Elsa F. Velazquez

Major Response to Imatinib Mesylate in KIT-Mutated Melanoma

addition, this is the first reported case of a synchronous malignant melanoma and a small-cell lung cancer metastasizing to the same anatomic location. This case illustrates the necessity of proper pathologic evaluation in a brain lesion assumed to be a metastasis. In this case, had the patient’s performance status improved after her motor vehicle accident, she would have been offered systemic chemotherapy for her extensive-stage smallcell lung cancer, which can markedly improve survival, even in advanced disease. If this brain lesion had proven to be solely melanoma, it is unlikely that any systemic treatment would have been offered.

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Bevacizumab plus Ipilimumab in Patients with Metastatic Melanoma

Hodi and colleagues report the safety and efficacy of targeting angiogenesis and CTLA-4 in a phase I trial of 46 patients with metastatic melanoma, which revealed the influence of VEGF-A blockade on inflammation, lymphocyte trafficking, and immune regulation, and their synergistic therapeutic effects. Ipilimumab improves survival in advanced melanoma and can induce immune-mediated tumor vasculopathy. Besides promoting angiogenesis, vascular endothelial growth factor (VEGF) suppresses dendritic cell maturation and modulates lymphocyte endothelial trafficking. This study investigated the combination of CTLA4 blockade with ipilimumab and VEGF inhibition with bevacizumab. Patients with metastatic melanoma were treated in four dosing cohorts of ipilimumab (3 or 10 mg/kg) with four doses at 3-week intervals and then every 12 weeks, and bevacizumab (7.5 or 15 mg/kg) every 3 weeks. Forty-six patients were treated. Inflammatory events included giant cell arteritis (n = 1), hepatitis (n = 2), and uveitis (n = 2). On-treatment tumor biopsies revealed activated vessel endothelium with extensive CD8+ and macrophage cell infiltration. Peripheral blood analyses demonstrated increases in CCR7+/−/CD45RO+ cells and anti-galectin antibodies. Best overall response included 8 partial responses, 22 instances of stable disease, and a disease-control rate of 67.4%. Median survival was 25.1 months. Bevacizumab influences changes in tumor vasculature and immune responses with ipilimumab administration. The combination of bevacizumab and ipilimumab can be safely administered and reveals VEGF-A blockade influences on inflammation, lymphocyte trafficking, and immune regulation. These findings provide a basis for further investigating the dual roles of angiogenic factors in blood vessel formation and immune regulation, as well as future combinations of antiangiogenesis agents and immune checkpoint blockade. Cancer Immunol Res; 2(7); 632–42. ©2014 AACR.

CTLA-4 blockade with ipilimumab induces significant clinical benefit in a female with melanoma metastases to the CNS

Background A 63-year-old female presented to her primary physician with numbness and weakness in her left leg, which progressed over several days to involve her entire lower extremities. MRI of the spine and brain revealed multiple metastases. The patient received ipilimumab and after 3 months experienced intermittent confusion and focal seizures.Investigations Electroencephalogram and MRI scans of the spine and brain, followed by surgical removal of a left frontal cortical brain metastasis and subsequent histological and pathological analyses.Diagnosis Metastatic melanoma from an unknown primary tumor.Management The patient was treated with ipilimumab on a compassionate-use program and dexamethasone, celecoxib, and levetiracetam to treat the symptoms and seizures. Postoperative stereotactic radiosurgery was initiated.

Lichen sclerosus in 68 patients with squamous cell carcinoma of the penis: frequent atypias and correlation with special carcinoma variants suggests a precancerous role.

Lichen sclerosus, an unusual chronic mucocutaneous condition of the penis, has been found in association with invasive penile cancer, and squamous cell carcinoma has been reported in patients with longstanding lichen sclerosus. The aim of this study was to determine the anatomic distribution and prevalence of lichen sclerosus in patients with squamous cell carcinoma of the penis and to search for a correlation with special types of carcinomas. Clinical and pathologic data from 207 penectomy and circumcision specimens with squamous cell carcinomas and giant condylomas were evaluated, and 68 patients with lichen sclerosus were identified. Mean age was 61 years. The preferential anatomic site of lichen sclerosus was the foreskin, but other sites (glans and coronal sulcus) including urethra were also involved. Grossly, the lesions showed white–gray smooth or irregular patches and plaques adjacent to invasive cancers. Microscopically, the lesion was stromal–epithelial with atrophic and hyperplastic epithelium and edematous or densely eosinophilic hyalinized lamina propria. A variable band of lymphocytic infiltration beneath the sclerosis was noted. Lichen sclerosus were preferentially associated with non-human papillomavirus variants of squamous cell carcinoma. When lichen sclerosus was associated with malignancy, it often showed, in addition to the hyperplastic epithelium, a low-grade squamous intraepithelial lesion. These findings suggest that lichen sclerosus may represent preneoplastic condition for at least some types of penile cancers, in particular those not related to human papillomavirus.

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

Histologic classification of penile carcinoma and its relation to outcome in 61 patients with primary resection.

A retrospective review of the clinical and pathologic features of 61 cases of penile squamous cell carcinoma (SCC), all treated by primary surgical resection at the Memorial Sloan Kettering Cancer Center during the period 1949-1992, was undertaken. Inguinal lymph node dissection material was evaluated in 40 cases. All carcinomas were of squamous cell type and were classified as follows: usual type, 36 cases (59%); papillary, not otherwise specified (NOS), 9 cases (15%), basaloid, 6 cases (10%); warty (condylomatous), 6 cases (10%); verrucous, 2 cases (3%), and sarcomatoid, 2 cases (3%). A high rate of nodal metastasis and poor survival were found for the basaloid and sarcomatoid neoplasms (5 of 7 patients with metastasis, 71%, and 5 of 8 dead of disease, 63%). Only 1 patient with a verruciform tumor (defined as a tumor of nonspecific papillary, warty, or verrucous type) had inguinal node metastasis and none died from penile cancer. An intermediate rate of metastasis and mortality (14 of 26, 54%, and 13 of 36, 36%, respectively) was found for typical SCC. Penile carcinomas are morphologically heterogeneous, and there is a correlation of histologic type and biologic behavior. This mandates accurate histologic subtyping by the pathologist.

Histologic grade and perineural invasion are more important than tumor thickness as predictor of nodal metastasis in penile squamous cell carcinoma invading 5 to 10 mm.

Penile squamous cell carcinomas (SCCs) invading to a depth inferior to 5 mm usually have very low risk for regional metastasis, whereas tumors thicker than 10 mm have a high metastatic potential. A significant number of squamous cell carcinomas, however, belong to an intermediate category (5 to 10 mm in thickness) in which the incidence of regional lymph node metastasis is very difficult to predict. Consequently, a frequent clinical dilemma is whether to perform or not inguinal dissection in this group of lesions. The objective of this study was to evaluate multiple risk factors for regional metastasis in tumors 5 to 10-mm thick. One hundred thirty-four partial penectomies with invasive carcinomas 5 to 10-mm thick, all of which with corresponding inguinal lymph node dissection, were evaluated. Factors evaluated were—patients age, anatomic site, histologic grade, tumor thickness, anatomic levels of invasion, and vascular and perineural invasion. Grades were classified as 1, well; 2, moderately; and 3, poorly differentiated. To evaluate independent significance of various prognostic factors, a logistic regression analysis was performed, and a nomogram was prepared to evaluate metastatic risk according to histologic grade and perineural invasion. Groin metastasis was found in 66 of 134 patients (49%). High histologic grade and perineural invasion were statistically significant pathologic factors associated with groin metastasis. Nodal metastases were found in 2 of 25 grade 1 (8%), 24 of 46 grade 2 (52%), and 40 of 63 grade 3 carcinomas (63%) (P value 0.0001). Of 48 patients with perineural invasion, metastasis was found in 33 cases (69%) (P value 0.001). The average tumor thickness, anatomic level of invasion, and presence of vascular invasion were not statistically significantly different in metastasizing and nonmetastasizing neoplasms. Fifty percent of tumors invading 5 to 10 mm were not associated with metastasis and may be spared a nodal dissection. In this subset of patients, high histologic grade and perineural invasion were significant and useful risk factors associated with regional metastasis. The probability of inguinal node metastasis in patients with grade 1 tumors without perineural invasion is almost nonexistent whereas for high-grade tumors associated with perineural invasion is near 80%.

Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.

Antitumor CD8+ T cells educated in vitro can persist as memory T cells and induce antitumor responses in humans without prior conditioning or cytokine treatment. Memory that Keeps Going and Going Senior scientists consoling their trainees about failed experiments tout the value of persistence. Yet, persistence is not only important for the scientist tirelessly pipetting at two in the morning, it is key for immunological memory as well. Melanoma is a malignant tumor of melanocytes, the pigment cells found in the skin. Advanced-stage melanoma has a poor prognosis, with patients on average surviving less than a year. A new promising therapy for advanced-stage melanoma patients harnesses the immune system to attack the tumor cells. In adoptive T cell therapy, cytotoxic cells specific for the tumor are transferred into patients, where they then traffic to and destroy the tumor cells. However, one limitation of this therapy is keeping the tumor-specific T cells alive in the patient, which has been largely unsuccessful in the absence of extensive treatment of the patient. Butler et al. now report a means to expand these T cells that allows them to persist in the absence of extra patient manipulation. The authors use artificial antigen-presenting cells to turn antitumor T cells into memory T cells, which survive longer and respond more quickly than normal effector T cells. These artificial antigen-presenting cells express molecules that “costimulate” the T cells, resulting in T cells that both look and act like memory T cells in the culture dish. These educated tumor-specific cells were then introduced into patients with advanced-stage melanoma. These cells functioned as memory cells in the patients as well: persisting for long periods of time, homing to the tumor site, and demonstrating tumor-specific activation and function, all in the absence of further patient manipulation. Although this is an early clinical trial with a small number of patients, there is some indication that this treatment may have clinical benefit for patients with this devastating disease. Persistence, both of the T cells and the scientists running the study, has finally paid off. Although advanced-stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of antitumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred antitumor CD8+ T lymphocytes (CTLs) have had limited life spans unless the host has been manipulated. To generate CTLs that have an intrinsic capacity to persist in vivo, we developed a human artificial antigen-presenting cell system that can educate antitumor CTLs to acquire both a central memory and an effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. We examined whether antitumor CTLs generated using this system could function and persist in patients. We showed that MART1-specific CTLs, educated and expanded using our artificial antigen-presenting cell system, could survive for prolonged periods in advanced-stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTLs trafficked to the tumor, mediated biological and clinical responses, and established antitumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.

PTEN Expression in Melanoma: Relationship with Patient Survival, Bcl-2 Expression, and Proliferation

Purpose: Inactivation of the tumor suppressor gene, phosphatase and tensin homologue (PTEN), is a major alteration in preclinical melanoma models. We investigated the clinical relevance of PTEN expression in the primary melanoma patients with extended follow-up. Experimental Design: We correlated PTEN expression with clinicopathologic variables and outcome in 127 primary melanomas (median follow-up, 12.8 years). We evaluated the associations between PTEN expression and proliferation and resistance to apoptosis (assessed by Ki-67 and Bcl-2, respectively). We also examined the effect of a favorable phenotype, defined as retained PTEN, low proliferative index, and low expression of Bcl-2 on disease-free survival and overall survival. Results: Altered PTEN, Bcl-2, and Ki-67 expressions were observed in 55 of 127 (43.3%), 61 of 127 (48%), and 43 of 114 (37.7%) of cases, respectively. Decreased PTEN expression correlated significantly with the ulceration (P = 0.01). Rates of disease-free survival and overall survival in patients with favorable phenotype were 72% and 74% at 5 years versus 64% and 64% in patients with an unfavorable phenotype. At 10 years, the rates of disease-free survival and overall survival were 72% and 68% for patients with a favorable phenotype but declined to 60% and 55% in patients with an unfavorable phenotype. However, relationships between both PTEN and Bcl2 and patient survival were not significant as well as the associations between PTEN and Bcl-2 or Ki-67. Conclusions: Our data suggest that altered PTEN expression is common in primary melanomas and is associated with aggressive tumor behavior. However, PTEN alone provided limited prognostic value. Our findings show the need to examine molecular alterations identified in preclinical studies using an adequately large cohort of patients with extended follow-up to better assess the magnitude of their clinical relevance.