Jessica S. Crystal

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.

Efficient Identification of Mutated Cancer Antigens Recognized by T Cells Associated with Durable Tumor Regressions

Purpose: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. Experimental Design: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors. Results: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. Conclusions: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis. Clin Cancer Res; 20(13); 3401–10. ©2014 AACR.

The public health burden of emergency general surgery in the United States: A 10-year analysis of the Nationwide Inpatient Sample--2001 to 2010.

BACKGROUND Emergency general surgery (EGS) represents illnesses of very diverse pathology related only by their urgent nature. The growth of acute care surgery has emphasized this public health problem, yet the true “burden of disease” remains unknown. Building on efforts by the American Association for the Surgery of Trauma to standardize an EGS definition, we sought to describe the burden of disease for EGS in the United States. We hypothesize that EGS patients represent a large, diverse, and challenging cohort and that the burden is increasing. METHODS The study population was selected from the Nationwide Inpatient Sample, 2001 to 2010, using the AAST EGS DRG International Classification of Diseases—9th Rev. codes, selecting all EGS patients 18 years or older with urgent/emergent admission status. Rates for operations, mortality, and sepsis were compiled along with hospital type, length of stay, insurance, and demographic data. The &khgr;2 test, the t test, and the Cochran-Armitage trend test were used; p < 0.05 was significant. RESULTS From 2001 to 2010, there were 27,668,807 EGS admissions, 7.1% of all hospitalizations. The population-adjusted case rate for 2010 was 1,290 admissions per 100,000 people (95% confidence interval, 1,288.9–1,291.8). The mean age was 58.7 years; most had comorbidities. A total of 7,979,578 patients (28.8%) required surgery. During 10 years, admissions increased by 27.5%; operations, by 32.3%; and sepsis cases, by 15% (p < 0.0001). Mortality and length of stay both decreased (p < 0.0001). Medicaid and uninsured rates increased by a combined 38.1% (p < 0.0001). Nearly 85% were treated in urban hospitals, and nearly 40% were treated in teaching hospitals; both increased over time (p < 0.0001). CONCLUSION The EGS burden of disease is substantial and is increasing. The annual case rate (1,290 of 100,000) is higher than the sum of all new cancer diagnoses (all ages/types): 650 per 100,000 (95% confidence interval, 370.1–371.7), yet the public health implications remain largely unstudied. These data can be used to guide future research into improved access to care, resource allocation, and quality improvement efforts. LEVEL OF EVIDENCE Epidemiologic study, level III.

Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor

Effective adoptive T-cell therapy requires multiple tumor-epitope reactive T-cell clones. Fresh TILs were found to frequently contain such cells. Their TCRs were rapidly isolated based only on their frequency and could be used for personalized TCR-gene therapy. Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA–TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734–43. ©2016 AACR.

Routine second-opinion cytopathology review of thyroid fine needle aspiration biopsies reduces diagnostic thyroidectomy

BACKGROUND Follicular thyroid carcinoma cannot be distinguished reliably from benign follicular neoplasia by fine needle aspiration (FNA) biopsy. Given an estimated 20% risk of malignancy, many patients with indeterminate FNA biopsies require thyroidectomy for diagnosis. Some centers have shown significant discordance when a second pathologist evaluates the same FNA biopsy. We sought to determine whether routine second-opinion cytopathology reduces the need for diagnostic thyroidectomy, especially in patients with indeterminate FNA biopsies. METHODS In all, 331 thyroid FNA biopsy specimens obtained from outside centers from 2004 to 2009 were reviewed at our institution. The FNA biopsy results were categorized into nondiagnostic (Bethesda I), benign (Bethesda II), indeterminate (follicular/Hurthle cell neoplasm, follicular/Hurthle cell lesion; Bethesda III & IV), and malignant (papillary or suspicious for papillary or other malignancy; Bethesda V and VI). Second-opinion cytology was compared with the initial opinion in 331 cases and with final operative pathology in the 250 patients who progressed to thyroidectomy. RESULTS The average patient age was 51 with a predominant number of female (79%) participants. The overall cytology concordance for all 331 FNA biopsies was 66% (218/331). Concordance was highest at 86% (74/86) with malignant FNA biopsies. Concordance in the 129 patients with indeterminate FNA biopsies was only 37% (48/129). Indeterminate FNA biopsies were reread as nondiagnostic in 21% (27/129) of patients and as benign in 42% (54/129) of patients. Twenty-two patients with an indeterminate FNA biopsy reread as benign progressed to operative therapy for reasons other than cytology (eg, symptomatic nodule and radiation exposure/high risk) and were found to be benign in 95% (21/22) of patients on operative pathology for a 95% negative predictive value. An additional 11 patients with an indeterminate FNA reread as benign had follow-up FNA biopsies, each of which was benign. Indeterminate FNA biopsies on initial cytology had a malignancy rate of 13% (17/129) on operative pathology compared with 29% (14/48) for indeterminate FNA biopsies from second opinion. A second opinion improved FNA biopsy accuracy from 60% to 74%. Overall, second-opinion cytology of indeterminate FNA biopsies avoided diagnostic operation in 25% (32/129). CONCLUSION Routine second opinion review of indeterminate thyroid FNA biopsies can potentially obviate the need for diagnostic thyroidectomy in 25% of patients without increases in false negatives.

Isolation of T cell receptors specifically reactive with mutated tumor associated antigens from tumor infiltrating lymphocytes based on CD137 expression.

Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patients tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs. Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12–24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNγ secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro. Dominant TCR α and β chains were identified in the enriched populations using a combination of 5′ rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens. Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells. Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer. Clin Cancer Res; 23(10); 2491–505. ©2016 AACR.

Characterization of an Immunogenic Mutation in a Patient with Metastatic Triple Negative Breast Cancer.

Purpose: The administration of autologous tumor-infiltrating lymphocytes (TILs) can mediate durable tumor regressions in patients with melanoma likely based on the recognition of immunogenic somatic mutations expressed by the cancer. There are limited data regarding the immunogenicity of mutations in breast cancer. We sought to identify immunogenic nonsynonymous mutations in a patient with triple-negative breast cancer (TNBC) to identify and isolate mutation-reactive TILs for possible use in adoptive cell transfer. Experimental Design: A TNBC metastasis was resected for TIL generation and whole-exome sequencing. Tandem minigenes or long 25-mer peptides encoding selected mutations were electroporated or pulsed onto autologous antigen-presenting cells, and reactivity of TIL was screened by upregulation of CD137 and IFNγ ELISPOT. The nature of the T-cell response against a unique nonsynonymous mutation was characterized. Results: We identified 72 nonsynonymous mutations from the tumor of a patient with TNBC. CD4+ and HLA-DRB1*1501–restricted TILs isolated from this tumor recognized a single mutation in RBPJ (recombination signal binding protein for immunoglobulin kappa J region). Analysis of 16 metastatic sites revealed that the mutation was ubiquitously present in all samples. Conclusions: Breast cancers can express naturally processed and presented unique nonsynonymous mutations that are recognized by a patients immune system. TILs recognizing these immunogenic mutations can be isolated from a patients tumor, suggesting that adoptive cell transfer of mutation-reactive TILs could be a viable treatment option for patients with breast cancer. Clin Cancer Res; 23(15); 4347–53. ©2017 AACR.

Enhancing the tissue donor pool through donation after death in the field.

INTRODUCTION Tissue transplantation is an important adjunct to modern medical care and is used daily to save or improve patient lives. Tissue allografts include bone, tendon, corneas, heart valves and others. Increasing utilization may lead to tissue shortages, and tissue procurement organizations continue to explore ways to expand the cadaveric donor pool. Currently more than half of all deaths occur outside the acute care setting. HYPOTHESIS Many who suffer prehospital deaths might be eligible for non-organ tissue donation. METHODS A retrospective review of electronic prehospital medical records was conducted from May 1, 2008 through December 31, 2009. All prehospital deaths were included irrespective of cause. Once identified, additional medical history was obtained from prehospital, inpatient, and emergency department records. Age, medical history, and time of death were compared to exclusion criteria for four tissue procurement organizations (MTF, LifeNet, LifeCell, EyeBank). After analysis, percentages of eligible donors were calculated. RESULTS Over 50,000 prehospital records were reviewed; 432 subjects died in the field and were eligible for analysis. Ages ranged from four to 103 years of age; the average was 68.3 (SD = 20.1) years. After exclusion for age, medical conditions, and time of death, 185 unique patients (42.8%) were eligible for donation to at least one of the four tissue procurement organizations (range 11.6%-34.3%). CONCLUSIONS After prehospital death, many individuals may be eligible for tissue donation. These findings suggest that future prospective studies exploring tissue donation after prehospital death are indicated. These studies should aim to clarify eligibility criteria, create protocols and infrastructure, and explore the ethical implications of expanding tissue donation to include this population.

Primary Leiomyosarcoma of the Colon: A Report of Two Cases, Review of the Literature, and Association with Immunosuppression for IBD and Rheumatoid Arthritis

Primary leiomyosarcomas (LMS) of the colon are rare and aggressive neoplasms and have been infrequently reported in the literature. These tumors are more aggressive and have poorer prognoses than adenocarcinoma of the colon and are often mistaken as such on initial evaluation. While the former has a clear association with inflammatory bowel disease (IBD), this correlation is not known to exist with LMS and IBD. Nor is there a known link between LMS and the immunosuppression for IBD, despite the known association between malignancy and immunosuppression for other diseases. Due to the low prevalence of this disease entity, there is limited knowledge and literature on the approach to diagnosing and treating these neoplasms, especially in the setting of the aforementioned comorbidities. Here, we describe two cases of this rare entity, presenting in two different circumstances: one in the setting of immunosuppression for IBD and arthritis, with a synchronous urothelial carcinoma, and the second appearing as the source of an acute abdomen. Both diagnoses were established following pathologic analysis.

Injury patterns and outcomes in late middle age (55–65): The intersecting comorbidity with high-risk activity – A retrospective cohort study

Background Late middle age (LMA), is a watershed between youth and old age, with unique physical and social changes and declines in vitality, but a desire to remain active despite increasing comorbidity. While post-injury outcomes in the elderly are well studied, little is known regarding LMA patients. We analyzed the injured LMA population admitted to a rural, regional Level 1 Trauma Center relative to outcomes for both younger and older patients. Materials and methods Our registry was queried retrospectively for patients admitted 7/2008- 12/2015; they were divided into three cohorts: 18–54, 55–65, and >65 years. Demographics, injury details, comorbidities, and outcomes were compiled and compared using ANOVA and Chi-square; p < 0.05 was significant. Results During the study period, 10,543 were admitted; 1419 (14%) were LMA who experienced overall injury mechanisms, severities and patterns that mirrored the younger cohort. However comorbidity rates were high (56.4%) and comparable to the elderly. LMA patients had the highest rates of alcohol abuse, morbid obesity, and psychiatric illness (p < 0.0001) and suffered the poorest outcomes: highest complications and hospital charges, and longest ICU and hospital LOS. LMA mortality (4.1%) was 41% higher than younger patients (2.9%; p < 0.02) and similar to the older cohort (4.7%; p = 0.32). Conclusions The LMA population has similar mechanisms and injury patterns to younger patients, while exhibiting comorbidity rates similar to the elderly. High-energy injuries exact a greater toll in LMA with poorer outcomes and greater resource utilization. Targeted outreach for injury prevention, and future studies, are needed to address high-risk behavior, substance abuse, and societal contributors.