Chyi-Chia Richard Lee

A Pilot Trial Using Lymphocytes Genetically Engineered with an NY-ESO-1–Reactive T-cell Receptor: Long-term Follow-up and Correlates with Response

Purpose: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1–reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers. Experimental Design: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report. Results: Eleven of 18 patients with NY-ESO-1+ synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1+ melanomas (55%) who received autologous T cells transduced with an NY-ESO-1–reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%. Conclusions: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments. Clin Cancer Res; 21(5); 1019–27. ©2014 AACR.

EZH2-dependent suppression of a cellular senescence phenotype in melanoma cells by inhibition of p21/CDKN1A expression

Polycomb group (PcG) proteins such as Enhancer of zeste homolog 2 (EZH2) are epigenetic transcriptional repressors that function through recognition and modification of histone methylation and chromatin structure. Targets of PcG include cell cycle regulatory proteins which govern cell cycle progression and cellular senescence. Senescence is a characteristic of melanocytic nevi, benign melanocytic proliferations that can be precursors of malignant melanoma. In this study, we report that EZH2, which we find absent in melanocytic nevi but expressed in many or most metastatic melanoma cells, functionally suppresses the senescent state in human melanoma cells. EZH2 depletion in melanoma cells inhibits cell proliferation, restores features of a cellular senescence phenotype, and inhibits growth of melanoma xenografts in vivo. p21/CDKN1A is activated upon EZH2 knockdown in a p53-independent manner and contributes substantially to cell cycle arrest and induction of a senescence phenotype. EZH2 depletion removes histone deacetylase 1 (HDAC1) from the CDKN1A transcriptional start site and downstream region, enhancing histone 3 acetylation globally and at CDKN1A. This results in recruitment of RNA polymerase II, leading to p21/CDKN1A activation. Depletion of EZH2 synergistically activates p21/CDKN1A expression in combination with the HDAC inhibitor trichostatin A. Since melanomas often retain wild-type p53 function activating p21, our findings describe a novel mechanism whereby EZH2 activation during tumor progression represses p21, leading to suppression of cellular senescence and enhanced tumorigenicity. Mol Cancer Res; 9(4); 418–29. ©2011 AACR.

Skin cancers, blindness, and anterior tongue mass in African brothers

HistoryTwo Northern African brothers presented to theNational Institutes of Health for evaluation of severedamage to sun-exposed areas of the skin, eyes, andmucosae; multiple skin cancers; a tongue mass; andphotophobia with loss of vision.The patients were born full term after uncompli-cated pregnancies and achieved age-appropriatedevelopmental milestones. Patient XP393BE (Fig 1,A), 23 years old, was noted to have freckle-likepigmented lesions on his face at 2.5 years andphotophobia by age 3 (Table I). A squamous cellcarcinoma(SCC)hadbeenremovedfromhisnoseatage 13 and the site was grafted with sun-shieldedskin from his thigh. His brother, patient XP394BE(Fig 1, D), 17 years old, developed freckle-likelesions on his face by 8 years of age. At age 13, anSCCwasexcisedfromhisrightcheek.Bothboyshadbilateral progressive loss of vision with unilateralblindness since the age of 12 to 14 years (Fig 1, B).PatientXP393BEhada10-year history ofaslowlyenlarging, painful, bleeding mass on the tip of histongue (Fig 2, A). He denied weight loss, excessiveconsumption of alcohol, or chewing of tobacco orbetel quid.LivinginKuwait,Sudan,Libya,andEgypt,neitherpatient had a history of sunburns or use of sunprotection. The patients have a 21-year-old unaf-fected brother. Their parents were second cousinsand members of the same tribe in the Sudan. Therewas no family history of cancer.Physical examinationSkin examination of both patients revealed nu-merous 1- to 5-mm hyperpigmented macules on thecheeksandscalp(seeFigs1and2)andsun-exposedportions of the chest and extremities with sparing ofsun-protected sites. Patient XP393BE had a 1.3- 31.7-cm stellate, indurated, black, brown and grayplaque on his left cheek (Fig 1, A). Dermatoscopyrevealed characteristic leaf-like structures and blue-gray ovoid nests that distinguish pigmented basalcell carcinoma from melanoma (Fig 1, C). A 1-cmnodular, ulcerated mass was present on the base ofhis nose, and there were multiple translucent blacklesions on his face. Patient XP394BE had a crusted,darklypigmentedplaqueontheleftalaandtipofthe

Homeostatic Tissue Responses in Skin Biopsies from NOMID Patients with Constitutive Overproduction of IL-1β

The autoinflammatory disorder, Neonatal-onset Multisystem Inflammatory Disease (NOMID) is the most severe phenotype of disorders caused by mutations in CIAS1 that result in increased production and secretion of active IL-1β. NOMID patients present with systemic and organ-specific inflammation of the skin, central nervous system and bone, and respond dramatically to treatment with IL-1 blocking agents. We compared the cellular infiltrates and transcriptome of skin biopsies from patients with NOMID (n = 14) before treatment (lesional (LS) and non-lesional (pre-NL) skin) and after treatment (post-NL) with the IL-1 blocker anakinra (recombinant IL-1 receptor antagonist, Kineret®, Swedish Orphan Biovitrum AB, SOBI), to normal skin (n = 5) to assess tissue responses in the context of untreated and treated disease. Abundant neutrophils distinguish LS skin from pre-NL and post-NL skin. CD11c+ dermal dendritic cells and CD163+ macrophages expressed activated caspase-1 and are a likely source of cutaneous IL-1 production. Treatment with anakinra led to the disappearance of neutrophils, but CD3+ T cells and HLA-DR+ cells remained elevated. Among the upregulated genes IL-6, IL-8, TNF, IL-17A, CCL20, and the neutrophil defensins DEFA1 and DEFA3 were differentially regulated in LS tissues (compared to normal skin). Important significantly downregulated pathways in LS skin included IL-1R/TLR signaling, type I and II cytokine receptor signaling, mitochondrial dysfunction, and antigen presentation. The differential expression and regulation of microRNAs and pathways involved in post-transcriptional modification were suggestive of epigenetic modification in the chronically inflamed tissue. Overall, the dysregulated genes and pathways suggest extensive “adaptive” mechanisms to control inflammation and maintain tissue homeostasis, likely triggered by chronic IL-1 release in the skin of patients with NOMID.

Pyoderma Gangrenosum–Like Ulcer in a Patient With X-Linked Agammaglobulinemia: Identification of Helicobacter bilis by Mass Spectrometry Analysis

BACKGROUND Pyoderma gangrenosum-like ulcers and cellulitis of the lower extremities associated with recurrent fevers in patients with X-linked (Bruton) agammaglobulinemia have been reported to be caused by Helicobacter bilis (formerly classified as Flexispira rappini and then Helicobacter strain flexispira taxon 8). Consistent themes in these reports are the difficulty in recovering this organism in blood and wound cultures and in maintaining isolates in vitro. We confirmed the presence of this organism in a patients culture by using a novel application of gene amplification polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry. OBSERVATION An adolescent boy with X-linked agammaglobulinemia presented with indurated plaques and a chronic leg ulcer whose origin was strongly suspected to be an H bilis organism. Histologic analysis demonstrated positive Warthin-Starry staining of curvilinear rods, which grew in culture but failed to grow when subcultured. They could not be identified by conventional techniques. A combination of gene amplification by polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry confirmed the identity of this organism. CONCLUSIONS This novel technology was useful in the identification of a difficult-to-grow Helicobacter organism, the cause of pyoderma gangrenosum-like leg ulcers in patients with X-linked agammaglobulinemia. Correct identification of this organism as the cause of pyoderma gangrenosum-like ulcers in patients with X-linked agammaglobulinemia is of great importance for the early initiation of appropriate and curative antibiotic therapy.

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.

Histologic variants of periungual fibromas in tuberous sclerosis complex.

To the editor: Periungual fibroma (PF) is a cutaneous manifestation of tuberous sclerosis complex (TSC). Although a benign tumor, it can bleed, cause pain, and distort the nail. Non-traumatic PFs are among the major diagnostic criteria of tuberous sclerosis complex (TSC).1 The lesions usually develop post-puberty, are more common on toenails than on fingernails, and occur more frequently in women than in men.2,3 We conducted a retrospective review of 32 PFs from 18 TSC patients seen at the NIH between 2000–2008. The clinical features of these cases are summarized in Table 1. All but one patient were females since they were enrolled in studies of lymphangioleiomyomatosis, a pulmonary disease in TSC patients that occurs almost exclusively in women.3 The average age of the patients included in this study was 49.8±7.7 years old. All but two of the PFs were obtained from the toes and the remaining were from the fingers. The duration of the lesions ranged from 2 to 40 years. Table 1 Clinical and histological features of periungual fibromas in TSC Grossly, most PFs are pink to red, firm, conical papules emerging from under the proximal nail fold (Figure 1A, 1C and 1E). A longitudinal nail groove extends from under the tumor to the free edge of the nail.4 The lesions usually measure 1–5 mm from the base to the tip. Histologically, they are non-encapsulated and are comprised of stellate shaped fibroblasts admixed with vertically oriented dense collagen and blood vessels. The lesions can be highly vascular, predominantly fibrotic, or show a mixed pattern. The epidermal changes are characterized by compact hyperkeratosis, acanthosis, thickened granular layer and irregular rete ridges (Figures 1B, 1D, and 1F). Parakeratosis is not observed. Figure 1 Variants of periungual fibroma, showing paired gross and microscopic images. Angiomatous The histological appearance of TSC PFs varied depending on the relative proportions of vascular proliferation and stromal fibrosis. The angiomatous subtype, observed in 5 samples, is characterized by numerous dilated vascular spaces, lined by plumped endothelial cells. The vascular lumens were large compared to that in normal skin. In between the vascular spaces are proliferation of stellate fibroblasts admixed with variable amounts of dense dermal collagen (Figure 1B). The fibrotic subtype, observed in 19 samples, featured predominantly vertically oriented thick collagen bundles in the dermis admixed with thick-walled small blood vessels (Fig. 1D). Elastic tissue (EVG) staining showed significantly decreased amounts of dermal elastic fibers, compared to the non-lesional skins obtained from the same patients (figure not shown). A third histologic subtype, observed in 8 samples, showed histological features intermediate between angiomatous and fibrotic subtypes and therefore was categorized as mixed subtype (Fig. 1F). Some of these changes were consistent with the observations of Nickle and Reed5, and of Kint and Barran6. Lesions categorized histologically as angiomatous subtype generally appeared more red, while the fibrotic subtype tended to be more white and firm; there was no obvious correlation of subtype with tumor location. Although the angiomatous and fibrotic subtypes can be distinguished histologically, they may represent a spectrum of changes of these tumors from angiomatous to fibrotic subtype over time. The proposed histologic sub-typing of periungal fibromas may provide helpful insights into the pathogenesis of these lesions.

Cutaneous metastasis of prostate cancer: a case report and review of the literature with bioinformatics analysis of multiple healthcare delivery networks.

Distant cutaneous metastases of prostate carcinomas are extremely rare, despite its high incidence and prevalence. Most cases are either a result of local extension, such as into seminal vesicles or distant metastases to bone. Few cases of true cutaneous metastatic prostate carcinoma exist in the literature. Clinically, cutaneous prostate carcinoma has been reported to mimic many other conditions, such as cellulitis, sebaceous cysts, zosteriform lesions, telangectasias and more, resulting in poor recognition. We report a case of distant cutaneous metastasis of prostate carcinoma and recent review of the literature with an analysis of de‐identified patient records from multiple healthcare delivery networks. A diagnosis of metastatic prostate carcinoma may have been easily overlooked given the location and nature of the rash. Reviewing case reports and using aggregated electronic health records (EHRs), we find that fewer than 0.1% of all prostate carcinomas result in cutaneous metastases, compared with much higher rates in other types of cancers. Coupled with the low frequency of metastases to skin, careful consideration must be taken when evaluating a rash in a patient with a prior history of cancer. A complete clinical history and strong suspicion would be required to make a diagnosis of cutaneous metastases of the prostate.

Palmoplantar pustules and osteoarticular pain in a 42-year-old woman

Key teaching points • Synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome is characterized by distinctive osteoarticular manifestations and a spectrum of neutrophilic dermatoses. • The most common dermatologic manifestations include palmoplantar pustulosis, acne conglobata, and acne fulminans. • SAPHO syndrome should be considered in patients presenting osteoarticular pain, particularly involving the anterior chest wall and/or spine, and neutrophilic skin lesions.

Genetic diversity in melanoma metastases from a patient with xeroderma pigmentosum.

TO THE EDITOR Melanoma is among the most severe and lethal forms of human skin cancer (Mackie, 2006; Miller and Mihm Jr, 2006; Fecher et al., 2007; Gray-Schopfer et al., 2007). For 2008, more than 110,000 new cases of melanoma (about 50,000 melanoma in situ (MIS) and 60,000 invasive melanomas) were estimated with more than 8,000 deaths (American Academy of Dermatology, 2008; Ries et al., 2008). To examine genetic diversity, we studied metastatic melanoma lesions from a patient with xeroderma pigmentosum (XP). XP is a rare genetic disease with defective DNA repair and a more than 1000-fold increase in melanoma frequency (Kraemer et al., 1987, 1994; Ruenger et al., 2008). The XP patient, (XP4BE), had no blistering reaction to sunlight despite living on a farm with unrestricted exposure to sunlight. By 8 years of age he had extensive freckling on his face (Figure 1a). At the age of 9 years a large warty tumor was excised from his nose. He subsequently developed more than 100 cancers in sun-exposed skin, primarily basal cell carcinomas and squamous cell carcinomas, which were treated with electrodessication and curettage or surgical excision followed by skin grafting (Figure 1b). At the age of 23 years a primary malignant melanoma of the right post-auricular area had been widely excised, and evidence of spread was found in two of the 35 posterior cervical nodes. Figure 1 Clinical appearance, histology, and DNA sequencing of metastatic melanoma lesions in patient XP4BE XP4BE was admitted to the NIH Clinical Center in 1969 at the age of 25 years (Robbins et al., 1974) and studied in accordance with the NIH human research guidelines then in effect. He was the first XP “variant” patient recognized, a form of XP with normal nucleotide excision repair (Robbins et al., 1974). His cells were subsequently found to have a defect in the error-prone polymerase, polymerase eta (Johnson et al., 1999; Masutani et al., 1999). At admission, his neurological examination was normal. In 1971, a melanoma nodule was present on his scalp (Figure 1c). He had no beneficial response to two courses of bis-choroethyl-nitrosourea (BCNU), steroids, or radiation to the brain and lumbar areas for treatment of the metastatic melanoma. He died at the age of 27 years in 1971. Autopsy performed at NIH revealed thromboemboli with pulmonary infarctions, aspergillus pneumonia and cerebral abscess, and metastatic melanoma involving his brain, spinal cord, stomach, small bowel, liver, gall bladder, adrenal glands, kidneys, lymph nodes, left testis, right lung, pancreas, thyroid gland, and soft tissue of the right thigh. The paraffin blocks from this autopsy were recently retrieved, sectioned, and stained. We do not have tissue from the primary lesion, which was removed before coming to NIH. We analyzed seven metastatic lesions from this patient, the metastatic scalp nodule obtained before BCNU treatment (Figure 1c–e), and six lesions obtained at autopsy (central nervous system (CNS) × 2, muscle, liver, kidney, and pancreas) (Table 1) as well as normal tissue. Using laser capture microdissection, we isolated about 500 tumor or normal cells for DNA analysis (Figure 1d and e) as described previously (Wang et al., 2009). To minimize formalin sequencing artifacts (Williams et al., 1999), we used more than 500 cells for analysis and repeated the sequencing on independent PCR products obtained from adjacent regions of the tumor for several tumors. Table 1 Mutations in PTEN, NRAS, and BRAF in metastatic melanoma lesions from patient XP4BE We studied the tumor suppressor gene PTEN (phosphatase and tensin homolog), which is one of the most frequently mutated genes in human cancer, including melanoma (Goel et al., 2006; Baker, 2007) as well as the NRAS and BRAF oncogenes that have been reported to be mutated in melanomas (Curtin et al., 2005). We used sequencing techniques previously described (Curtin et al., 2005; Wang et al., 2009 and references therein). Previously, we screened 59 primary cutaneous melanomas from 8 other XP patients and 56% had PTEN base substitution mutations. There were 1–4 mutations in individual melanomas, including MIS, the earliest stage of melanoma (Wang et al., 2009). Table 1 summarizes the pathological and mutational features of all seven metastatic melanoma lesions tested. Samples from six (86%) of the melanomas showed base substitution mutations in the PTEN tumor suppressor gene. No insertions or deletions were observed. Fifteen base substitution mutations were detected and 14 of these were different from each other. There was 1 nonsense mutation (Gln245X), 12 missense mutations, and 2 synonymous mutations that did not alter the amino-acid sequence. Individual metastatic lesions showed marked genetic diversity with 1–4 different PTEN base substitution mutations: the liver lesion had one mutation; a CNS and a skeletal muscle metastasis each had two mutations; kidney and pancreas metastases each had three mutations; and the metastatic melanoma lesion on the scalp had four different mutations. Thirteen (87%) of the mutations were present at dipyrimidine sites, a feature of UV-induced mutations (Wang et al., 2009). Loss of heterozygosity was found in two of the metastatic melanomas that also had PTEN missense mutations. A Thr167Ala missense mutation was reported previously in an astrocytoma (Raffel et al., 1999). The missense mutations were located in the dual-specificity protein phosphatase domain (amino acids 25–179) as well as in the calcium/lipid binding region (amino acids 190–347). Two of four (50%) of the metastatic lesions tested had NRAS base substitution mutations. One mutation in NRAS was found in the kidney metastasis and two NRAS mutations were found in the pancreas metastasis. However, these three NRAS mutations were all synonymous. None of the five metastatic lesions tested had BRAF base substitution mutations. The low frequency of mutations in NRAS and BRAF contrasts with the higher frequency of mutations in the PTEN gene obtained from the same samples and suggests that PTEN mutations were not the result of formalin artifacts that would be expected to similarly affect all genes (Williams et al., 1999). Thus, mutations in the NRAS and BRAF oncogenes did not appear to have a major role in metastasis of melanomas in this XP patient. Patient XP4BE presented with metastatic lesions. In our study of early melanomas in other XP patients (Wang et al., 2009), we found the UV type mutations, gGc131gAc and aGg164aAg, in MIS lesions. These same mutations were also present in three of the metastatic lesions (liver, CNS, and kidney) (Table 1) and might have been present in the primary melanoma(s) in XP4BE. These mutations result in amino-acid alterations (Gly44Asp and Arg55Lys) in the protein phosphatase domain of PTEN and may represent persistent UV-induced genetic alterations associated with metastasis. Surprisingly, there was no single mutation present in all of the metastatic lesions, as would be expected if all of the metastases arose from a single primary melanoma (Wang et al., 2006; Sabatino et al., 2008). Multiple tumor samples from the same patient had different mutations, indicating the presence of different clonal populations of tumor cells in different metastatic lesions. Independent PCR amplification of laser capture microdissected tumor tissue from adjacent areas of tumors 1, 2, and 4 revealed the identical mutations in PTEN exon 2 as shown in Table 1, with no new mutations found, which is evidence that these mutations were not formalin artifacts (Williams et al., 1999). In addition, normal PTEN sequence was obtained for exons 4, 6, 7, and 8 for tumors 1–6, suggesting that there were local sequence variations in these metastatic melanomas. These metastatic lesions with different PTEN mutations might have arisen from different primary melanomas. The high frequency of UV type mutations in these metastatic lesions (87 vs 54% expected (Wang et al., 2009)) is consistent with their origin from sunlight-induced primary melanomas. Alternatively, alterations in other gene(s) might have induced the metastases (Wang et al., 2006; Sabatino et al., 2008). As in the primary melanomas (Wang et al., 2009), the finding of multiple PTEN mutations in metastatic lesions indicates that there is a marked genetic diversity in these tumors, perhaps reflecting the hypermutability of XP variant cells (Waters et al., 1993; Stary et al., 2003; Wang et al., 2007). A similar genetic heterogeneity of metastatic melanomas from non-XP patients was reported based on the studies of loss of heterozygosity and X-chromosome inactivation (Katona et al., 2007). This genetic diversity may affect melanoma development, progression toward metastasis, and response to therapy.