Long-term Surviving Research Articles

Strong single-agent and PD-1/PD-L1 combination antitumor activity of antisense oligonucleotides suppressing Neuropilin 1 expression.

e14592 Background: While the introduction of immune checkpoint inhibitors (ICIs) is considered as a breakthrough in cancer therapy, large proportions of patients still do not respond or develop resistance, indicating a need for new targets and therapies to provide long-term therapeutic benefits. Neuropilin-1 (NRP1) is a membrane-bound multi domain protein expressed by different cell types in the tumor microenvironment. NRP1 promotes cell migration, survival, and angiogenesis via distinct domains. Moreover, it mediates immunosuppression by stabilization of regulatory T cells, attraction of immunosuppressive myeloid cells into the tumor microenvironment, and exhaustion of effector T cells. Therapeutic benefit is expected to be superior by simultaneous suppression of all functions and consequently all domains, which can be optimally achieved by suppression of its expression, for example mediated by antisense oligonucleotides (ASOs), instead of e.g. domain specific antibodies. Methods: Using our in-house bioinformatics and screening systems, we have identified potent and specific locked nucleic acid (LNA) modified ASOs specifically targeting NRP1. Mice bearing established syngeneic MC38 or EMT6 tumors were treated by intraperitoneal injection of unformulated ASO either as monotherapy or in combination with ICIs. Tumor growth and survival were determined as well as target engagement. Results: After systemic administration of different NRP1-specific ASOs we observed a potent downregulation of NRP1 expression in different cell types in tumors, including myeloid cells, T cells and non-immune cells as well as a strong reduction of soluble NRP1 plasma levels. Suppression of NRP1 correlated with both a delayed tumor growth or even complete tumor regression and prolonged survival as a monotherapy which was further improved in combination with ICIs. Particularly in an EMT6 breast cancer model, combination of NRP1 ASOs with PD-1 antibody resulted in tumor rejection and long-term survival in most animals. Rechallenge of long-term surviving mice with EMT6 tumor cells did not result in tumor growth. Conclusions: We could show that ASO-mediated suppression of NRP1 expression has strong potential as a novel therapeutic strategy, especially in combination with ICIs. Additional studies to further elucidate the mechanisms underlying the observed anti-tumor efficacy as well as IND-enabling activities are underway to progress the program towards clinical testing.

Long term survival in patients with classic infantile Pompe disease reveals a spectrum with progressive brain abnormalities and changes in cognitive functioning.

The aim of this longitudinal cohort study, is to provide more insight into the pattern of brain abnormalities, and possible consequences for cognitive functioning, in patients with classic infantile Pompe disease. We included 19 classic infantile Pompe patients (median age last assessment 8.9 years, range 1.5-22.5 years; 5/19 CRIM negative), treated with ERT. Using MR imaging of the brain (T1, T2, and FLAIR acquisitions), we classified progression of brain abnormalities on a 12-point rating scale at multiple time points throughout follow-up. Additionally we noted specific white matter patterns and examined atrophy. Cognitive development was studied using Wechsler IQ assessments obtained by certified neuropsychologists. The association between age and cognitive functioning, and MRI ratings and cognitive functioning was assessed by linear regression models. All but one patient developed brain abnormalities. The abnormalities progressed in a similar pattern throughout the brain, with early involvement of periventricular white matter, later followed by subcortical white matter, gray matter structures, and juxtacortical U-fibers. We found a significant decline (p < 0.01), with increasing age for full scale IQ, performance IQ and processing speed, but not for verbal IQ (p = 0.17). Each point increment in the 12-point MRI rating scale was associated with a significant decline (3.1-6.0 points) in all the IQ index scores (p < 0.05). The majority of long-term surviving patients in our cohort develop incremental brain MRI abnormalities and decline in cognitive functioning. This highlights the need for new therapies that can cross the blood-brain barrier in order to treat this CNS phenotype.

Long-term survival of stage IV melanoma patients: evaluation on 640 melanoma patients entering stage IV between 2014 and 2017

PurposeSince the introduction of immune checkpoint inhibitors (ICI) and targeted therapies (TT), survival rates of metastatic melanoma patients have increased significantly and complete remissions are no longer rarities. Consequently, there is an increasing number of long-term survivors who have not yet been comprehensively characterized.MethodsWe included melanoma patients who entered stage IV between 2014 and 2017 and survived at least 5 years after entering stage IV. Descriptive statistics were performed to characterize the applied systemic therapies, response rates and to report which of these patients are still alive today.Results640 patients entered stage IV at the University Hospital Tuebingen. Of these, 207 patients (32%) were still alive at least 5 years after entering stage IV. Details of applied therapies and response rates were available in 176 patients (85%). About 90% of patients (n = 159) were still alive at the time of analysis. Median survival since first stage IV diagnosis was 6.0 years (range 5–9 years). An impressive majority of patients (n = 146, 83%) were no longer receiving systemic therapy at the time of evaluation. Complete remission under first line systemic therapy was seen in 36% of the patients.ConclusionThis dataset comprises the largest available cohort of long-term surviving stage IV melanoma patients. Since 90% of patients in our cohort are still alive today, we expect an increasing number of long-term survivors in the future. Our data indicate the need for specific follow-up programs addressing the needs of long-term survivors.

Neurosurgical treatment of pediatric brain tumors - results from a single center multidisciplinary setup

ObjectiveThe challenge of pediatric brain tumor surgery is given due to a relative low prevalence but high heterogeneity in age, localization, and pathology. Improvements of long-term overall survival rates were achieved during the past decades stressing the importance of a multidisciplinary decision process guided by a national treatment protocol. We reviewed the entire spectrum of pediatric brain tumor surgeries from the perspective of an interdisciplinary pediatric neuro-oncology center in Germany.MethodsEvery patient who underwent brain tumor surgery from January 2010 to June 2017 in our Pediatric Neurosurgery department was retrospectively included and evaluated regarding the course of treatment. Perioperative data such as tumor localization, timing of surgery, extent of resection, neuropathological diagnosis, transfusion rates, oncologic and radiation therapy, and neurological follow-up including morbidity and mortality were evaluated.ResultsTwo hundred ninety-three pediatric brain tumor patients were applicable (age: 8.28 ± 5.62 years, 1.22:1.0 m:f). A total of 531 tumor surgical interventions was performed within these patients (457 tumor resections, 74 tumor biopsies; mean interventions per patient 1.8 ± 1.2). Due to a critical neurologic status, 32 operations (6%) were performed on the day of admission. In 65.2% of all cases, tumor were approached supratentorially. Most frequent diagnoses of the cases were glial tumors (47.8%) and embryonal tumors (17.6%). Preoperative planned extent of resection was achieved in 92.7%. Pre- and postoperative neurologic deficits resolved completely in 30.7%, whereas symptom regressed in 28.6% of surgical interventions. New postoperative neurologic deficit was observed in 10.7%, which resolved or improved in 80% of these cases during 30 days. The mortality rate was 1%.ConclusionWe outlined the center perspective of a specialized pediatric neuro-oncological center describing the heterogeneous distribution of cases regarding age-related prevalence, tumor localization, and biology, which requires a high multidisciplinary expertise. The study contributes to define challenges in treating pediatric brain tumors and to develop quality indicators for pediatric neuro-oncological surgery. We assume that an adequate volume load of patients within a interdisciplinary infrastructure is warranted to aim for effective treatment and decent quality of life for the majority of long-term surviving pediatric tumor patients.

Local Delivery of Adipose Stem Cells Promotes Allograft Survival in a Rat Hind-Limb Model of Vascularized Composite Allotransplantation.

Adipose stem cells (ASCs) are a promising cell-based immunotherapy because of their minimally invasive harvest, high yield, and immunomodulatory capacity. In this study, the authors investigated the effects of local versus systemic ASC delivery on vascularized composite allotransplant survival and alloimmune regulation. Lewis rats received hind-limb transplants from Brown Norway rats and were administered donor-derived ASCs (passage 3 or 4, 1 × 10 6 cells/rat) locally in the allograft, or contralateral limb, or systemically at postoperative day 1. Recipients were treated intraperitoneally with rabbit anti-rat lymphocyte serum on postoperative days 1 and 4 and daily tacrolimus for 21 days. Limb allografts were monitored for clinical signs of rejection. Donor cell chimerism, immune cell differentiation, and cytokine expression in recipient lymphoid organs were measured by flow cytometric analysis. The immunomodulation function of ASCs was tested by mixed lymphocyte reaction assay and ASC stimulation studies. Local-ASC-treated recipients achieved significant prolonged allograft survival (85.7% survived >130 days; n = 6) compared with systemic-ASC and contralateral-ASC groups. Secondary donor skin allografts transplanted to the local-ASC long-term surviving recipients accepted permanently without additional immunosuppression. The increases in donor cell chimerism and regulatory T-cells were evident in blood and draining lymph nodes of the local-ASC group. Moreover, mixed lymphocyte reaction showed that ASCs inhibited donor-specific T-cell proliferation independent of direct ASC-T-cell contact. ASCs up-regulated antiinflammatory molecules in response to cytokine stimulation in vitro. Local delivery of ASCs promoted long-term survival and modulated alloimmune responses in a full major histocompatibility complex-mismatched vascularized composite allotransplantation model and was more effective than systemic administration. ASCs are a readily available and abundant source of therapeutic cells that could decrease the amount of systemic immunosuppression required to maintain limb and face allografts.

A Lesson of Immunosuppression in Renal Transplant: Retreat or Hold?

Background: Aiming at understanding whether there are cases of near-tolerance among long-term surviving kidney transplant recipients in our center, or even operant tolerance can be attempted based on their immune status, we analyzed changes of immune cell subsets and cytokines in various groups, and evaluated immune status of long-term survival recipients. Methods: A real-world, observational, retrospective cohort study was conducted in our hospital. Twenty-eight long-term recipients were selected as study subjects, 15 recent postoperative stable recipients, and 15 healthy subjects as controls. T and B lymphocyte subsets, MDSCs, and cytokines were detected and analyzed. Results: Treg/CD4 T cells, total B and B10 cells in long-term and recent renal recipients were lower than healthy controls (HC). The level of IFN-γ and IL-17A in long-term survival patients was obviously higher than that in recent postoperative stable recipients and HC, while TGF-β1 level was significantly lower in long-term survival group than in short-term postoperative group and HC. Notably, compared with short-term recipients, it has been found that the IL-6 level in both positive and negative HLA groups were obviously lower (all P<0.05). In the long-term survival group, 43% of recipients were positive for urinary protein and 50% were positive for HLA antibody. Conclusion: This "real-world" study validates the findings of real status of long-term survival recipients observed in clinical trials. Contrary to a state of proper tolerance as expected, the group recipients in long-term survival were accompanied by the increased indicators of immune response, while those related to immune tolerance were not significantly increased. Long-term survival recipients with stable renal function may be in an immune equilibrium state where immunosuppression and rejection coexist under the action of low-intensity immune agents. If immunosuppressive agents are reduced or even removed, rejection may occur.

59. Long-term survival and clinical outcomes in patients managed with emergent surgery for a spinal epidural abscess with associated sepsis and neurologic decline

<h3>BACKGROUND CONTEXT</h3> Patients with a spinal epidural abscess (SEA) and associated sepsis and neurologic deterioration require immediate evaluation and prompt surgical intervention. The goals of surgery are life preservation, eradication of the infectious nidus, and restoration of neurologic function. The focus of treatment in these critically ill patients is to achieve these immediate goals. There has been limited attention given to the long-term status of patients who have survived this life threatening condition. <h3>PURPOSE</h3> To identify the survivability and long-term outcome of patients who underwent emergent surgical management for a SEA with associated sepsis and neurologic decline. <h3>STUDY DESIGN/SETTING</h3> A retrospective review of all consecutive patients with a SEA, sepsis and neurologic decline treated at a single, tertiary referral medical center from 2000-2010. <h3>PATIENT SAMPLE</h3> Fifty-four patients. <h3>OUTCOME MEASURES</h3> Survival/mortality, neurologic improvement (ASIA Grade), need for further spine surgery. <h3>METHODS</h3> All patients were clinically septic and had documented neurologic deterioration directly related to a spinal epidural abscess. Surgery was performed emergently from the time of notification of the spine service. Surgical approach was determined by the location of the abscess in relation to the thecal sac; 8 occurred in the cervical spine (all anterior), 20 in the thoracic spine (7 anterior, 13 posterior) and 26 in the lumbar spine (9 anterior, 17 posterior). All cervical SEA underwent an anterior decompression and fusion with structural grafting and plating, while anterior SEA within the thoracic or lumbar spines underwent anterior decompression and fusion with structural grafting followed by a staged, posterior instrumented fusion. Posterior SEA in the thoracic or lumbar spine underwent decompression and abscess evacuation only. Preoperative neurologic status was ASIA B in 20 patients, C in 20, and D in 10. The mean time of neurologic deterioration documented in the medical record was 24 hours (range: 6 hours-5 days). Stepwise multiple regression analysis was performed to identify risk factors for survival and neurologic improvement. Long-term survival was assessed using the institution's electronic medical record and subsequently confirmed by direct patient contact. <h3>RESULTS</h3> All patients survived the surgery, although 6 (11%) died within one month. Significant risk factors for early mortality included age >70 (p=.01), hospitalization >5 days prior to surgery (p=.02), ASIA D (p=.02), diabetes (p=.02), end stage renal disease (p=.02), and MRSA sepsis (p=.02). Of the surviving patients, the mean neurologic improvement was 2.4 ASIA grades. Of surviving patients, 92% (44/48) regained the ability to ambulate. Significant factors for neurologic improvement included age <70 (p=.01), documented neurologic decline <24 hours (p=.01), non-diabetic (p=.02), a cervical abscess (p=.01), a thoracic or lumbar abscess not requiring fusion (p=.02), and preoperative ASIA D (p=.01). At the 10-year follow-up, 88% (42/48) of the patients who survived their perioperative course were still alive. The six non-surviving patients had died of causes unrelated to their spine infections. At the 10-year follow-up, all 42 patients were noted to have maintained their level of neurologic recovery, with none having evidence of recurrent neurologic deficits or loss of ambulatory ability. Ten percent (4/42) of the long-term surviving patients had undergone additional spine surgery, 2 for removal of painful segmental instrumentation and 2 for extension of fusions for symptomatic adjacent level disease. None of the long-term survivors required additional surgery for a recurrent spine infection. Advanced imaging (MRI or CT scan) was available for 30 of the surviving patients. Aside from the 2 patients requiring surgery for adjacent level stenosis, there were no instances of residual infectious spinal column involvement, segmental instability or sagittal plane deformity in any patient. <h3>CONCLUSIONS</h3> Spinal epidural abscesses in patients with sepsis and neurologic deterioration can be effectively managed when diagnosed promptly and treated with emergent surgery. In this series, the majority of patients survived, had neurologic recovery and regained independent ambulation. However, there was a notable early mortality rate (11%), with older, diabetic patients with prolonged neurologic deficits most susceptible. Long-term survival occurred in 88% of patients, with evidence of sustained neurologic improvement, permanent eradication of the spine infection, and relatively uncommon need for additional surgery. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Allogeneic stem cell transplantation for mantle cell lymphoma\u2014update of the prospective trials of the East German Study Group Hematology/Oncology (OSHO#60 and #74)

Mantle cell lymphoma (MCL) is a non-Hodgkin’s lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

Zika virus-based immunotherapy enhances long-term survival of rodents with brain tumors through upregulation of memory T-cells.

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiation attenuated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiation attenuated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an increase in the number of T-cells, including CD4+ and tissue-resident effector/ effector memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

Human kidney graft survival correlates with structural parameters in baseline biopsies: a quantitative observational cohort study with more than 14 years' follow-up.

This prospective cohort study evaluates associations between structural and ultrastructural parameters in baseline biopsies from human kidney transplants and long-term graft survival after more than 14 years' follow-up. Baseline kidney graft biopsies were obtained prospectively from 54 consecutive patients receiving a kidney transplant at a single institution. Quantitative measurements were performed on the baseline biopsies by computer-assisted light microscopy and electron microscopy. Stereology-based techniques estimated the fraction of interstitial tissue, the volume of glomeruli, mesangial fraction, and basement membrane thickness of glomerular capillaries. The fraction of occluded glomeruli and scores according to the Banff classification were achieved. Kidney graft survival was analyzed by Kaplan-Meier estimates and Cox regression. Association to long-term kidney function was also analyzed. The long-term surviving kidney transplants were characterized at implantation by less arteriolar hyaline thickening (P < 0.001) and less interstitial fibrosis (P = 0.001), as well as a lower fraction of occluded glomeruli (P = 0.004) and lower glomerular volume (P = 0.03). At the latest follow-up, eGFR was decreased by 12 ml/min/1.73 m2 per unit increase in the score for arteriolar hyalinosis at implantation (P = 0.02), and eGFR was decreased by 19 ml/min/1.73 m2 per 106 μm3 increase in glomerular volume at baseline (P = 0.03). The unbiased Cavalieri estimate of glomerular volume and the ultrastructural parameters are the first to be evaluated in a cohort study with prospective follow-up for more than 14 years. The study shows that baseline biopsies from human kidney grafts contain extraordinary long-term prognostic information, and it highlights the importance of these intrinsic graft factors.

Abstract IA05: Molecular analysis of exceptional response in high-grade serous ovarian cancer

Abstract A majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of approximately 40%, and less than 10% survive more than 10 years. Profound genomic instability, treatment with DNA-damaging drugs, and a large tumor burden promote the development of acquired resistance in a large proportion of patients, including multiple convergent resistant events within individuals. Despite the propensity of HGSC to develop drug resistance, a small proportion of patients with advanced disease at diagnosis become long-term survivors. These exceptional patients include those who had suboptimal surgical clearance, and therefore were not cured surgically, and others who have had no evidence of disease recurrence following initial surgery and adjuvant chemotherapy. Our study, which is part of the international collaborative Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG), seeks to identify molecular, immunologic, and epidemiologic factors that influence long-term survival in HGSC. This presentation describes our genomic and immunologic analysis of exceptional survival of this deadly disease. Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterized by RNA sequencing, DNA methylation profiling, and immunohistochemistry. Thirty-eight (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemosensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls. Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related), and Signatures 5, 8, and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P &amp;lt; 0.001) compared to patients with RB1-positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P &amp;lt; 0.001). Multiplexed immunohistochemical analysis with immune cell panels demonstrated distinct associations with long-term survivors. This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations are associated with enhanced host immune responses and long-term survival. Citation Format: Dale Garsed, Ahwan Pandey, Sian Fereday, Kathryn Alsop, Maartje Wouters, Flurina Saner, Catherine Kennedy, Celeste Pearce, Malcolm Pike, Susan Ramus, Martin Kobel, Anna deFazio, Ellen Goode, Brad Nelson, David Bowtell. Molecular analysis of exceptional response in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA05.

Does Osteoarthritis of the Ipsilateral Hip Develop After Knee Rotationplasty? A Radiologic Study on Patients with Long-term Survival Undergoing Surgery for Bone Tumors.

During routine check-ups of long-term surviving knee rotationplasty patients referred to our institute, we noted that the operated limb radiograph showed signs of hip osteoarthritis in some patients, and that one patient reported hip pain. We wondered whether radiographic changes and symptoms are common in patients undergoing rotationplasty due to mechanical or overloading problems during gait. Based on these considerations, we wished to determine how frequently arthrosis or arthritis would arise in the ipsilateral hip to a knee rotationplasty at long-term follow-up. To our knowledge, this issue has not been explored. Clinicians and patients should be aware of any problems that may affect the hip over time that could result in treatment. At a minimum of 10 years after rotationplasty, in a single-center series, we therefore asked: (1) What proportion of patients reported hip pain at rest and with activity? (2) What proportion have radiographically visible arthritic changes in the ipsilateral or contralateral hip? (3) What proportion of patients have changes in the hip morphology (femoral rotation, cervicodiaphyseal angle, acetabular angle, osteopenia)? Between 1986 to 2009, 42 patients underwent rotationplasty in our institute. They represent the 16.5% of a series of 254 children (age 3 to 14 years) affected by high-grade bone sarcomas located in the distal half of the femur. Three more patients were adolescents older than 15 years and were treated with rotationplasty because of the tumor volume and extracompartmental involvement. Of these 45 patients, 14 died of disease at a mean of 37 months; 31 patients were survivors at the time the study was done. Three of these long-term survivors were known to be alive but did not come for a clinic visit in the last 5 years. The remaining 28 patients were invited to participate in a research study on the long-term assessment of rotationplasty, and all agreed to participate. Unfortunately, 10 patients living far from our Institute could not come in the 2 days established for the assessment due to family or work constraints. AP plain radiographs of the pelvis were taken in 17 patients with long-term survival after knee rotationplasty. Patients were positioned in a standard weightbearing position, wearing their own prosthesis. Hip osteoarthritis was assessed using the Croft grading system. The presence of hip pain was assessed with the self-reported Numeric Rating Scale (NRS), with scores ranging from 0 to 10, at rest and during daily life activities. Femoral rotation, osteopenia, the cervicodiaphyseal and the acetabular angles were measured on radiographs. The median (range) age at follow-up was 32 years (22 to 45), the median age at the time of the intervention was 9 years (6 to 17), and the median follow-up duration after surgery was 25 years (11 to 30). One of the 17 patients complained of mild pain (NRS score of 2) in the ipsilateral hip during walking. No other patient claimed to have hip pain at rest or during walking or other daily life activities. Eleven of 17 patients had signs of OA according to the Croft grading system: one had Grade 1 OA, seven had Grade 2, and three had Grade 3. Six patients did not show any sign of OA in the ipsilateral hip. Two patients had Grade 1 OA and one had Grade 2 OA of the contralateral hip. Sixteen patients had femoral external rotation. An increased valgus cervicodiaphyseal angle and acetabular angle were present in 10 and 9 patients, respectively. Osteopenia at the ipsilateral hip when compared with the contralateral, was present in all patients but one, with varying degrees of severity. In this small series, signs of osteoarthritis of the hip ipsilateral to the rotationplasty were present in a high proportion of patients at a minimum follow-up interval of 10 years after surgery, although only one participant reported hip pain. The finding of arthrosis in the hip above a rotationplasty is potentially important because it may be related to gait abnormalities that may modify the hip's morphology over time. It is encouraging that symptomatic hip pain was uncommon in our patients, but this could become more of a clinical problem with longer follow-up and result in therapeutic intervention. Patients (or the parents of patients) undergoing this procedure should be informed that they might develop hip arthritis during young adulthood. Level IV, therapeutic study.

Worldwide First Successful and Reproducable Long-Term Survival up to Half a Year: Completed Preclinical Study with Life-Supporting Orthotopic Pig-to-Baboon Cardiac Xenotransplantation (oXHTx) Fullfilling the ISHLT Prerequisite for Clinical Cardiac Xenotransplantation

Major hurdles in oXHTx are the delayed xenograft rejection, the early perioperative cardiac xenograft dysfunction (PCXD) and the pig heart overgrowth, which were solved in this study with a costimulation blockade, a new non-ischemic cold preservation and a growth inhibition by anti-proliferative drugs. Aim was to achieve a 90-days-survival of minimal 60% (6 of 10 baboons) in this life-supporting orthotopic pig-to-baboon model (oXHTx), because this is the recommendation of the ISHLT to begin a clinical cardiac XT program. We transplanted 8 GalKO/hCD46/hTM transgenic (tg) pig hearts orthotopically into baboons with using a basic immunosuppression consisting of ATG, rituximab, mycophenolate (MMF), cortisone and a costimulation blockade CD40mAb (high dose: 50 mg/kg). To prevent PCXD, we used instead of the crystalloid solution a new non-ischemic 8°C cold perfusion technique with oxygenated erythrocytes. Additional antihypertensive drugs and an mTOR inhibitor (temsirolimus) were applied to inhibit pig xenograft growth and hypertrophy. In comparison to our previous group with crystalloid cardioplegia (Längin et al. Nature. 2018;564:430-433) in this group with cold perfusion preservation (non-ischemic) no PCXD was found. One baboon died of a pancreatitis on day 14, another of sepsis on day 26. By using the antiproliferative therapy, 6 of 8 recipient baboons reached the end of study, were long-term surviving (4 were actively terminated after 90 days according to the guidelines of our government). With special permit two further experiments could be prolonged to half a year and the animals were terminated on day 182 and 195. All baboons lived under excellent physical conditions and no hyperacute rejection or DXR occurred. First time in a life-supporting oXHT of multi-tg pig hearts here was a consistent reproduceable long-term survival of 3 - 6 months achieved, which is a major progress after 25 years of research. This is an essential milestone and breakthrough and meets the prerequisite according to the ISHLT to begin a clinical phase I study with patients in terminal heart failure. This paves the way to clinical cardiac XT in the next 2 to 5 years.

Identifying Long-Term Survival Candidates among Patients with Isolated Locoregionally Recurrent Breast Cancer: Implications of the Use of Systemic Chemotherapy.

PurposeWe aimed to investigate the clinicopathologic factors associated with distant metastasis (DM) and post-recurrence overall survival (OS) after salvage treatments for isolated locoregional recurrence (ILRR) of breast cancer and identify long-term surviving patients for providing a more personalized therapy.MethodsWe analyzed 125 patients who underwent salvage local treatments for ILRR after initial curative breast surgery.ResultsFifty-two (41.6%) patients experienced secondary recurrence or disease progression, of which 20 (38.5%) experienced a secondary locoregional recurrence and 40 (76.9%) experienced DM as the first site of failure. In multivariate analysis of distant metastasis free survival (DMFS) and post-recurrence OS, the initial pN2-3 stage, a disease-free interval of < 36 months, and non-curative resection for recurrent disease were independently poor prognosticators. The score for patients stratified according to the number of risk factors increased from 0 to 3; the corresponding 5-year DMFS rates were 91.4%, 53.0%, 35.9%, and 0% and the 5-year OS rates were 97.3%, 70.4%, 32.7%, and 25.0%, respectively (p < 0.001). Systemic chemotherapy reduced DM in patients with a score of 2–3, but it did not in those with a score of 0-1.ConclusionOur collective stratification can help with prognosis prediction for ILRR of breast cancer. Depending on the DM risk of patients, the potential combination of systemic therapy should be discussed further.

Clonal Dynamics of Early Responder and Long-Term Surviving CAR-T Cells in Humans

CD19 CAR-T cells show unprecedented responses in relapsed/refractory Acute Lymphoblastic Leukaemia, but long-term persistence appears critical for their use as a stand-alone therapy. The origin of long-term persisting CAR-T cells has yet to be defined and will be critical in designing manufacturing protocols to optimise long-term persistence. Previous data are conflicting with groups showing prolonged persistence of CAR-T cells from cell products with a predominantly effector memory (TEM) phenotype, whereas others suggesting that the dominant clones originate instead from infused stem cell memory (TSCM) and central memory (TCM) T cells. To date, it has not been possible to isolate long-term (&amp;gt; 1 year) persisting CAR-T cells in patients. In the CARPALL Phase I study, the use of an improved low-affinity CD19 CAR resulted in enhanced expansion and persistence of CAR-T cells in vivo (Ghorashian et al, Nature Medicine, in press). Combining this unique experimental setting with our well-established clonal tracking platform based on high-resolution integration sites (IS) analysis has enabled us to track the fate of the infused CAR-T cells. We analysed 2 patients with long-term persistent CAR-T cells detectable by flow cytometry in peripheral blood. CAR-T cells comprised 13 and 53% circulating CD3+ cells respectively at day 14 post-infusion, 7.1 and 7.7% circulating at 1 month, 0.7 and 1.3% at 6 months and 0.1% for both at the latest follow-ups (24-28 months). Blood samples taken at early (14d, 30d) and later (6m to 28m) time points were flow-sorted for CAR+ TCM/TEM mixed population and TSCM T cells, while the corresponding infused gene-modified products were separated into three subpopulations: TSCM, TCM and TEM. The integration profile of each sorted cell populations was established using linear amplification mediated-PCR (LAM-PCR) combined with high throughput sequencing. We identified a total of 7,105 and 4,692 IS from 2 patients overtime before infusion and up to 28 months after infusion. The infused CAR-T cell population was highly polyclonal before infusion. Although the total number of CAR-T cell clones decreased substantially upon in vivo selection, we did not observe any sign of aberrant clonal drifts and diversity was preserved long-term. Early after infusion during the response peak, TSCM underwent two waves of transient oligoclonal expansion. In both patients two distinct sets of individual TSCM clones contributed to the 73% and 97% of the whole analysed TSCM population at day 14 and 74% and 99% at day 30. Conversely, the largest memory/effector clones detected at the same timepoints spanned from 4% to 21% of the total TCM/TEM population. These TSCM clones subsequently contracted and were not observed at 6-28 months after infusion suggesting that different clones are responsible for the early response and prolonged immune surveillance. After 6 months post-infusion, when the IS profile of circulating CAR-T cells was compared with selected populations from the infused product, only 1.8%-6.1% of long term clones were derived from the infused TCM population, despite this accounting for the majority of IS in the products (72.7/75.8% of clones). Conversely, in both patients the majority of IS associated with long term persistence (90.7%/55.5%) were derived from the TSCM compartment. Our preliminary results raise two hypotheses on the clonal dynamics of infused CAR T cells: 1) There is an early expansion of a defined group of clones during the first 30 days, which is more pronounced in the precursor TSCM compartment. These early waves do not seem to be originated from clones that have substantially expanded in vitro such that their clonal mark could not be retained in the batch of the infused cell product analysed. Further, these clones rapidly disappear after the early anti-tumour response. 2) The long-term population of CAR-T cells seem to have a higher relation with TSCM clones that have expanded in vitro before infusion, supporting the notion that such cells in the infused batch would be the one primarily responsible for the preservation of circulating CAR-T cells in the treated patients. This study suggests for the first time that anti-leukemic response occurs along rapid waves of clonal succession and that TSCM are primarily responsible for the long-term survival of CAR-T cells. Disclosures Ghorashian: novartis: Honoraria; UCLB: Patents &amp; Royalties: UCLB; Celgene: Honoraria. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Thrasher:4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Amrolia:UCLB: Patents &amp; Royalties.

Clinical silence of pulmonary lymphoepithelioma-like carcinoma with subcutaneous metastasis: a case report

BackgroundDissemination of lung cancer to cutaneous sites usually results in a poor prognosis. Pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare tumor, and no therapeutic strategy for it has yet been established. We present herein an extremely rare case of a long-term surviving patient with PLELC showing subcutaneous metastasis.Case presentationA 76-year-old woman was diagnosed unexpectedly as having PLELC based on a nodule on her back. After surgical resection of the primary and metastatic lesions, she has remained alive with no recurrence for over 5 years without any additional therapy.ConclusionEven in the case of PLELC with subcutaneous metastasis, surgical management may afford a prognosis of long-term survival.

Abstract 2722: The genomic landscape of high-grade serous ovarian cancer in long-term survivors

Abstract Purpose: The majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of ~30%. However, a subset of patients have an extraordinary response to treatment and ~15% survive more than ten years (long-term survivors). The Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG) aims to uncover factors that influence long-term survival of HGSC patients. Here, we investigated the genomic and immunologic determinants of exceptional survival of this deadly disease. Experimental Design: Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterised by RNA sequencing, DNA methylation profiling and immunohistochemistry. Results: A total 38 (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemo-sensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression-free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls (316 unselected HGSC patients in The Cancer Genome Atlas). Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related) and Signatures 5, 8 and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). Staining of adjacent tumor tissue revealed that RB1 loss was associated with increased numbers of PD-1+ tumor-infiltrating lymphocytes (P = 0.015) and MHC class I on tumor cells (P = 0.002). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P &amp;lt; 0.001) compared to patients with RB1 positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P &amp;lt; 0.001). Conclusions: This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations might be associated with enhanced host immune responses and long-term survival. Citation Format: Dale W. Garsed, Ahwan Pandey, Sian Fereday, Kathryn Alsop, Maartje C. Wouters, Flurina Saner, Jessica A. Beach, Katy Milne, Catherine J. Kennedy, Joy Hendley, Nadia Traficante, Celeste L. Pearce, Malcolm C. Pike, Multidisciplinary Ovarian Cancer Outcomes Group, Susan J. Ramus, Martin Köbel, Brad H. Nelson, Ellen L. Goode, Anna deFazio, David D. Bowtell. The genomic landscape of high-grade serous ovarian cancer in long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2722.

Abstract 3776: Spatially resolved immunogenomic analyses reveal diverse sub tumoral microenvironments in the context of melanoma immunotherapy

Abstract Sustained periods of apparent clinical benefit despite lack of objective response are well known in a subpopulation of advanced melanoma patients. Inter-individual heterogeneity in response of separate tumors is common, characterizing complex overall response patterns. The molecular and cellular dynamics facilitating such long-term survival and heterogeneous response is poorly understood, particularly in the era of exposure to multiple potentially active therapies. We studied an exceptional case of long-term survival in a patient with non-responding metastatic melanoma in order to characterize the clonal and microenvironmental factors active across 3 time points. We performed immunogenomic analyses of 3 metachronous tumors, including a systemic therapy-naïve mass, 67 intratumor sub-regions of a non-responding mass during PD-1 inhibitor therapy, and a post-PD-1 inhibitor mass. We profiled samples using whole exome sequencing, RNA-sequencing (RNA-seq), immunohistochemistry (IHC), and T cell receptor sequencing. Longitudinal, spatial, and cross-modal analyses were performed. Longitudinal analyses identified mutations in several genes known to be associated with targeted or immune therapy resistance affecting distinct metastases. Genomic intratumoral heterogeneity (ITH) was primarily driven by subclonal copy number alterations that showed evidence of spatially-distinct evolution which may be in response to selective pressures at the tumor margin. RNA-seq revealed an unexpectedly high degree of ITH characterized by limited group-level gene or pathway associations with physical or immune characteristics of each site. Spatially-distinct pockets of immune activation and suppression were observed throughout the PD-1 inhibitor resistant metastasis despite a largely immune-excluded phenotype seen on IHC. A specific T cell Vβ CDR3 rearrangement was identified as dominant and recurrent not only across multiple spatial points within a single tumor mass, but also across metachronous tumors spanning the patient’s disease course. Immunophenotyping of the T cell population with single-cell RNA-seq suggested repeated T-cell priming events leading to the persistence of both activated and exhausted T cells bearing the same TCR-β at any given time. Our findings highlight an unexpected level of genomic and immune heterogeneity in metastatic melanoma tumors of a long-term surviving patient. The observed degree of ITH across local tumor microenvironments reiterates the inherent limitations to identifying robust and reproducible predictive biomarkers of therapy response based on limited physical sampling of tumors. Further spatiotemporal analysis of metastatic lesions in the context of immune checkpoint blockade will be required to determine how the mechanisms driving convergent microenvironmental phenotypes may be harnessed for therapeutic gain. Citation Format: Akash Mitra, Miles C. Andrews, Whijae Roh, Mariana P. de Macedo, Alexandre Reuben, Fernando Carapeto, Feng Wang, Sangeetha M. Reddy, Khalida Wani, Christine Spencer, John Miller, Aislyn Schalck, Latasha D. Little, Donald A. Sakellariou-Thompson, Curtis Gumbs, Wen-Jen Hwu, Chantale Bernatchez, Jianhua Zhang, Patrick Hwu, Nicholas Navin, Padmanee Sharma, James P. Allison, Jennifer Wargo, Alexander J. Lazar, Philip A. Futreal. Spatially resolved immunogenomic analyses reveal diverse sub tumoral microenvironments in the context of melanoma immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3776.

Abstract A144: The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine

Abstract Melanoma (MM) cells leave primary tumors early and evolve at different sites in parallel. However, timing in human MM is unknown. Lymphatic dissemination is fast shortly after the dermal invasion, but metastases may occur late. Such delay in metastases formation suggests possible host defense mechanism, or particular nature of non-actively proliferative (dormant) MM stem cell (MSC) population. T lymphocytes may also induce tumor dormancy. The proposed mechanism involves a cytostatic mode of tumor silencing resulting from the suppression of both neoplastic cell proliferation and cell cycle progression. Significant progress in the development of novel advanced MM therapies was made. However, not all patients benefit from the therapy, developing late metastases decades after treatment ends. Moreover, using the immunotherapy-based model of MM dormancy in mouse, vaccination against MM prevented tumor growth but failed to prevent tumor cell dissemination or elimination of all MM cells. We have developed therapeutic gene modified allogenic MM vaccine (AGI-101H) that has been tested since 1997 and resulted in long-term survival of a substantial fraction of patients. Genetic modification of vaccine cells to express designer cytokine cDNA encoding Hyper-IL-6 protein (comprising of IL-6 linked with a soluble IL-6 receptor) has altered AGI-101H cells phenotype towards MSC-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). We have found significant induction of anti-MSCs response in immunized patients by a generation of functionally active cytotoxic CD8+T-cells and antibodies specific for ALDH1A1 in circulation. However, the AGI-101H mode of action is complex: besides inducing a direct anticancer response to MSC population, long-term vaccination may induce MM dormancy through T-cell mediated immunity. The above hypothesis is based on clinical observations of AHI-101H treated patient who was progression-free for many years, but injury induced metastases formation in the bruise. This patient developed skin MM in 1999. In 2002 in transit metastases appeared (resected). The patient was enrolled into AGI-101H program. In 2003 metastases in liver (resected), 2004 metastasis in ovary (resected). 11 years (2004-2015) no progression. In Nov. 2015 she had bicycle accident, trauma, and MM metastasis presenting with a bruise (resected). The treatment continues with no progression. The aim was to determine the molecular mechanisms of peripheral T-cell-mediated induction of MM dormancy induced by vaccination. Therefore, T-cell mRNA expression profiling of long-term surviving patients was carried out. PBMCs were isolated from treated patients (18), untreated MM patients (13) and healthy controls (8). Untouched T-cells were separated by magnetic beads and total RNA isolated. The transcriptome profiling was performed with Affymetrix HG U219 microarray (19,285 markers). Differential gene expression (DGE) analysis between all groups was conducted and validated with RT-qPCR and FACS. The transcriptomic results were further analyzed with Gene Set Enrichment Analysis (GSEA) tool. DGE analyses comparing AGI-101H vaccinated (AV) and nonimmunized (C) patients revealed 538 differentially expressed genes (DEGs), with 373 downregulated and 165 upregulated in AV (adj p &amp;lt; 0.05). Among these 538 markers, the expression of 14 (with |log2FC| &amp;gt; 1) is now being validated with RT-qPCR and FACS. Also, GSEA analysis revealed significant enrichment of “TNFa_signaling_via_NFkB”, “TGFb_signaling” and “G2/M_checkpoint” hallmark processes (MSigDB Hallmark gene sets) in AGI-101H vaccinated patients, that indicates considerable activation of antitumor response and prevention of conversion of MM dormanT-cells into proliferative metastases. Transcriptome profiling of peripheral T-cells in long-surviving MM patients immunized with AGI-101H suggests activation of the molecular mechanism that sustains MM cells in the non-actively proliferative dormant state. Citation Format: Andrzej A. Mackiewicz, Partycja Czerwinska, Marcin Rucinski, Katarzyna Gryska, Iga Grzadzielewska, Anna Jaworska, Jacek Mackiewicz. The transcriptomic profile of peripheral T-cells that maintain dormant state of melanoma cells in patients treated with allogenic melanoma vaccine [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A144.

Prolonged cell persistence with enhanced multipotency and rapid angiogenesis of hypoxia pre-conditioned stem cells encapsulated in marine-inspired adhesive and immiscible liquid micro-droplets

Stem cell therapies are emerging regenerative treatments for ischemic and chronic diseases. Although high cell retention and prompt angiogenesis are prerequisites to improving efficacy, advancements have not yet been developed. Here, we proposed long-term surviving and angiogenesis-inducing stem cell with high cell retention thanks to fluid immiscible liquid micro-droplets bio-inspired by a glue modality ‘complex coacervate’ found in the sandcastle worm. Formed by the Coulombic force between polycationic MAP and polyanionic hyaluronic acid, the exploited coacervate micro-droplets enabled the encapsulation of stem cells. The underwater adhesiveness facilitated integrating the encapsulated stem cells onto various surfaces with impressive cell retention after facile injection. Stem cells encapsulated in the coacervate platform formed cell clusters capable of pre-adjusting to hypoxia by expressing hypoxia-inducible factor 1α (HIF-1α), increasing viability and reducing apoptosis under hypoxia and ischemia as well as normoxia. Interestingly, multipotent and angiogenic factors were significantly enhanced by HIF-1α expression. In the in vivo evaluation, the coacervate platform showed impressive angiogenesis with biocompatibility and long-term cell retention capacity with sustainable release as protein factories. Therefore, the proposed MAP-based water-immiscible, injectable, sticky, and bioactive 3D coacervate micro-droplets offers a promising tool for chronic diseases in body fluid-rich environments. Statement of SignificanceHigh cell retention, long-term survival, and rapid angiogenesis are prerequisites of successful stem cell therapy. However, no previous advancements have simultaneously satisfied all of these requirements. In this work, we clearly developed a novel, revolutionary stem cell carrier platform with underwater adhesiveness from a mussel-derived glue protein and water immiscibility from a sandcastle-worm-inspired glue modality via ‘complex coacervation’. To the best of our knowledge, no report has emerged employing coacervate as a stem cell therapeutic platform. This fluid-immiscible, injectable, sticky, and bioactive 3-dimensional stem cell micro-droplets demonstrated the excellent stem cell retention and viability under hypoxia environments and enhanced multipotent and angiogenic effects with minimal immune response.