Neoadjuvant-Adjuvant or Adjuvant-Only Pembrolizumab in Advanced Melanoma.

BACKGROUND: Whether pembrolizumab given both before surgery (neoadjuvant therapy) and after surgery (adjuvant therapy), as compared with pembrolizumab given as adjuvant therapy alone, would increase event-free survival among patients with resectable stage III or IV melanoma is unknown. METHODS: In a phase 2 trial, we randomly assigned patients with clinically detectable, measurable stage IIIB to IVC melanoma that was amenable to surgical resection to three doses of neoadjuvant pembrolizumab, surgery, and 15 doses of adjuvant pembrolizumab (neoadjuvant-adjuvant group) or to surgery followed by pembrolizumab (200 mg intravenously every 3 weeks for a total of 18 doses) for approximately 1 year or until disease recurred or unacceptable toxic effects developed (adjuvant-only group). The primary end point was event-free survival in the intention-to-treat population. Events were defined as disease progression or toxic effects that precluded surgery; the inability to resect all gross disease; disease progression, surgical complications, or toxic effects of treatment that precluded the initiation of adjuvant therapy within 84 days after surgery; recurrence of melanoma after surgery; or death from any cause. Safety was also evaluated. RESULTS: At a median follow-up of 14.7 months, the neoadjuvant-adjuvant group (154 patients) had significantly longer event-free survival than the adjuvant-only group (159 patients) (P = 0.004 by the log-rank test). In a landmark analysis, event-free survival at 2 years was 72% (95% confidence interval [CI], 64 to 80) in the neoadjuvant-adjuvant group and 49% (95% CI, 41 to 59) in the adjuvant-only group. The percentage of patients with treatment-related adverse events of grades 3 or higher during therapy was 12% in the neoadjuvant-adjuvant group and 14% in the adjuvant-only group. CONCLUSIONS: Among patients with resectable stage III or IV melanoma, event-free survival was significantly longer among those who received pembrolizumab both before and after surgery than among those who received adjuvant pembrolizumab alone. No new toxic effects were identified. (Funded by the National Cancer Institute and Merck Sharp and Dohme; S1801 ClinicalTrials.gov number, NCT03698019.).

Quantitative histopathology identifies patients with thin melanomas who are at risk for metastases.

This small exploratory study was designed to test the hypothesis that thin melanoma lesions contain nuclei of two similar phenotypes, in different proportions. In lesions likely to progress to metastatic disease, one of these phenotypes predominates. Histopathological sections from 18 cases of thin melanomas which did not progress to metastasis, and from 10 cases which did progress were imaged and digitized at high resolution, with a total of 2084 and 1148 nuclei, respectively, recorded. Five karyometric features were used to discriminate between nuclei from indolent and from potentially metastatic lesions. For each case, the percentage of nuclei classified by the discriminant function as having come from a potentially metastatic lesion was determined and termed as case classification criterion. Standard histopathological criteria, such as ulceration and high mitotic index, indicated in this material the need for intensive therapy for only one of the 10 participants, as compared with 7/10 identified correctly by the karyometric measure. Using a case classification criterion threshold of 40%, the overall accuracy was 86% in the test set. The proportion of nuclei of an aggressive phenotype may lend itself as an effective prognostic clue for thin melanoma lesions. The algorithm developed in this training set appears to identify those patients at high risk for metastatic disease, and demonstrates a basis for a further study to assess the utility of prognostic clues for thin melanomas.

Phase IIB Randomized Study of Topical Difluoromethylornithine and Topical Diclofenac on Sun-Damaged Skin of the Forearm.

Prevention of nonmelanoma skin cancers remains a health priority due to high costs associated with this disease. Diclofenac and difluoromethylornithine (DFMO) have demonstrated chemopreventive efficacy for cutaneous squamous cell carcinomas. We designed a randomized study of the combination of DFMO and diclofenac in the treatment of sun-damaged skin. Individuals with visible cutaneous sun damage were eligible. Subjects were randomized to one of the three groups: topical DFMO applied twice daily, topical diclofenac applied daily, or DFMO plus diclofenac. The treatment was limited to an area on the left forearm, and the duration of use was 90 days. We hypothesized that combination therapy would have increased efficacy compared with single-agent therapy. The primary outcome was change in karyometric average nuclear abnormality (ANA) in the treated skin. Individuals assessing the biomarkers were blinded regarding the treatment for each subject. A total of 156 subjects were randomized; 144 had baseline and end-of-study biopsies, and 136 subjects completed the study. The ANA unexpectedly increased for all groups, with higher values correlating with clinical cutaneous inflammation. Nearly all of the adverse events were local cutaneous effects. One subject had cutaneous toxicity that required treatment discontinuation. Significantly more adverse events were seen in the groups taking diclofenac. Overall, the study indicated that the addition of topical DFMO to topical diclofenac did not enhance its activity. Both agents caused inflammation on a cellular and clinical level, which may have confounded the measurement of chemopreventive effects. More significant effects may be observed in subjects with greater baseline cutaneous damage.

Oral administration of soluble guanylate cyclase agonists to rats results in osteoclastic bone resorption and remodeling with new bone formation in the appendicular and axial skeleton.

Orally administered small molecule agonists of soluble guanylate cyclase (sGC) induced increased numbers of osteoclasts, multifocal bone resorption, increased porosity, and new bone formation in the appendicular and axial skeleton of Sprague-Dawley rats. Similar histopathological bone changes were observed in both young (7- to 9-week-old) and aged (42- to 46-week-old) rats when dosed by oral gavage with 3 different heme-dependent sGC agonist (sGCa) compounds or 1 structurally distinct heme-independent sGCa compound. In a 7-day time course study in 7- to 9-week-old rats, bone changes were observed as early as 2 to 3 days following once daily compound administration. Bone changes were mostly reversed following a 14-day recovery period, with complete reversal after 35 days. The mechanism responsible for the bone changes was investigated in the thyroparathyroidectomized rat model that creates a low state of bone modeling and remodeling due to deprivation of thyroid hormone, calcitonin (CT), and parathyroid hormone (PTH). The sGCa compounds tested increased both bone resorption and formation, thereby increasing bone remodeling independent of calciotropic hormones PTH and CT. Based on these studies, we conclude that the bone changes in rats were likely caused by increased sGC activity.

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer.

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides.

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure-toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure-toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

Colon and pancreas tumors enhance coagulation: role of hemeoxygenase-1.

Colon and pancreatic cancer are associated with significant thrombophilia. Colon and pancreas tumor cells have an increase in hemeoxygenase-1 (HO-1) activity, the endogenous enzyme responsible for carbon monoxide production. Given that carbon monoxide enhances plasmatic coagulation, we determined if patients undergoing resection of colon and pancreatic tumors had an increase in endogenous carbon monoxide and plasmatic hypercoagulability. Patients with colon (n = 17) and pancreatic (n = 10) tumors were studied. Carbon monoxide was determined by the measurement of carboxyhemoglobin (COHb). A thrombelastographic method to assess plasma coagulation kinetics and formation of carboxyhemefibrinogen (COHF) was utilized. Nonsmoking patients with colon and pancreatic tumors had abnormally increased COHb concentrations of 1.4 ± 0.9 and 1.9 ± 0.7%, respectively, indicative of HO-1 upregulation. Coagulation analyses comparing both tumor groups demonstrated no significant differences in any parameter; thus the data were combined for the tumor groups for comparison with 95% confidence interval values obtained from normal individuals (n = 30) plasma. Seventy percent of tumor patients had a velocity of clot formation greater than the 95% confidence interval value of normal individuals, with 53% of this hypercoagulable group also having COHF formation. Further, 67% of tumor patients had clot strength that exceeded the normal 95% confidence interval value, and 56% of this subgroup had COHF formation. Finally, 63% of all tumor patients had COHF formation. Future investigation of HO-1-derived carbon monoxide in the pathogenesis of colon and pancreatic tumor-related thrombophilia is warranted.

Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

A phase 2 trial of complete resection for stage IV melanoma: results of Southwest Oncology Group Clinical Trial S9430.

BACKGROUND: On the basis of retrospective experience at individual centers, it appears that patients with stage IV melanoma who undergo complete resection have a favorable outcome compared with patients with disseminated stage IV disease. The Southwest Oncology Group (SWOG) performed a prospective trial in patients with metastatic melanoma who were enrolled before complete resection of their metastatic disease and provided prospective outcomes in the cooperative group setting. METHODS: Based on their physical examination and radiologic imaging studies, patients with a stage IV melanoma judged amenable to complete resection underwent surgery within 28 days of enrollment. All eligible patients were followed with scans (computed tomography or positron emission tomography) every 6 months until relapse and death. RESULTS: Seventy-seven patients were enrolled from 18 different centers. Of those, 5 patients were ineligible; 2 had stage III disease alone; and 3 had no melanoma in their surgical specimen. In addition, 8 eligible patients had incompletely resected tumor. Therefore, the primary analysis included 64 completely resected patients. Twenty patients (31%) had visceral disease. With a median follow-up of 5 years, the median relapse-free survival was 5 months (95% CI, 3-7 months) whereas median overall survival was 21 months (95% CI, 16-34 months). Overall survivals at 3 and 4 years were 36% and 31%, respectively. CONCLUSIONS: In a prospective multicenter setting, appropriately selected patients with stage IV melanoma achieved prolonged overall survival after complete surgical resection. Although median relapse-free survival was only 5 months, patients could still frequently undergo subsequent surgery for isolated recurrences. This patient population is appropriate for aggressive surgical therapy and for trials evaluating adjuvant therapy.

A phase 2a study of topical perillyl alcohol cream for chemoprevention of skin cancer.

The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene-induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary.

Giant epidermal cyst of the gluteal region.

Epidermal cysts are common, benign, intradermal or subcutaneous, typically asymptomatic masses, ranging from 1 to 4 cm in size. They may occur anywhere in the body, with a predilection for the face, neck, and trunk. Transformation to squamous-cell carcinoma is rare. We present a case of a 61-year-old male patient with a large, growing mass in his posterior left gluteal region. Given the concern for a malignancy, he was referred to a surgical oncologist. Magnetic resonance imaging (MRI) without contrast was performed due to poor renal function and revealed a large cystic mass in the left gluteal subcutaneous soft tissues that was subsequently excised. Pathological examination revealed an epidermal inclusion cyst that measured 17.8 × 13.18 × 5.8 cm. To our knowledge, this is the largest epidermal inclusion cyst reported in the English literature.

Melanoma high-risk families' perceived health care provider risk communication.

BACKGROUND: Families with a melanoma history are at risk of melanoma. Melanoma survival improves when people are aware of their risk and ways to modify it. We explored at-risk families' perceived risk communication from healthcare providers. METHODS: Qualitative description. RESULTS: Participants perceived: (1) few provider discussions of melanoma risk or risk-modifying behaviors; (2) a desire to trust information from providers; (3) the healthcare system constrains communication; and (4) concerns about provider competence and caring regarding worrisome lesions. CONCLUSIONS: Providers should provide clear, comprehensive, accurate, and consistent messages about melanoma to persons at high risk; messages also convey competence and caring.

Notch1 is an effector of Akt and hypoxia in melanoma development.

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

Phase 1 study of topical perillyl alcohol cream for chemoprevention of skin cancer.

Perillyl alcohol (POH) is a natural product derived from plants such as cherry and lavendin. Previous studies have indicated that topical POH inhibits ultraviolet (UV) B-induced skin carcinogenesis in vivo, and it may be an effective chemopreventive agent for skin cancer. We performed a 1-mo, first-in-man, Phase 1 trial of topically administered POH cream in human subjects. Endpoints included safety and evaluation of any histopathological changes in skin after 1 mo use of POH cream. We randomized 25 subjects with normal, healthy skin with little or no sun damage and no history of skin cancer in a double-blind fashion to receive topical POH (0.76% wt/wt) on 1 forearm with placebo cream applied to the other forearm twice daily for 30 days. Subjects were monitored for toxicity, and a 4 mm punch biopsy in the treated area was performed at the end of study for histopathological evaluation. The topical cream was well tolerated. No serious cutaneous toxicities, systemic toxicities, or histopathological abnormalities were observed. A total of 8 subjects (32%) reported mild adverse events possibly or probably related to use of cream including reversible appearance of 1 to 2 small papules. However, there was no significant difference between lesions appearing on the POH treated forearm vs. the placebo-treated forearm.

Using optical coherence tomography to evaluate skin sun damage and precancer.

BACKGROUND AND OBJECTIVES: Optical coherence tomography (OCT) is a depth resolved imaging modality that may aid in identifying sun damaged skin and the precancerous condition actinic keratosis (AK). STUDY DESIGN/MATERIALS AND METHODS: OCT images were acquired of 112 patients at 2 sun protected and 2 sun exposed sites, with a subsequent biopsy. Each site received a dermatological evaluation, a histological diagnosis, and a solar elastosis (SE) score. OCT images were examined visually and statistically analyzed. RESULTS: Characteristic OCT image features were identified of sun protected, undiseased, sun damaged, and AK skin. A statistically significant difference (P<0.0001) between the average attenuation values of skin with minimal and severe solar elastosis was observed. Significant differences (P<0.0001) were also found between undiseased skin and AK using a gradient analysis. Using image features, AK could be distinguished from undiseased skin with 86% sensitivity and 83% specificity. CONCLUSION: OCT has the potential to guide biopsies and provide non-invasive measures of skin sun damage and disease state, possibly increasing efficiency of chemopreventive agent trials.

Reduced Pms2 expression in non-neoplastic flat mucosa from patients with colon cancer correlates with reduced apoptosis competence.

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

Patchy field defects of apoptosis resistance and dedifferentiation in flat mucosa of colon resections from colon cancer patients.

BACKGROUND: Abnormal areas in normal-appearing flat colonic mucosa (field defects) may predispose individuals to colon cancer. Markers of field defects would indicate cancer risk. METHODS: We evaluated apoptosis capability, dedifferentiation, frequency of simple aberrant crypts, aberrant crypt foci, microadenomas, and total nicotinamide adenine dinucleotide levels at locations within normal-appearing flat mucosa obtained from colon resections. RESULTS: Among goblet cells from colonic mucosa samples of individuals without colonic neoplasia, there was a high mean deoxycholate-induced apoptotic index (AI) of 59.1% and high Dolichos biflorus agglutinin (DBA) lectin reactivity (differentiation) in 85.0% of samples. In contrast, flat mucosa samples from colon cancer patients had a significantly (P <.01) lower average AI of 37.4%, and a significantly (P =.03) lower percentage (40.5%) had high DBA reactivity. For colon cancer patients, AI and DBA reactivity values were patchy within a resection. Nicotinamide adenine dinucleotide levels were highly variable among individuals without neoplasia, and aberrant crypt foci and microadenomas were rare. CONCLUSIONS: AI and aberrant DBA reactivity are promising indicators of colon cancer risk. Our results attest to the importance of obtaining multiple samples to assess colon cancer risk because of the patchy nature of field defects.