

When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared to publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, while UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line’s description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC.

Abstract CT079: Durable responses to avelumab (anti-PD-L1) in patients with Merkel cell carcinoma progressed after chemotherapy: 1-year efficacy update

Background: Merkel cell carcinoma (MCC), a rare, aggressive skin cancer, is a chemosensitive disease, but responses are seldom durable. Avelumab is a fully human anti-PD-L1 monoclonal antibody. In a phase 2 trial of avelumab in patients with previously treated metastatic MCC (mMCC), the objective response rate (ORR) after ≥6 months of follow-up was 31.8%, including complete response (CR) in 9.1%, the estimated proportion with duration of response (DOR) ≥6 months was 92%, and the 6-month progression-free survival (PFS) rate was 40% (95% CI 29-50) (Kaufman et al., Lancet Oncol 2016). Here we present updated efficacy data with ≥1 year of follow-up in all patients. Methods: Patients with distant mMCC and prior progression on chemotherapy received avelumab 10 mg/kg IV Q2W until confirmed progression, unacceptable toxicity, or withdrawal. Tumors were assessed every 6 weeks (RECIST v1.1 by independent review). ORR, DOR, PFS, and overall survival (OS) were evaluated. Time-to-event endpoints were analyzed using Kaplan-Meier methodology. Safety data were not analyzed for this update. Results: Patients with mMCC (N=88) were treated with avelumab. Median age was 72.5 years (range 33-88), and 53% had visceral disease. As of Sep 3, 2016, median follow-up was 16.4 months (range 12.0-25.3), and treatment was ongoing in 22% (n=19); main reasons for discontinuations were disease progression (n=44; 50%), death (n=7; 8%), adverse event (n=7; 8%), or withdrawal (n=4; 5%). ORR was 33.0% (95% CI 23.3-43.8) with 10 (11.4%) CRs and 19 (21.6%) partial responses, including 1 new CR and 1 patient improving from PR to CR since the 6-month analysis. The 6-month durable response rate was 30.6% (95% CI 20.9-40.3). Median DOR has not been reached (range 2.8-23.3+ months; 95% CI 18.0-not estimable), and responses were ongoing in 21/29 patients (72.4%) at the time of analysis. The estimated proportion of responders with ≥1-year duration of response was 74% (95% CI 53-87). Estimated 1-year PFS rate was 30% (95% CI 21-41) and 1-year OS rate was 52% (95% CI 41-62). Median OS was 12.9 months (95% CI 7.5-not estimable). Conclusion: In longer-term follow-up from this study of avelumab in patients with distant metastatic MCC progressed after chemotherapy, the majority of responses were durable beyond 1 year and 2 new CRs were reported. Maturing PFS and OS data suggest long-term benefit in a proportion of patients. Clinical trial information: NCT02155647. Citation Format: Howard L. Kaufman, Jeffery S. Russell, Omid Hamid, Shailender Bhatia, Patrick Terheyden, Sandra P. D’Angelo, Kent C. Shih, Celeste Lebbe, Michele Milella, Isaac Brownell, Karl D. Lewis, Jochen H. Lorch, Anja von Heydebreck, Lisa Mahnke, Paul Nghiem. Durable responses to avelumab (anti-PD-L1) in patients with Merkel cell carcinoma progressed after chemotherapy: 1-year efficacy update [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT079. doi:10.1158/1538-7445.AM2017-CT079