Merkel Cell Carcinoma Outcomes: Does AJCC8 Underestimate Survival?

INTRODUCTION: The eighth edition of the American Joint Committee on Cancer (AJCC8) Staging Manual provides important information for staging and prognostication; however, survival estimates for patients with Stage I-III Merkel cell carcinoma (MCC), a rare disease, may be as practical using data from large-volume centers as that collated for the AJCC analysis. As such, we compared our institutional outcomes to AJCC8. METHODS: Patients who presented from 2005 to 2017 with MCC to two high-volume centers were included. Demographics, clinicopathologic characteristics, survival and recurrence data were compiled, and outcomes compared to AJCC8. RESULTS: A total of 409 patients were included. Median age was 75 (range 29-98) years, and 68% were male. Median follow-up was 16 months (0-157). Five-year overall survival (OS) was 70%; 5-year disease-specific survival (DSS) was 84%. When stratified by extent of disease, 5-year OS was higher for patients with local disease compared to those with nodal disease (72.6% vs 62.7%, p=0.005). Similarly, patients with local disease had higher 5-year DSS than those with nodal disease (90.1% vs 76.8%, p=0.002). Five-year recurrence-free survival was 59.2% for all patients, 65.0% for local disease and 48.3% for nodal disease (p=0.033). CONCLUSIONS: Here, MCC patients with local or nodal disease have substantially higher OS rates than predicted in AJCC8 (5-year: 72.6% vs 50.6%; 62.7% vs 35.4%, respectively). Importantly, 5-year DSS was significantly better than the OS rates reported presently and in AJCC8. As clinicians and patients rely on AJCC to accurately prognosticate and guide treatment decisions, these estimates should be reassessed and updated to more accurately predict survival outcomes.

Correction to: Merkel Cell Carcinoma Outcomes: Does AJCC8 Underestimate Survival?

C.R. Farley and M.C. Perez contributed equally to this publication and are co-first authors. J.S. Zager and M.C. Lowe contributed equally to this publication and are co-corresponding authors.

Cross-sectional associations between cutaneous viral infections and regulatory T lymphocytes in circulation.

BACKGROUND: Cutaneous viral infections and immune suppression are risk factors for some forms of nonmelanoma skin cancer; however, their interrelationship is poorly understood. OBJECTIVES: To examine cross-sectional associations between cutaneous viral infections and circulating forkhead-box P3 (FOXP3)-expressing T-regulatory (Treg) cells, suppressive cells that dampen effective antitumour immunity. MATERIALS AND METHODS: Blood, eyebrow hair (EBH) and skin swab (SSW) samples were collected from 352 patients 60 years and older undergoing skin screening, without prevalent skin cancer, while participating in an ongoing prospective cohort study of cutaneous viral infections and skin cancer. DNA corresponding to 98 cutaneous human papillomavirus (HPV) types and five human polyomaviruses (HPyV) was assessed in EBH and SSW. Distinct classes of circulating Treg-cell subpopulations were defined by flow cytometry including cutaneous lymphocyte antigen (CLA) and CCR4high Treg cells, both previously associated with cutaneous diseases. Age- and sex-adjusted associations between circulating T-cell populations and infection were estimated using logistic regression. RESULTS: Total Treg-cell proportion in peripheral blood was not associated with ß HPV or HPyV infection. However, the proportion of circulating CLA+ Treg cells was inversely associated with γ HPV EBH infection [odds ratio (OR) 0·54, 95% confidence interval (CI) 0·35-0·84]. Interestingly, circulating Treg cells expressing markers indicative of antigen activation (CD27- CD45RA- FOXP3+ CD4+ ) were also inversely associated with γ HPV infection in SSW (OR 0·55, 95% CI 0·30-0·99) and EBH (OR 0·56, 95% CI 0·36-0·86). CONCLUSIONS: Inverse associations between circulating Treg cells and γ HPV infection suggest that localized viral infection may promote immunosuppressive cell migration into skin.

Pathological assessment of resection specimens after neoadjuvant therapy for metastatic melanoma.

Background: Clinical trials have recently evaluated safety and efficacy of neoadjuvant therapy among patients with surgically resectable regional melanoma metastases. To capture informative prognostic data connected to pathological response in such trials, it is critical to standardize pathologic assessment and reporting of tumor response after this treatment. Methods: The International Neoadjuvant Melanoma Consortium meetings in 2016 and 2017 assembled pathologists from academic centers to develop consensus guidelines for pathologic examination and reporting of surgical specimens from AJCC (8th edition) stage IIIB/C/D or oligometastatic stage IV melanoma patients treated with neoadjuvant-targeted or immune therapy. Patterns of pathologic response are provided context to inform these guidelines. Results: Based on our collective experience and guided by efforts in well-established neoadjuvant settings like breast cancer, procedures directing handling of pre- and post-neoadjuvant therapy-treated melanoma specimens are provided to facilitate comparison of findings across different trials and centers. Definitions of pathologic response are provided together with guidelines for reporting and quantifying the extent of pathologic response. Finally, the spectrum of histopathologic responses observed following neoadjuvant-targeted and immune-checkpoint therapy is described and illustrated. Conclusions: Standardizing pathologic evaluation of resected melanoma metastases following neoadjuvant-targeted or immune-checkpoint therapy allows more robust stratification of patient outcomes. This includes recognizing the spectrum of histopathologic response patterns to neoadjuvant therapy and a standard approach to grading pathologic responses. Such an approach will facilitate comparison of results across clinical trials and inform ongoing correlative studies into the mechanisms of response and resistance to agents applied in the neoadjuvant setting.

Phase I clinical trial of the Src inhibitor dasatinib with dacarbazine in metastatic melanoma.

BACKGROUND: Src inhibitors sensitise melanoma cells to chemotherapy in preclinical models. The combination of dasatinib and dacarbazine was tested in a phase I trial in melanoma. METHODS: Patients had ECOG performance status 0-2 and normal organ function. Dacarbazine was administered on day 1 and dasatinib on day 2 through 19 of each 21-day cycle. Both were escalated from 50 mg b.i.d. of dasatinib and 800 mg m(-2) of dacarbazine. Available pre-treatment biopsies were sequenced for BRAF, NRAS, and C-Kit mutations. RESULTS: Dose-limiting toxicity was reached at dasatinib 70 mg b.i.d./dacarbazine 1000 mg m(-2), and was predominantly haematological. In 29 patients receiving dasatinib 70 mg b.i.d., the objective response rate (ORR) was 13.8%, the clinical benefit rate (ORR+SD) was 72.4%, the 6-month progression-free survival (PFS) was 20.7%, and the 12-month overall survival (OS) was 34.5%. Two out of three patients who were wild type for BRAF, NRAS, and c-KIT mutations had confirmed partial responses, and one had a minor response. CONCLUSION: The recommended phase II dose is dasatinib 70 mg b.i.d with dacarbazine 800 mg m(-2). PFS and OS data for dasatinib at 70 mg b.i.d. with dacarbazine compared favourably with historical controls. Preliminary data support evaluating tumour mutation status further as a biomarker of response.

Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy.

BACKGROUND: Resistance to BRAF inhibitors is an emerging problem in the melanoma field. Strategies to prevent and overcome resistance are urgently required. METHODS: The dynamics of cell signalling, BrdU incorporation and cell-cycle entry after BRAF inhibition was measured using flow cytometry and western blot. The ability of combined BRAF/MEK inhibition to prevent the emergence of resistance was demonstrated by apoptosis and colony formation assays and in 3D organotypic cell culture. RESULTS: BRAF inhibition led to a rapid recovery of phospho-ERK (pERK) signalling. Although most of the cells remained growth arrested in the presence of drug, a minor population of cells retained their proliferative potential and escaped from BRAF inhibitor therapy. A function for the rebound pERK signalling in therapy escape was demonstrated by the ability of combined BRAF/MEK inhibition to enhance the levels of apoptosis and abrogate the onset of resistance. CONCLUSION: Combined BRAF/MEK inhibition may be one strategy to prevent the emergence of drug resistance in BRAF-V600E-mutated melanomas.

Proliferating trichilemmal cysts of the scalp on CT.

Proliferating trichilemmal cysts, also known as pilar tumors, are slow-growing lobulated masses most commonly found on the scalp of elderly women. We present the case of a 69-year-old woman with a 25-year history of multiple enlarging scalp masses. The patient was evaluated for surgical consultation after the dominant mass presented with malignant degeneration. A CT of the head revealed multiple large, subcutaneous, cystic masses with calcifications.

Fibronectin induction abrogates the BRAF inhibitor response of BRAF V600E/PTEN-null melanoma cells.

The mechanisms by which some melanoma cells adapt to Serine/threonine-protein kinase B-Raf (BRAF) inhibitor therapy are incompletely understood. In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF mutant and PTEN null. Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN dependent. These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs. Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment. Analysis of a melanoma tissue microarray showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis. Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5ß1 integrin. This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1. The protection conveyed by the induction of FN expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Identification of beta-actin sequences necessary for induction by phorbol esters and calcium ionophores.

Transcription of the cytoskeletal beta-actin gene is rapidly induced by phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore, A23187, in cultured H4IIE hepatoma (H4) cells. PMA directly activates protein kinase C (PKC) and activation of PKC is necessary for the cellular actions of PMA, including induction of beta-actin gene transcription. In the present study, we determined the DNA sequence requirements for induction of the beta-actin gene by PMA and A23187. Constructs containing progressive deletions of normal and mutated human beta-actin 5' sequences fused to the reporter gene, bacterial chloramphenicol acetyltransferase, were analysed in transient transfections of H4 cells. We delineated the PMA response DNA element of the human beta-actin gene to the proximal CCArGG box (-62 to -53) in the 5' flanking region. In contrast, A23187 did not induce expression of transfected gene constructs containing this CCArGG box. Additionally, we demonstrated that CCArGG boxes from two other PMA-induced genes in H4 cells, c-fos and gamma-actin, could confer PMA inducibility to a heterologous promoter. This CCArGG box specifically interacts with one or more proteins present in nuclear extracts of H4 cells. These results indicate that in cultured cells, PMA-dependent induction of the beta-actin gene is mediated through the proximal CCArGG box. This suggests that the CCArGG box is a target for PKC action and may be involved in the control of other PKC regulated genes.

Rapid regulation of albumin transcription by insulin and phorbol esters in rat hepatoma cells.

The short-term effects of insulin and phorbol esters on the regulation of the albumin gene in rat H4IIE (H4) hepatoma cells were investigated and compared to the expression of a gene known to be inhibited by these agents, phosphoenol pyruvate carboxykinase (PEPCK). Both insulin and phorbol esters inhibited transcription of the albumin gene in a rapid, dose-dependent manner. Within 15 min, albumin transcription was reduced by approx. 80%. The inhibitory effects of insulin were evident at concentrations of insulin as low as 5.10(-11)M, suggesting that these effects were mediated through insulin-specific pathways. The ability of both phorbol esters and insulin to inhibit albumin transcription suggests that the negative control of this gene is a stable feature in H4 cells. The effect of phorbol esters to mimic insulin action on the albumin gene, and on several other genes in this cell line, implies that a common pathway may be shared by both insulin and phorbol esters.

Induction of cytoskeletal gene expression by insulin.

Insulin has rapid pleiotropic effects on cellular metabolism. In certain cell types, insulin can cause morphological changes by inducing rearrangements of cytoskeletal components, but the regulation of cytoskeletal gene expression by insulin has not been previously described. In the present work insulin was found to rapidly, but transiently, increase transcription of the cytoskeletal beta-actin and alpha-tubulin genes in rat H4IIE hepatoma cells. Insulin-induced transcription of beta-actin mRNA was evident within 5 min and was maximal by 10-15 min at 1000% above control levels. beta-Actin transcription was induced at insulin concentrations as low as 5 x 10(-12) M insulin and was maximal at 5 x 10(-9) M. Transcription of the alpha-tubulin gene was also rapidly stimulated by physiological concentrations of insulin, but only to 300-400% above basal levels. For both the beta-actin and alpha-tubulin genes, the induction of transcription was transient, with a return to basal levels by 60-120 min. Transcription of neither the skeletal or cardiac alpha-actin gene nor the beta-tubulin gene was altered by insulin administration. Messenger RNA levels for the beta-actin and alpha-tubulin genes increased, but to a lesser extent than transcription, since these mRNAs were abundant and stable before the transient induction of transcription. Inhibitors of protein synthesis, in the presence or absence of insulin, also acutely stimulated transcription of these genes.

Insulin and dexamethasone regulation of a rat hepatoma messenger ribonucleic acid: insulin has a transcriptional and a posttranscriptional effect.

Experiments were conducted to investigate the actions of glucocorticoids and insulin on the induction of a specific mRNA (p33-mRNA) expressed in rat H4 hepatoma cells. Previous studies have found a 10-fold increase in this mRNA following 60 min of insulin addition. In the present study, dexamethasone (Dex) induced the cellular concentration of p33-mRNA 10- to 15-fold. This effect was time and dose dependent. The effect of Dex could be accounted for by a 10- to 15-fold increase in p33-mRNA transcription. However, insulin administration resulted in only a 3-fold increase in the transcription of p33-mRNA. The insulin induction of transcription was time and dose dependent and was blocked by the addition of alpha-amanitin. There was no increase in the transcription of a control gene, beta-tubulin, by either insulin or Dex. Neither insulin nor Dex altered the stability of p33-mRNA. Since the cellular concentration of p33-mRNA was induced to a greater extent than was transcription, insulin must be regulating at least one other step in the sequence between RNA synthesis and RNA stability.

Transcriptional regulation of a rat hepatoma gene by insulin and protein kinase-C.

Both insulin and phorbol esters rapidly stimulated the cytoplasmic accumulation of a specific mRNA (designated p33) in a time- and dose-dependent manner in serum-deprived rat H4 hepatoma cells. When cells were pretreated with phorbol esters to produce a deficiency in protein kinase-C, the ability of further phorbol ester addition to stimulate p33 mRNA accumulation was abolished. However, after pretreatment of H4 cells with phorbol esters, insulin still induced cellular p33 mRNA concentrations, but to a lesser degree. The primary effect of phorbol esters was to increase transcription of the p33 gene, and this was abolished after pretreatment with phorbol esters. In previous work, insulin was shown to stimulate p33 gene transcription, but this effect was insufficient to account for the level of insulin-induced p33 mRNA production. The transcriptional effect of insulin was further reduced by phorbol ester pretreatment. Insulin must, therefore, regulate p33 gene expression by at least two pathways, at least one of which may be modulated by protein kinase-C.

Evidence for diverse roles of protein kinase-C in the inhibition of gene expression by insulin: the tyrosine aminotransferase, albumin, and phosphoenolpyruvate carboxykinase genes.

We have previously shown that insulin is less effective in inducing expression of several genes in H4 hepatoma cells with reduced functional protein kinase-C (PKC) activity. However, other reports suggest that insulin regulation of gene transcription is not PKC dependent. Insulin and phorbol 12-myristate 13-acetate (PMA) rapidly inhibit transcription of the tyrosine aminotransferase and albumin genes. Prolonged PMA pretreatment, to desensitize cells to PMA, resulted in a loss of insulin ability to inhibit albumin transcription. Insulin was still able to inhibit tyrosine aminotransferase transcription, but less than in non-PMA-pretreated cells, and there was also a slight decrease in the ability of insulin to inhibit phosphoenolpyruvate carboxykinase transcription. We previously demonstrated decreased responsiveness of PMA-induced gene expression in insulin-desensitized cells. In the present work, using insulin-desensitized H4 cells (insulin pretreatment for 24 h), subsequent treatment with PMA did not alter phosphoenolpyruvate carboxykinase transcription rates, whereas PMA did inhibit tyrosine aminotransferase transcription rates to an extent similar to observed in nonpretreated cells. Unexpectedly, there was a significant increase in albumin transcription after PMA addition to insulin-pretreated cells. These findings support our hypothesis that the role of PKC in the regulation of gene expression by insulin varies for different insulin-regulated genes.

Positive interaction between insulin and phorbol esters on the regulation of a specific messenger ribonucleic acid in rat hepatoma cells.

With Northern blot analysis, we demonstrate that phorbol 12-myristate 13-acetate (PMA) stimulated P-33 messenger RNA (mRNA)-accumulation in rat hepatoma cells in a time- and concentration-dependent manner similar to insulin and plant lectins. No effect of PMA on P-33 mRNA half-life was detected when mRNA synthesis was inhibited with either actinomycin D or 5.6-dichloro-1-beta-D-ribofuranosyl benzimidazole. The effects of insulin and PMA were additive at submaximal concentrations and no additivity was observed under these conditions at maximal concentrations. Thus PMA has a marked insulin-like effect on the accumulation of P-33 mRNA in rat hepatoma cells.

The regulation of p33 gene expression by insulin and calcium ionophores.

We have previously shown that insulin induces p33 transcription and mRNA levels in serum-deprived rat H4 hepatoma cells. In the current study, the effects of the calcium ionophores A23187 and ionomycin on the regulation of p33 gene expression were examined. When H4 cells were incubated with A23187 or ionomycin (1 microM) for 90-300 min, 700-900% and 400-600% increases in p33 mRNA levels were observed. The effects of ionophore and insulin together on p33 mRNA levels were not additive. Insulin-induced increases in p33 mRNA levels were diminished at low concentrations of extracellular calcium, but were unchanged by the calcium channel blocker verapamil. The chelation of intracellular calcium using 20 and 60 microM quin2-AM resulted in 50% and 90% reductions in insulin-induced p33 mRNA levels. When transcription assays were performed, A23187 treatment for 15-180 min increased p33 transcription 300-400%. The lesser effect of A23187 on transcription compared to that on mRNA levels was also true for insulin treatment. Similar results were obtained using ionomycin. Insulin- and A23187-induced p33 transcription was reduced by quin2-AM to below basal levels. These studies show that calcium ionophores stimulate p33 gene expression and suggest that changes in intracellular calcium can alter insulin's induction of this gene.

One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element.

The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.

Synergistic induction of gene 33 expression by retinoic acid and insulin.

The expression of gene 33 in rat liver and hepatoma cells is regulated by multiple hormones and other bioactive agents. Previous studies have demonstrated a 15-fold increase in gene 33 mRNA after 1 h of insulin treatment. We demonstrate in this report that retinoic acid (RA) also controls the expression of this gene. Gene 33 mRNA levels are rapidly elevated by RA, with maximal accumulation (19-fold over control) attained after just 1 h of RA treatment. The transcription rate of gene 33 was increased by RA to a maximum level 6-fold greater than control values. Studies with inhibitors of RNA synthesis demonstrated no increase in the stability of gene 33 mRNA in response to RA or insulin. In addition, a synergistic induction of both gene 33 mRNA levels and the transcription rate of gene 33 was observed when both RA and insulin were added together. In the presence of both hormones, the transcription rate was induced almost 20-fold in 30 min, followed by a 49-fold increase in mRNA levels after 1 h. Thus, gene 33 represents the first example of a gene whose transcription rate is elevated directly by both insulin and RA, and synergistically elevated by treatment with both hormones together.

Pathologic examination of the sentinel lymph node in malignant melanoma.

Sentinel lymphadenectomy is gaining increasing popularity in the staging and treatment of patients with melanoma at risk for metastases. As a result, pathologists are encountering these specimens more frequently in their daily practice. The pathologic status of the sentinel lymph node is pivotal to the patient's care because it provides staging information that dictates the need for further therapy, and therefore detailed pathologic assessment is warranted. A standard pathology protocol to handle these nodes has been developed at our institution and involves complete submission of all tissue with routine use of immunohistochemical staining for S-100 protein. By using this protocol, 838 sentinel lymph nodes from 357 patients have been examined, and metastases were found in 16% of patients. Although the metastasis was clearly seen on sections stained with hematoxylin and eosin in 55% of the positive patients, the immunostain showed metastatic disease not appreciable on initial hematoxylin and eosin screening in an additional 28 lymph nodes (45% of node-positive patients). Intraoperative touch preparation cytology may be used as an adjunct technique in sentinel lymph nodes grossly suspicious for metastatic disease. This technique has been performed on 23 sentinel lymph nodes, with no false positives and an overall sensitivity of 62%. The thorough pathologic evaluation of sentinel lymph nodes in patients with malignant melanoma requires complete submission of all tissue, routine use of immunohistochemistry, and touch preparation cytology in selected cases.