The list of immunotherapeutic agents that show positive results in renal cell carcinoma (RCC) continues to grow, with researchers exploring different combinations, including the addition of vaccines, in this arena. In particular, findings from a phase I study of alpha-1, 3 galactosyltransferase-expressing allogeneic RCC immunotherapy are encouraging, according to Hans J. Hammers, MD, PhD, associate professor, University of Texas Southwestern Medical Center, Dallas.

In the small study, patients with metastatic RCC were treated with HyperAcute Renal (HAR) immunotherapy. This vaccine consists of 2 allogeneic renal cancer cell lines, which have been genetically modified to express alpha-1,3-galactosyltransferase, a carbohydrate that humans have an inherent pre-existing immunity. By targeting alpha-1,3-galactosyltransferase, the immune system can begin to identify antigens expressed by the patient&rsquo;s own tumor cells, says Hammers. &ldquo;The idea is that cell lines that have been modified in this particular manner can jump-start an immune response.&rdquo;

In this phase I dose escalation study, patients received a weekly injection of HAR (150 x 106 cells/300 x 106 cells intradermally) for 4 weeks, followed by biweekly injections for the next 10 weeks. Results showed tolerability and suggested that RCC is an appropriate tumor type regarding combination immunotherapy including HAR.

Hammers discussed the role of vaccines, potential next steps, and the future of immunotherapy in RCC.

Could you provide an overview of the phase I study on alpha-1,3- galactosyltransferase?

The treatment is an allogeneic renal cell carcinoma vaccine, meaning these are cell lines derived from different patients than the patient being treated. They have been genetically altered to express a certain sugar molecule, alpha-1,3-galac- tosyltransferase, that we see, for example, in pigs. In fact, most of the antibodies that we have circulating in our blood, actually are formed against alpha-1,3-galactosyltransferase. So, if you either transplant an organ from a pig or inject cancer cells that have been spiked with alpha-1,3-galac- tosyltransferase, they would be rapidly destroyed, leading to active inflammation.

These were 2 different cell lines, and the patient received subcutaneous injections in essentially 2 different doses. Drug development for vaccines is much less complicated than it is for targeted agents. The trial was designed in a way that patients initially received injections once a week for 1 month and then every 2 weeks thereafter. It was also designed in a way that patients were not allowed to have any concurrent therapy for the first 2 months, but then could combine it with any standard-of-care therapy thereafter, such as tyrosine, but also immune checkpoint inhibitors as they became available.

What we presented at the GU [Genitourinary Cancers] Symposium are safety data on the 15 patients that we have more details on. And overall, there were no big surprisesit is very well tolerated, with mostly local reactions, and in the end, we have a dose that can move forward into phase II studies.

Are there any next steps for future clinical trials?

We know it&rsquo;s safe to do, but we still have to wait for some dataa subset of patients was actually treated with nivolumab (Opdivo) after 2 months. We stand to see what kind of impact it has&mdash;if there is some sort of clinical activity that might be enhanced. But I do think it deserves at least testing in combination with nivolumab, but to some degree it&rsquo;s also a commercial decision by the company, if there is enough promising data. The goal of this phase I trial is achieved &mdash; we have a safe dose. Now there are several other aspects that play into this.

What do you want community oncologists to take away from this study?

I think the main message is that maybe it is time to revisit vaccines. We&rsquo;ve spent a lot of time on the ridiculed recent trials that have been negative. There was a very large phase III trial that was negative, but vaccines have not really been tested in the context of immune checkpoint inhibition, and to some degree, that&rsquo;s a natural marriage. We do know that vaccines will face challenges, in particular with what we&rsquo;ve learned about immune checkpoints. So, combining vaccines with immune checkpoint makes a lot of sense.

There will be different approachesallogeneic vaccines can be taken off the shelf very easily, but may not be as individualized as if you were to focus on analyzing a patient&rsquo;s tumor and created tailor-made vaccines for the individual patients. But, that&rsquo;s a more advanced approach. I think we will see several of these concepts tested in the clinic, I don&rsquo;t know if they are going to work, but I do think that vaccines need to be visited and that&rsquo;s what we will see.

What is the current role of immunotherapy in RCC, and where do you see it in the future?

I think it is a really exciting time in kidney cancer. We have had an immune checkpoint inhibitor nivolumab&mdash;approved since November 2015; nobody is unfamiliar with these agents anymore. The question really is, where do we go from here? It is an agent, if used alone, that has moderate activity. Response rates are around 20%; these can be durable responses but they are simply not enough if you want to treat patients effectively with immunotherapy. I think there are several different approaches&mdash;one is to combine it with other agents such as targeted agents.

One marriage that I think is quite natural is combining VEGF [vascular endothelial growth factor] inhibitors with nivolumab. What we&rsquo;ve learned is that it is probably better to use selective agents such as axitinib (Inlyta), rather than sunitinib (Sutent) or pazopanib (Votrient), and those combinations are moving forward in phase III trials. We have some data emerging from smaller phase II trials with very encouraging activity and very high response rates, as presented at ESMO [European Society for Medical Oncology] in the combination of axitinib and pembrolizumab (Keytruda).

The questions are going to be how durable are those responses, what&rsquo;s the quality of response, and do we really see true synergy in an immunological perspective? Another combination, that is a competitor to some degree, is the combination of programmed cell death protein-1 inhibitors and cytotoxic T-lymphocyte-associated protein 4 inhibitors. There is a very large phase III trial that we are looking forward to seeing data on as early as this year, which compared the combination of nivolumab and ipilimumab (Yervoy) versus sunitinib. This could also be a changing standard of care. We know with this particular combination, the response rates are highwe expect to see response rates around 40%&mdash; and they tend to be quite durable with a median duration of almost 2 years.

One data set that I am very curious about is the combination of atezolizumab (Tecentriq) and bevacizumab (Avastin). I think this might give us the first hint of synergy between VEGF inhibitors and PD-L1 [programmed death-ligand 1] inhibitorsand how durable might those responses be. I think it is a very important trial, and we will see if that is going to guide us in a certain direction.

Hammers HJ, Drake CG, Zakharia Y, et al. A phase 1 study of alpha-1,3- galactosyltransferase-expressing allogeneic renal cell carcinoma immunotherapy in patients with metastatic renal cell cancer. J Clin Oncol. 2017;35(suppl 6S; abstr 528).

Articles

Clinical Articles

Hans J. Hammers, MD, PhD, discusses&nbsp;findings from a phase I study of alpha-1, 3 galactosyltransferase-expressing allogeneic RCC immunotherapy.

Hammers Recommends Revisiting Vaccines in Renal Cell Carcinoma

As clinicians have gained an improved understanding of the biology of soft-tissue sarcoma (STS) malignancies, the ability to better distinguish and identify STS subtypes has extended the hope that exploiting the molecular characteristics of each subtype will lead to the emergence of targeted treatment options. For patients who experience STS in the extremities, radiation therapy is used to facilitate limb- and function-preserving surgeries while maintaining 5-year local control above 85%. For STS originating from nonextremity sites, in contrast, the rate of 5-year local control is only about 50%. What follows is a review of several studies that have evaluated the role of adjuvant or neoadjuvant chemotherapy for the treatment of localized STS.

In the United States, approximately 15,000 new cases of sarcoma of the bone and soft tissues are diagnosed annually, according to data from the Surveillance, Epidemiology, and End Results database.

The incidence of sarcoma is within the same order of magnitiude as other malignancies such as myeloma, cervical, glioma, and esophageal, but more frequent than Hodgkin lymphoma or testicular cancer. The disease affects all age groups, and 15% are found in children aged 15 years and younger, and 40% in those 55 years and older.

These tumors encompass a variety of histological subtypes. They appear to arise from the supporting connective tissues, including smooth and skeletal muscles, tendons, fat, brous tissue, synovial tissue, vessels, and nerves.

Approximately 60% of soft-tissue sarcoma (STS) malignancies arise in the extremities, most frequently in the lower extremity.

Despite STS being a relatively uncommon malignancy, research investigating the genetic and molecular characteristics of its subtypes has enhanced our understanding of cancer biology overall, and it has led to the development of targeted therapeutics with applicability to a broad range of other malignancies.

For example, the pioneering use of neoadjuvant chemotherapy was first realized in the treatment of pediatric osteosarcoma

; the first demonstration of a chromosomal translocation manifesting as a pathogenic fusion gene and protein product in solid tumors

; and the effectiveness of agents like imatinib mesylate (Gleevec) in treating gastrointestinal stromal tumors (GISTs) was first learned during research conducted in sarcoma. Here, we discuss the state of the science for the treatment of sarcoma.

Historically, the standard of therapy for STS affecting the extremities was amputation. Function-preserving limb-conserving resection with negative microscopic margins is now considered the standard of care today. The goal of neoadjuvant therapy is to improve both local anddepending on modality&mdash;distant control. Risk of recurrence will vary based on histologic subtype, tumor grade, and size. The potential benefit from neoadjuvant therapies, however, must be weighed against the risk of disease progression due to delayed resection during treatment, as well as an increased risk of postoperative complications.

Surgery, Radiation Therapy, and Chemotherapy Research Conducted by Cooperative Groups Cooperative groups and several institutions of research have been the vanguard of sarcoma research for the past 30 years, particularly in the clinical and translational arena. Notable advancements in the treatment of adult STS, from 1970 to 2000, include improvements in pathologic definition through identification of specific translocations and growing improvements in immunohistochemistry; new imaging modalities, including positron emission tomography (PET) scans; refinements in prognosis and staging; advances in the precision of radiotherapy techniques; surgical advances in functional preservation; and the emergence of doxorubicin and ifosfamide as active drugs, as well as the development of more effective systemic agents.

Superior local control with the addition of adjuvant radiation therapy (RT) after limb-sparing surgery in STS was demonstrated in 2 randomized controlled trials. The first was reported by Yang and colleagues, from the National Cancer Institute (NCI) of Canada, in which the investigators assessed the impact of postoperative external-beam radiation therapy on local recurrence (LR), overall survival (OS), and quality of life after limb-sparing resection of extremity sarcomas.

Patients with high-grade STS received concurrent doxorubicin and cyclophosphamide with external beam radiation therapy (EBRT). For patients with high-grade tumors, 10-year local control (LC) was significantly higher for those treated with RT versus those who were not (100% vs 78%), but there was no difference in 10-year distant metastasis-free survival (DMFS) or overall survival (OS). For patients with low-grade tumors, LC was similarly statistically higher for those treated with RT, and again, there was no difference in DMFS or OS.

In the second study, Pisters and colleagues conducted a trial in which patients with high- and low-grade STS who had a complete resection were randomized while in surgery to receive RT in the form of an Iridium-192 brachytherapy implant (42 to 45 Gray [Gy]) over the next 4 to 6 days, or no RT. For patients with high-grade tumors, 5-year LC favored the RT arm (89% vs 66%, respectively), but there was no OS difference.

For patients with low-grade tumors, neither LC nor OS were significantly impacted by RT.

Interestingly, the NCI Canada trial randomized patients with extremity STS to preoperative RT (50 Gy in 25 fractions [fx] 16 to 20 Gy boost treatment for positive surgical margins) versus postoperative RT (66 Gy in 33 fx). With respect to radiation treatment field design, the initial treatment field used 5-cm proximal and distal margins beyond the tumor (preoperative RT) or tissues at risk (postoperative RT). Additionally, the boost was designed with 2-cm proximal and distal margins. The primary endpoint of this study was the rate of major wound complications within 120 days of surgery.

The trial closed after accruing 190 of the planned 266 patients because of a significantly higher rate of wound complications with preoperative RT (35%) versus postoperative RT (17%), with the highest rates of complications in the upper leg (45%). In this study, function at 6-weeks was better with postoperative RT.

At a median follow-up of 6.9 years, there was no significant difference in LC (93% for preoperative RT vs 92% for postoperative RT), recurrence-free survival (RFS; 58% vs 59%), or OS (73% vs 67%).

With respect to significant adverse predictors for outcome, positive surgical margins were significant for LC; large tumor size and high grade were signficant for RFS and OS.

With respect to long-term toxicity, postoperative RT was associated with worse fibrosis and joint stiffness compared with preoperative RT.

Given that the efficacies of preoperative and post- operative RT are deemed equivalent, decisions regarding which approach to use are driven by the difference in toxicity profiles. Preoperative RT is associated with a higher rate of wound complications, which are typically reversible, whereas post-operative RT is associated with higher rates of limb edema, brosis, and stiffness, which are typically not reversible.

The role of adjuvant or neoadjuvant chemotherapy in localized STS has been investigated in several studies. Specifically, the National Cancer Institute performed a trial that randomized 43 patients with high-grade extremity STS to amputation at the joint proximal to the tumor versus limb-sparing resection and postoperative RT. Randomization favored limb sparing (2:1). The RT dose was 45 to 50 Gy, plus a boost dose to 60 to 70 Gy. RT was delivered with concurrent doxorubicin, cyclophosphamide, and high-dose methotrexate. With respect to margin status, 4 of 27 patients in the RT group had positive margins. There was increased LR with limb-sparing and adjuvant RT compared with amputation (20% vs 0%, respectively), but the impressive limb preservation rate established limited surgery and RT as the new standard of care.

There were no significant differences in 5-year DFS (78% for the amputation group vs 71% for the limb-sparing and RT group) or in OS (88% vs 83%, respectively). In a separate trial, Rosenberg and colleagues reported in the same publication, 65 patients with extremity STS were randomized to receive surgery alone (either limb- sparing or amputation) versus surgery and adjuvant doxorubicin, cyclophosphamide, and high-dose methotrexate. Compared with patients who did not receive chemotherapy, those who received adjuvant chemotherapy enjoyed both improved DFS (92% vs 60%; P = .0008) and OS (95% vs 74%; P = .004).

A study from Massachusetts investigated neoadjuvant chemoradiation therapy (CRT) for large STS included 48 patients with >8 cm STS of the extremities. Patients underwent interdigitated sequential CRT as follows: mesna/doxorubicin/ifos- famide/dacarbazine (MAID) followed by RT (22 Gy in 11 fx), followed by MAID, followed by RT (22 Gy in 11 fx), followed by MAID. The researchers from Harvard University found that if surgical margins were positive, patients received an additional 16 Gy boost postoperatively. Five-year LC was 92%, DFS was 86%, and OS was 44%. Compared with historical controls, there was a significant decrease in distant metastases. and significant increases in DFS and OS. There was a 29% wound complication rate and 2% treatment-related deaths.

The findings of this study led to a multi-institutional cooperative group follow up trial, Radiation Therapy Oncology Group (RTOG) 9514. This was a phase II trial of 64 patients with >8 cm grade 2 or 3 STS of the extremity or torso. Investigators found that 44% of patients had malignant brous histiocytoma, 13% had leiomyosarcoma, and 88% had STS of the extremity. Patients underwent interdigitating chemoradiation, which was defined as 1 cycle of MAID, followed by RT (22 Gy in 11 fx), followed by a second cycle of MAID, a second course of RT (22 Gy in 11 fx), and then a third cycle of MAID. Patients then underwent resection followed by fourth and fth cycles of adjuvant MAID, preceded by a 14 Gy postoperative boost in the setting of positive surgical margins.

The researchers reported that 91% achieved R0 re- sections and 59% of patients received the full chemotherapy course. The 3-year locoregional failure was 18% (if amputation was considered a failure; 10%, if not). The 3-year DFS was 57%, distant DFS was 64%, OS was 75%, and there was a 92% amputation-free rate. There was a 5% rate of treatment-related deaths (including 2 secondary acute myeloid leukemias), and 84% of patients had grade 4 toxicity (mostly hematologic). The authors concluded that the regimen was effective, but the substantial toxicity encountered has precluded widespread adoption of this regimen.

Of note, the RTOG 9514 trial used a more intense version of MAID (employing a higher dose of cyclophosphamide) than was used in the preceding single institution study; it also employed very generous radiation fields, with 9 cm proximal and distal margins on tumors that were all >8 cm. Both of these factors were likely contributors to the observed toxicity.

The European Organization for Research and Treatment of Cancer (EORTC) undertook 2 important trials to assess the role of neoadjuvant chemotherapy with unresolved results. EORTC/Soft Tissue and Bone Sarcoma Group 62871 was a randomized phase II trial that enrolled 134 patients with STS >8 cm or grade 2 or 3. Patients were randomized to surgery alone or to surgery plus neoadjuvant doxorubicin/ifosfamide. Postoperative RT was given for marginal surgery, positive surgical margins, or LR. There were no differences in 5-year DFS (52% vs 56%) or OS (64% vs 65%), but the study was closed early due to poor accrual, and thus it was not sufficiently powered to detect a difference.

In addition, EORTC 62961 randomized 341 patients with >5 cm, grade 2 or 3, deep and extracompartmental STS to receive neoadjuvant etoposide/ifosfamide/doxorubicin (EIA) versus neoadjuvant EIA plus hyperthermia. With respect to this study, hyperthermia resulted in improved median (3.8 vs 2 years; P = .044) and median DFS (2.6 vs 1.4 years; P =.040).

The Sarcoma Meta-Analysis Collaboration published an analysis in 2008 that included 18 randomized trials with 1953 patients treated with (primarily) adjuvant or neoadjuvant chemotherapy.

Findings included statistically significant absolute reductions of 4% for LR and 9% for distant recurrence, and an absolute improvement of 6% for survival attributable to chemotherapy. However, the fact that this update did not include the largest negative trial renders the results less conclusive.

Perhaps the most dramatic advance for the treatment of sarcomas over the past 2 decades comes from basic research in GISTs, which are the most common mesenchymal tumors in the gastrointestinal tract. Historically, these tumors had been treated by surgery alone, were refractory to systemic therapy, and were associated with a poor prognosis. In 1998, Hirota and colleagues reported that molecular analyses on GISTs identified constitutively active mutations in c-kit, which encodes a tyrosine kinase receptor.

The tyrosine kinase inhibitor imatinib mesylate (Gleevec), which was developed to inhibit BCR-ABL in chronic myelogenous leukemia, is also a potent inhibitor of KIT.

In a preclinical cell culture model, imatinib mesylate showed activity against GIST.

Therefore, Demetri and colleagues performed a multi-institutional clinical trial using imatinib mesylate in patients with metastatic disease and observed an objective response in more than half of the patients.

A randomized phase III double-blind, placebo-controlled trial of adjuvant imatinib mesylate after surgery, versus surgery alone, for GISTs that were at least 3 cm in size and positive for KIT protein expression showed improved RFS at 1 year (98% vs 83%; P <.0001).

RTOG 0132/American College of Radiology Imaging Network 6665 was among the prospective studies to investigate the effectiveness of imatinib mesylate on GIST and to contribute data to the management guidelines for GIST. Different from other studies, this multi-institutional prospective phase II study was designed to assess the clinical outcomes and tolerability of imatinib (600 mg/day) given for 8 to 12 weeks either as preoperative therapy prior to a planned resection of intermediate- to high-risk primary GIST or as a cytoreductive agent prior to planned resection of metastatic and/or recurrent GIST. All patients planned to continue postoperative imatinib (600 mg/day) therapy for an extended post-operative period of 2 years. The short-term analysis of the patients enrolled into this clinical trial showed encouraging results of outcomes with a low rate of toxicity.

The long-term analysis of results from this study suggests that a high percentage of patients developed progressive disease after discontinuation of 2-year maintenance imatinib therapy following surgery.

Based on these and other studies, imatinib mesylate is now a standard treatment for many patients with GIST.

Combining Immunotherapy With Radiation Therapy for Sarcoma

Finkelstein and colleagues have explored combin- ing radiation therapy and immunotherapy for sarcoma.

His group determined the effect of combination of intratumoral administration of dendritic cells (DCs) and fractionated EBRT on tumor-specific immune responses in patients with STS. Targeted accrual was achieved with 17 patients with large (>5 cm) high-grade STS enrolled in the phase I/ II study. Patients were treated in the neoadjuvant, preoperative setting with 50.4 Gy of EBRT, delivered in 28 fx, 5 days per week, combined with intratumoral injection of 107 DCs followed by complete surgical resection. DCs were injected on the second, third, and fourth Fridays of the treatment cycle to maximize time before re-exposure to radiation field. Detailed clinical outcome evaluation and immunological assessments were performed. With respect to outcomes, the treatment was found to be well tolerated. Of note, no patient had a tumor-specific immune response before combined EBRT/ DC therapy; following therapy, 9 patients (52.9%) developed tumor-specific immune responses, which lasted from range of 11 to 42 weeks.

With respect to clinical outcomes, 12 of 17 patients (70.6%) were progression-free after 1 year. Treatment caused a dramatic accumulation of thymus cells in the tumor. The presence of CD4+ T cells in the tumor was positively correlated with tumor-specific immune responses that developed following combined therapy. Accumulation of myeloid-derived suppressor cells but not regulatory T cells was negatively correlated with the development of tumor-specific immune responses. Experiments with 111Indium labeled DCs demonstrated that these antigen-presenting cells need at least 48 hours to start migrating from the tumor site. Thus, these data suggest the combination of intratumoral DC administration with EBRT was safe, and it resulted in induction of antitumor immune responses. These results are promising and have generated further testing to assess clinical efficacy more broadly.

In conclusion, conducting meaningful trials in sarcoma has been increasingly challenging due to funding constraints coupled with both the rarity of the disease and the large variety of sarcoma histologic subtypes. As such, the study of local management with surgery and/or RT is additionally challenging given the variety of anatomic sites where sarcomas occur. Indeed, each anatomic site is associated with specific and varied potential toxicities related to resection and RT. Despite these factors, the aforementioned work continues to advance the state of science for this important area. We must continue to be vigilant in a such low-incidence disease such as sarcoma as breakthrough work will pave the foundations for continued work in rare disease states.

Ferrari A, Sultan I, Huang TT, et al. Soft tissue sarcoma across the age spectrum: a population-based study from the Surveillance Epidemiology and End Results Database. Pediatr Blood Cancer. 2011;57(6):943-949. doi: 10.1002/pbc.23252.

Bui MM, Han G, Acs G, et al. Connexin 43 is a potential prognostic biomarker for ewing sarcoma/primitive neuroectodermal tumor. Sarcoma. 2011;2011:971050. doi: 10.1155/2011/971050.

Jaffe N, Murray J, Traggis D, et al. Multidisciplinary treatment for childhood sarcoma. Am J Surg. 1977;133(4):405-413.

Kaneko Y, Kobayashi H, Handa M, et al. EWS-ERG fusion transcript produced by chromosomal insertion in a Ewing sarcoma. Genes Chromosomes Cancer. 1997;18(3):228-231.

Yang JC, Chang AE, Baker AR, et al. Randomized prospective study of the bene t of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity. J Clin Oncol. 1998;16(1):197-203.

Pisters PW, Harrison LB, Leung DH, et al. Long-term results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma. J Clin Oncol. 1996;14(3):859-868.

O&rsquo;Sullivan B, Davis AM, Turcotte R, et al. Preoperative versus postoperative radiotherapy in soft-tissue sarcoma of the limbs: a randomised trial. Lancet. 2002;359(9325):2235-2241.

O&rsquo;Sullivan B, Davis A, Turcotte R, et al. Five-year results of a randomized phase III trial of pre-operative vs post-operative radiotherapy in extremity soft tissue sarcoma. [abstract] J Clin Oncol. 2004;22:s819

Davis AM, O&rsquo;Sullivan B, Turcotte R, et al; Canadian Sarcoma Group; NCI Canada Clinical Trial Group Randomized Trial. Late radiation morbidity following randomization to preoperative versus postoperative radiotherapy in extremity soft tissue sarcoma. Radiother Oncol. 2005;75(1):48-53.

Rosenberg SA, Tepper J, Glatstein E, et al. The treatment of soft-tissue sarcomas of the extremities: prospective randomized evaluations of (1) limb- sparing surgery plus radiation therapy compared with amputation and (2) the role of adjuvant chemotherapy. Ann Surg. 1982;196(3):305-315.

DeLaney TF, Spiro IJ, Suit HD, et al. Neoadjuvant chemotherapy and radiotherapy for large extremity soft-tissue sarcomas. Int J Radiat Oncol Biol Phys. 2003;56(4):1117-1127.

Kraybill WG, Harris J, Spiro IJ, et al; Radiation Therapy Oncology Group Trial 9514. Phase II study of neoadjuvant chemotherapy and radiation therapy in the management of high-risk, high-grade, soft tissue sarcomas of the extremities and body wall: Radiation Therapy Oncology Group Trial 9514. J Clin Oncol. 2006;24(4):619-625.

Gortzak E, Azzarelli A, Buesa J, et al; E.O.R.T.C. Soft Tissue Bone Sarcoma Group and the National Cancer Institute of Canada Clinical Trials Group/ Canadian Sarcoma Group. A randomised phase II study on neo-adjuvant chemotherapy for &lsquo;high-risk&rsquo; adult soft-tissue sarcoma. Eur J Cancer. 2001;37(9):1096 -1103.

Angele MK, Albertsmeier M, Prix NJ, et al. E ectiveness of regional hyperthermia with chemotherapy for high-risk retroperitoneal and abdominal soft-tissue sarcoma after complete surgical resection: a sub-group analysis of a randomized phase-III multicenter study. Ann Surg. 2014; 260(5):749-754; discussion 754-756. doi: 10.1097/SLA.0000000000000978.

Pervaiz N, Colterjohn N, Farrokhyar F, et al. A systematic meta-analysis of randomized controlled trials of adjuvant chemotherapy for localized resectable soft-tissue sarcoma. Cancer. 2008;113(3):573-581. doi: 10.1002/cncr.23592.

Bramwell V, Rouesse J, Steward W, et al. Adjuvant CYVADIC chemotherapy for adult soft tissue sarcoma--reduced local recurrence but no improvement in survival: a study of the European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group. J Clin Oncol. 1994;12(6):1137-1149.

Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998;279(5350):577-580.

Heinrich MC, Gri th DJ, Druker BJ, et al. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood. 2000;96(3):925-932.

Tuveson DA, Willis NA, Jacks T, et al. STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications. Oncogene. 2001;20(36):5054-5058.

Demetri GD, von Mehren M, Blanke CD, et al. E cacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med. 2002;347(7):472-480.

Dematteo RP, Ballman KV, Antonescu CR, et al; American College of Surgeons Oncology Group (ACOSOG) Intergroup Adjuvant GIST Study Team. Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebo-controlled trial. Lancet.2009;373(9669):1097-1104. doi: 10.1016/S0140-6736(09)60500-6.

Eisenberg BL, Harris J, Blanke CD, et al. Phase II trial of neoadjuvant/adjuvant imatinib mesylate (IM) for advanced primary and metastatic/recurrent operable gastrointestinal stromal tumor (GIST): early results of RTOG 0132/ ACRIN 6665. J Surg Oncol. 2009;99(1):42-47. doi: 10.1002/jso.21160.

Wang D, Zhang Q, Blanke CD, et al. Phase II trial of neoadjuvant/adjuvant imatinib mesylate for advanced primary and metastatic/recurrent operable gastrointestinal stromal tumors: long-term follow-up results of Radiation Therapy Oncology Group 0132. Ann Surg Oncol. 2012; 19(4):1074-1080. doi: 10.1245/s10434 - 011-2190 - 5.

Finkelstein SE, Iclozan C, Bui MM, et al. Combination of external beam radiotherapy (EBRT) with intratumoral injection of dendritic cells as neo- adjuvant treatment of high-risk soft tissue sarcoma patients. Int J Radiat Oncol Biol Phys. 2012;82(2):924-932. doi: 10.1016/j.ijrobp.2010.12.068.

Researchers offer a review of the role of adjuvant or neoadjuvant chemotherapy, targeted therapy, and immunotherapy in localized soft tissue sarcoma.&nbsp;

Radiation Therapy, Chemotherapy, Targeted Therapy, and Possibly Immunotherapy in Localized Sarcoma

This study evaluates low-dose radiotherapy in combination with docetaxel and cetuximab in patients with recurrent, unresectable squamous cell carcinoma of the head and neck initially treated with definitive chemoradiation therapy.

In this phase II study, cetuximab 400 mg/m2 was given in the usual loading dose only on day 1 of week 1 and then, subsequently, in 250 mg/m2 weekly doses on day 1 of each following week (weeks 2-7). Docetaxel 20 mg/m2 was administered weekly on day 1 from weeks 2-7. Radiotherapy consisted of 0.5 Gy/fraction twice a day at least 6 hours apart on days 2 and 3 of weeks 2-7 for a total dose of 12 Gy.

Nine patients were screened, 5 patients were enrolled, and 4 patients were treated between October 2013 and February 2015. After completion of salvage treatment per protocol, at 1-month follow-up, partial response was seen in 2 patients (50%), stable disease in 1 patient (25%), and progressive disease in 1 patient (25%). By 6 months, all patients had progressive disease, 1 patient (25%) completed a full course of re-irradiation, and 1 patient (25%) received further chemotherapy for progression. Treatment-related morbidity was limited to difuse grade 1 skin toxicity reported in all patients. The Data Safety and Monitoring Board suggested closing this protocol as clinical response approached the prede ned futility endpoint for complete response.

Low-dose radiotherapy in addition to docetaxel and cetuximab was well tolerated, although treatment effects were transient. More durable treatment options may exist for definitive treatment; however, this protocol may offer palliation to select patients.

Local/regional failure after definitive radiotherapy (RT) remains a significant problem, particularly in advanced-stage head and neck cancer.

Approximately 50% to 60% of patients who fail initial definitive treatment will die as a direct consequence of locally or regionally recurrent disease.

Surgical salvage is sometimes successful, but it is not always feasible due to disease extent, location, or patient comorbidities.

For these patients, systemic therapy, reirradiation, and supportive care may be the only remaining options.

Historically, prior to when reirradiation was not yet commonly used, systemic therapy alone was considered the remaining palliative treatment option for inoperable patients with recurrent squamous cell carcinoma of the head and neck (SCCHN). Use of platinum-based chemotherapy has been associated with response rates of less than 50% and a median overall survival (OS) of 6 to 8 months.

More recently, cetuximab therapy has demonstrated improved response rates and survival in platinum-refractory SCCHN compared with historical controls.

In a study of 442 patients randomized to receive a platinum-based chemotherapy and fluorouracil with or without cetuximab, the addition of cetuximab significantly prolonged the median OS to 10.1 versus 7.4 months in the control arm.

Furthermore, the same authors reviewed 3 separate phase II trials in patients with recurrent or metastatic SCCHN who progressed on platinum-based therapies, and found that the addition of cetuximab led to additional improvements in response rates from 10% to 13%.

The authors concluded that cetuximab may prolong survival in patients with SCCHN who fail first-line therapy.

Reirradiation of the head and neck may be fraught with difficulties in relation to concerns regarding normal tissue tolerance and, sometimes, tumor radioresistance.

More recently, preclinical and clinical data have shown that reirradiation is feasible and may provide improved disease control, translating into superior survival in subsets of patients; that is, however, in the setting of a potentially high toxicity profile.

Various types of RT delivery, such as con- ventionally fractionated therapy, hyperfractionation, brachytherapy, and stereotactic body RT, have been attempted with and without concurrent chemotherapy in this group of patients. In appropriately selected patients who are able to undergo reirradiation, there is usually an improved disease response compared with systemic therapy alone; however, there also continues to be a notable risk of treatment-related morbidity with such combination therapy.

In contrast to the paucity of treatment options for patients in the loco-regionally recurrent and unresectable setting, there have been numerous developments in the primary treatment of SCCHN. Low-dose fractionated RT (LDFRT) is a novel approach that has been utilized in the treatment of loco-regionally very advanced SCCHN as a means of decreasing the toxicity of reirradiation. LDFRT resulted in more effective cell-kill than was predicted by the linear quadratic model in preclinical studies, possibly through both the hyperradiation sensitivity (HRS) phenomenon and/or through potentiating the effect of chemotherapy.

Early work by groups such as Joiner and colleagues showed an initial phase of HRS in head and neck cell lines that were exposed to doses less than 1 Gy, specifically around 0.5 Gy.

In this low-dose region, there exists a paradoxical increase in tumor cell kill compared with higher doses. It is hypothesized that HRS does not cause the initiation of DNA repair mechanisms that would otherwise arrest these cells for repair in the second gap (G2) phase. As such, these damaged cells ultimately undergo cellular death.

The use of LDFRT along with chemotherapy has also demonstrated a chemo-potentiating role. In vitro data initially revealed that LDFRT potentiates the effects of the paclitaxel in both wild type and mutant p53 head and neck tumor cell lines.

Furthermore, LDFRT in both cell lines caused an up-regulation in the pro-apoptotic protein, BAX, and a down-regulation in the anti-apoptotic proteins BCL-2 and NF-&kappa;B. The combined effects of LDFRT and docetaxel therapy on the growth of SCCHN were also evaluated in the nude mouse model to elicit the HRS phenomenon present in G2/mitosis (M) cell cycle.

Combination therapy showed a significant improvement in both tumor regression and local control. Additionally, molecular analysis of resected tumor specimens demonstrated that BAX levels were elevated with concomitant increase in cytochrome c oxidase release to the cytosol in the LDFRT and docetaxel group. These findings strongly suggest that LDFRT can be used in combination with docetaxel to potentiate the effects of docetaxel on tumor regression through an apoptotic mode of death. As such, the G2/M cell cycle arrest by docetaxel appears to be an important component of the enhanced apoptotic effect of LDFRT + docetaxel combined treatment.

The aforementioned in vitro and in vivo studies also led to a phase II clinical trial. Gleason and colleagues evaluated 39 patients with loco-regionally advanced SCCHN who were treated with induction paclitaxel, carboplatin, and LDFRT every 21 days for 2 cycles.

RT consisted of 80 centigray (cGy) twice a day on days 1 and 2 to a total dose of 640 cGy. Initial results were promising and showed a response rate of 90% at the primary disease site and 69% at the nodal site.

Five-year follow-up was similar to historical controls, showing an OS of 62% and progression-free survival (PFS) of 58%. In this study, treatment-related toxicity was minimal.

Our novel approach to treat recurrent SCCHN was based on the following 5 hypotheses, extrapolated from the aforementioned data: 1) LDFRT would be utilized to sensitize chemotherapy, 2) low-dose HRS response would be significantly enhanced in docetaxel-induced G2/M cell cycle arrest, 3) LDFRT would render an enhanced BAX-activationmediated mode of apoptotic cell death, 4) cetuximab would help arrest the cells in the inactive, noncycling G1/ G0 state leading to a p21-mediated mode of cell death; and 5) the toxicity profile would be expected to be minimal. The purpose of this study is to describe our initial exploratory experience with LD- FRT in addition to docetaxel and cetuximab in the setting of recurrent SCCHN.

Study and patient characteristics/eligibility criteria

This study was Institutional Review Boardapproved, Investigational New Drug&ndash;exempt, and Health Insurance Portability and Accountability Act&ndash;compliant. In addition to having loco-regional recurrence in the head and neck, all patients fulfilled all the following inclusion criteria: 1) Eastern Cooperative Oncology Group performance status of 0-2, 2) histo-pathologically confirmed recurrence of SCCHN, 3) not candidates for surgical salvage treatment due to unresectability, and 4) a period of at least 6 months from the completion of prior RT had passed. Exclusion criteria included distant metasta- ses outside the head and neck, primary disease in the nasopharynx or salivary glands, other invasive malignancies, or medical illnesses impairing ability to receive treatment.

The plan was to enroll 25 evaluable patients and follow them for a minimum of 2 additional years. The primary objective was to show an improvement of response rate, from 40% with chemotherapy alone to 70% with the addition of LDFRT. Considering the possibility that 5% of study-eligible patients will not be evaluable, we aimed to enroll 27 patients to ensure an analysis set of 25 patients who would be evaluable for efficacy. This sample size would provide reasonable precision in estimating the overall response rate (ORR) as preliminary evidence that the proposed docetaxel-cetuximab-LDFRT treatment is efficacious in this population. ORR is defined as rate of patients achieving complete response (CR) or partial response (PR). Secondary objectives consisted of a safety profile along with an estimate of PFS and OS.

Cetuximab 400 mg/m2 IV was administered as a loading dose 1 week prior to RT and then cetuximab 250 mg/m2 IV was given weekly on day 1 of weeks 2 to 7. Docetaxel 20 mg/m2 IV was given weekly on day 1 of week 2 to 7. LDFRT consisted of 0.5 Gy/ fraction given twice a day via intensity-modulated radiotherapy at least 6 hours apart on days 2 and 3 of weeks 2 to 7 for a total dose of 12 Gy. Total therapy lasted for a period of 7 weeks. The Figure reviews the treatment schema.

Patients were assessed weekly in the Department of Radiation Oncology, Sylvester Comprehensive Cancer Center, University of Miami Health System. Every week, complete blood counts and chemistries were evaluated to evaluate hematologic toxicity. The National Cancer Institute&rsquo;s Common Terminology Criteria for Adverse Events, version 4.0, was utilized for documentation of toxicity.

Follow-up visits consisting of history and physical examination were performed at 1, 3, 6, and 9 months. Four weeks after treatment, a computed tomography or magnetic resonance imaging scan of the head and neck was obtained. Response was evaluated by the treating physician defined as follows: CR was defined as disappearance of the target lesions; PR was defined as at least a 30% decrease in size of the lesion; progressive disease (PD) was defined as at least a 20% increase in the size of the lesion; and stable disease (SD) was defined as insuficient changes to qualify for the aforementioned endpoints of PR or PD. Patients who had less than a CR were recommended for further treatment with RT or chemotherapy at the discretion of the treating physician.

Proposals were set in place for the Data Safety Monitoring Committee (DSMC) at the Sylvester Comprehensive Cancer Center to stop the trial based on response or toxicity data. These guidelines were developed using Bayesian methods that could be applied at any phase of enrollment without advance specification of the number of interim analyses to be performed. Under these methods, a prior probability is assigned to possible values for response and toxicity data. As data on treated patients become available, the probability distributions are revised and the resulting posterior probability becomes the basis for recommending either continuation or termination of the study.

Between October 2013 and February 2015, 9 patients were screened for this institutional study to receive LDFRT, cetuximab, and docetaxel. Four patients were found to be ineligible for the study as 1 patient was found upon further work-up to have metastatic disease, 1 patient had a poor performance status, 1 patient elected hospice, and 1 patient died prior to enrollment. Five patients were enrolled in this study; however, 4 patients were ultimately treated because 1 patient was found to have a poor performance status and was not recommended for further treatment at the discretion of the treating physician. Table 1 lists the charac- teristics of the patients included in this study. All patients were male. Primary disease sites were the larynx in 2 patients, floor of mouth in 1 patient, and the tonsillar fossa in 1 patient. Only 1 patient received surgery as treatment for his primary tumor, followed by RT consisting of 66 Gy, whereas 3 patients received definitive chemoradiation consisting of cisplatin 100 mg/m2 every 3 weeks and RT dose of 70 Gy to their primary tumor. Time from end date of definitive primary treatment to therapy for recurrent disease ranged from 13 to 26 months. Four patients completed the reirradiation protocol and were able to tolerate treatment.

All the patients had grade 1 fatigue attributed to both chemotherapy and radiotherapy treatment. All patients had grade 1 rash ascribed to chemotherapy. There were no grade 2 or higher toxicities.

At 1-month follow up, PR was seen in 2 patients (50%), SD in 1 patient (25%), and PD in 1 patient (25%). By 6 months, all patients had PD. One patient (25%) underwent a second course of reirradiation consisting of 58 Gy in 2 Gy/fraction as salvage treatment at progression, and 1 patient (25%) underwent chemo- therapy. Table 2 summarizes treatment response. Given that the accumulating data suggested that the ORR was no better than 40%, the DSMC recommended early discontinuation due to lack of efficacy as previously outlined.

To our knowledge, this is the first study to evaluate the use of LDFRT with chemotherapy in patients with recurrent SCCHN. The primary endpoint for assessing efficacy was ORR. The secondary objectives were treatment-related acute and long-term toxicities and estimate of PFS and OS. In this exploratory analysis, we found an initial 75% rate of stable or responsive disease. The treatment effect was transient, with all patients experiencing PD at 6 months. The treatment was tolerated well with all patients experiencing grade 1 fatigue and rash and no reported grade 2 or higher toxicities. The DSMC suggested closing this protocol early as results were approaching the predefined futility endpoint.

In the setting of nonsurgical treatment options for patients with recurrent SCCHN, there is a paucity of randomized data evaluating the comparative benefit of treatment options. Provider treatment biases as demonstrated in part by selection of healthier patients into surgical series, as well as the heterogeneity present in the remaining patient population, may account for this dearth of comparative clinical data. Consequently, national phase III studies such as Radiation Therapy Oncology Group 0421, a randomized trial designed to evaluate concurrent reirradiation and chemotherapy versus chemotherapy alone in patients with recurrent head and neck cancer, were closed early due to inadequate patient accrual.

Even after overcoming the challenge of enrolling patients, there are numerous difculties in maintaining a clinical trial within this select, chronically ill, and unstable population. Nine patients were originally screened, but only 4 were treated due to rapidly deteriorating functional status of the excluded patients. Furthermore, this trend of incomplete clinical trial data continues as this trial was closed early.

Prior to our protocol design, a study evaluating LDFRT, paclitaxel, and carboplatin in patients with primary locally advanced SCCHN was initially reported by Arnold and colleagues.

In their study, the response rate was initially high at the primary site and nodal site. Our study was in part motivated by these developments; however, more recently these originally promising results were published as a 5-year follow-up with the mature data matching historic data with respect to disease-free survival and OS.

In the recurrent SCCHN setting, the treatment effect with LDFRT and chemotherapy appears to be more transient, as our experience demonstrates. This may be in part due to a lower threshold for DNA repair due to previous treatment, or even an inherent and selected radio- resistance in cells surviving a previous full course of radiotherapy.

Early work with LDFRT unveiled the concept of HRS, in which low doses of radiation paradoxically increased tumor cell kill as compared with conventional doses.

Hypothetically, HRS-related damage is below the threshold of DNA repair initiation that would otherwise be recognized (eg, double-strand damage that occurs after conventional doses). As such, the cell undergoes an apoptotic death.

Depending on the study, this area of HRS is described as being in the <1 Gy range, usually described broadly from 0.1 to 0.8 Gy region of the cell survival curve.

Notably, this area of HRS is swiftly followed by an area of increased radioresistance (IRR). IRR is thought to represent repair mechanism initiation after a dose-active increase in serine/threonine kinase activity and G2 checkpoint function.

As such, radiation-induced damage triggers activation of repair mechanisms in the region of IRR. Some studies have described this area of IRR being present in the 0.3 to 1 Gy range.24,27 As such, it is plausible that 0.5 Gy may be too high of a dose to take advantage of HRS, and may even, conversely, fall into an area of IRR.

This is also the first study to use cetuximab and docetaxel in conjunction with LDFRT in patients with recurrent SCCHN. We based the use of cetuximab on the favorable response seen in patients receiving cetuximab in the recurrent setting, while docetaxel was chosen due to the potentiating effect of LDFRT based upon the aforementioned in vitro and in vivo clinical data.

Although the potentiating effect of docetaxel is well described, there could be more of a potentiation role with other chemotherapeutic agents. It appears that LDFRT and both chemotherapeutic agents were very well tolerated compared with published experiences of multidrug chemotherapy regimens alone.

From our single institutional experience, patients with loco-regionally recurrent unresectable SCCHN should be treated with more established RT techniques, as they may not optimally benefit from this protocol.

In patients with more limited options, as those presenting with metastatic disease, LDFRT with cetuximab and docetaxel may offer transient palliation with minimal morbidity.

Although there have been numerous improvements in the primary treatment of SCCHN, treatment of recurrent disease in inoperable patients is challenging. From our single-institutional experience, low-dose RT in addition to cetuximab and docetaxel is well tolerated in patients with recurrent SCCHN. Additional long-term treatment options may exist for patients with recurrent SCCHN; however, for a select group of patients, LDFRT and chemotherapy may offer transient palliation in the setting of low morbidity.

The use of low-dose radiotherapy may offer transient benefits; nonetheless the potential for palliative care exists.

A Phase II Trial of Cetuximab and Docetaxel With Radiation for Head and Neck Cancer

Cancer vaccines comprise a multitude of approaches. The major goal of any approach is to amplify or generate an adaptive immune response against specific tumor antigens. One of the major hurdles of cancer vaccine development is the challenge of identifying an optimal target antigen that could lead to therapeutic immune response in each patient. In situ vaccination approaches using a variety of intratumoral treatment strategies attempt to circumvent this problem by using the patient&rsquo;s own tumor as a vaccine. These locoregional approaches operate in 2 ways: through localized tumor lysis that releases tumor antigens and danger signals, and through provision of appropriate immunostimulatory signals to activate tumor recognition by the immune system. When combined with systemic immunomodulatory antibody therapy, these strategies present an opportunity to generate and amplify systemic anti-tumor immune response, leading to both local and abscopal therapeutic effects.

Activation of antitumor immune response involves a close interaction between the innate and the adaptive immune systems. Recognition of cancer by the immune system begins with processing and presentation of tumor antigens (TAs) by specialized antigen-presenting cells (APCs), such as dendritic cells (DCs). Antigenic peptides are processed and presented on the surface of APCs within the context of major histocompatibility complex (MHC). The MHC-peptide complex is recognized and bound by a thymus (T) cell expressing a unique T-cell receptor. In the environment of appropriate additional immune costimulatory signals and cytokines, this leads to T-cell activation, proliferation, and differentiation, with eventual migration to the tumor site where the T cells mediate the killing of the tumor cells expressing the specific antigen.

Cancer vaccines comprise a multitude of approaches. The major goal of any approach is to amplify or generate an adaptive immune response against specific tumor antigens.

Traditionally, cancer vaccines have involved immunization with a specific tumor antigen, typically a shared tumor antigen that is expressed by tumors of different patients (eg, NY-ESO-1, MAGE A3).

Such approaches proved challenging, as development of anti-vaccine immune response rarely led to clinical benefit, suggesting that perhaps these vaccine targets are not optimal or that other immune pathways need to be targeted in combination. With advances in sequencing and bioinformatic tools, the latest studies have extended these strategies to personalized vaccination approaches using tumor-specific antigens (ie, neoantigens) resulting from DNA mutations, deletions, insertions, and translocations in cancer cells. Studies are currently ongoing to determine whether these approaches would be more successful.

A major limitation of the traditional vaccination approaches remains the uncertainty of adequate prediction of the optimal antigens that would be appropriately recognized on tumors by T cells. In situ vaccination approaches include locoregional treatment strategies that strive to use the patient&rsquo;s own tumor as a vaccine through the release of tumor antigens and the provision of appropriate immune signals to activate tumor recognition by the immune system. Such strategies are agnostic to specific TAs and instead rely on the immune system to choose the most immunogenic antigens as targets. These approaches span a multitude of approved and experimental techniques, most of which are summarized in this review.

In addition to direct cytotoxic effects on tumor cells, radiation has been extensively studied for its ability to activate immune response. While direct killing of tumor cells results in tumor antigen release, radiation also leads to upregulation and release of additional inflammatory mediators, collectively known as damage-associated molecular patterns (DAMPs), which include upregulation of cell surface calreticulin and heat shock proteins, and release of intracellular contents such as adenosine triphosphate and high mobility group box 1 protein. In addition, localized cell death results in expression of pro-inflammatory cytokines and chemokines. These components serve as maturation signals for APCs and can thus promote presentation of TAs to the immune system and production of pro-inflammatory cytokines and chemokines.

The overall process has been named immunogenic cell death (ICD) and is thought to be responsible for occasional regression of distant lesions in patients receiving radiotherapy to a single lesion, a process known as the abscopal effect.

Localized radiotherapy alone, however, is rarely enough for effective abscopal clearance.

Similar to radiation therapy, other ablative therapies, such as radiofrequency ablation (RFA), high intensity-focused ultrasound (HIFU), and cryoablation, can be used as in situ vaccines.

RFA causes tumor necrosis through localized heating, inducing inflammation and circulation of tumor-specific immune cells. HIFU has been shown to increase cytotoxic T-cell response, cause tumor embolization, and induce circulating CD4+ T cells.

Cryoablation causes tumor necrosis through intracellular crystal formation and microvascular injury,

which also leads to ICD and generation of antitumor immune response.

The effects of chemotherapy on the immune system are very heterogeneous, as systemic chemotherapy has both negative and positive influence on systemic immune cells. Although chemotherapy is, in general, associated with immunosuppression, multiple studies have demonstrated positive effects of chemotherapy on the immune system. Systemically, such effects include preferential depletion of specific immunosuppressive cell subsets, such as regulatory T cells and myeloid-derived suppressor cells.

On the tumor level, chemotherapy acts as an in situ vaccine through induction of ICD.

It is important to note that different chemotherapy agents can lead to different immune effects and that not all agents have been associated with ICD. For example, oxaliplatin, but not cisplatin, has been demonstrated to stimulate antitumor immune response in mouse tumor models.

Like radiotherapy and many others on this list, immunotherapeutic efficacy of chemotherapy is limited when used alone.

Advances in understanding of tumor immune recognition and escape have led to identification of multiple mechanisms governing T-cell activation and inhibition. Such mechanisms include a range of immune stimulatory (eg, a co-stimulatory transmembrane glycoprotein receptor on activated T cells [OX40], glucocorticoid-induced tumor necrosis factor receptor related gene [GITR], CD40, CD137), and inducible co-stimulator (ICOS) and inhibitory receptors (eg, cytotoxic T lymphocyte antigen 4 [CTLA-4], programmed cell death protein 1 [PD-1], lymphocyte-activation gene 3 [LAG-3], T-cell immunoglobulin and mucin protein-3 [TIM- 3]), which are currently being explored in clinical trials using immunomodulatory antibodies targeting these receptors alone or in combination. Agents targeting PD-1 and its ligand programmed death-ligand 1 (PD-L1) are the most advanced in clinical development and have already been approved for a variety of indications.

Some of these agents, in particular CTLA-4 blocking antibodies and CD40 agonists, can be associated with significant toxicities with systemic administration. Several studies have demonstrated that intratumoral administration of these agents, such as anti-CTLA-4, anti-OX40, and anti-CD40, can be successful in regressing distant tumors in animal models through induction of systemic antitumor immunity.

Such therapies given intratumorally can thus be considered in situ vaccines and have a potential to offer the benefit of immunomodulatory antibody therapy while mitigating systemic toxicity of these drugs. Early data in patients with melanoma combining intratumoral interleukin-2 (IL-2) and the anti-CTLA-4 antibody ipilimumab demonstrated good safety and signal of efficacy.

Intratumoral injections of cytokines involved in immune modulation can induce in situ vaccination effects. Several cytokines have been explored in this setting, with IL-2, IL-12, and granulocyte- macrophage colony-stimulating factor (GM-CSF) as examples that have undergone extensive clinical testing. IL-2 is normally produced by T cells and controls the expansion and differentiation of T cells. It is approved for systemic treatment of metastatic melanoma and renal cell cancer, although its administration is limited by significant systemic toxicity. Intratumoral administration of IL-2 has shown significant activity in preclinical models, and several studies with intratumoral IL-2 using different delivery methods are currently ongoing.

IL-12 is produced by myeloid APCs and promotes T cell and natural killer (NK) cell activation, which has been demonstrated to result in significant antitumor activity in pre-clinical models.

While systemic treatment with the cytokine had significant toxicities, intratumoral administration of IL-12 using different vector strategies has demonstrated promising activity in preclinical and clinical studies.

GM-CSF is normally produced by a variety of immune cells, including macrophages, T cells, and NK cells, as well as non-immune cells such as endothelial cells and broblasts. GM-CSF controls DC maturation and function and has been explored for anti-cancer therapy. Intratumoral injection of GM-CSF has been investigated in several trials in melanoma with promising activity in early studies.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) found on leukocytes and APCs that bind to danger signals, such as pathogen-asso- ciated molecular patterns and DAMPs, resulting in APC activation, enhanced antigen presentation, and initiation of immune responses.

Intratumoral TLR agonists targeting different TLRs have been explored in many preclinical and clinical studies.

The TLR7 agonist, imiquimod, is approved for treatment of genital warts and basal cell carcinoma.

Intratumoral TLR9 agonist, in combination with radiation, demonstrated promising activity against injected and distant lesions in lymphoma.

The stimulator of interferon genes (STING) pathway is responsible for expression of interferon-&beta; by tumor-resident DCs, which is required for spontaneous tumor-initiated T-cell priming. Intratumoral injection of STING agonists, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), has yielded promising results in pre-clinical studies, providing tumor regression and long-lived immunologic memory.

Induced bacterial infection has been used since 1891, when Dr William Coley injected tumors with live Streptococcus pyogenes cultures after linking spontaneous tumor regression with concurrent bacterial infection.

Activation of PRRs by bacterial components induces activation and recruitment of innate and adaptive immune responses that can result in tumor recognition and systemic antitumor immunity. To this end, Bacillus Calmette-Guérin is approved for treatment of nonmuscle-invasive bladder cancer

and has been explored for intralesional therapy of other cancers. Several bacterial agents have been explored for intratumoral therapy in preclinical models.

Intralesional administration of Clostridia novyi spores has demonstrated anti-tumor response and regression in patients, partly mediated by cytotoxic T cells and DC activation.

Oncolytic viruses selectively infect and lyse tumor cells, releasing TAs and danger signals, eliciting a strong innate immune response and maturation of APCs. Additionally, engineering of oncolytic viruses to express genes for cytokines or TAs can further increase their efficacy. In a phase III trial, talimogene laherparepvec (T-VEC), an engineered herpes virus expressing GM-CSF, was shown to be superior to GM-CSF alone in advanced melanoma patients, with objective response rates (ORRs) of 26% versus 6% and durable response rates of more than 6 months in 16% of patients.

CAVATAK is a nonen- gineered cocksackievirus A21, the activity of which relies on potent activation of innate immune responses in the infected tumors. In a phase II trial in 57 patients with stage IIIC-IV melanoma who were treated intralesionally with weekly CAVATAK, a 28% ORR and a 1-year OS of 75% was observed,

with abscopal effects among the responders.

Promising results with DNX-2401, an engineered adenovirus, have also been achieved in patients with glioblastoma. A single intratumoral injection of DNX-2401 resulted in median survival of 11 months and durable complete responses (CRs) of more than 1.5 years in 3 of 37 patients.

Synergy of Immunomodulatory Antibodies and in situ Vaccines

Recent studies have demonstrated that the efficacy of immunomodulatory antibodies, such as the ones targeting PD-1 and CTLA-4, can be potentiated when combined with in situ vaccines. In preclinical models, combination with oncolytic viruses elicited responses in immunologically silent tumors.

In patients with advanced melanoma, the combination of ipilimumab (antiCTLA-4) and T-VEC showed a response rate of 50%, while being well tolerated.

Concomitant intratumoral application of ipilimumab and IL-2 resulted in abscopal effects in 8 of 9 patients evaulated.

When ipilimumab was combined with cryoablation, it increased cytokine levels and the amount of intratumoral CD4+ effector cells in stage I-II breast cancer, which has inherently low immune infiltration.

Combinations of immune checkpoint blockade and radiotherapy are currently subject to approximately 70 open trials and were recently reviewed.

Combination of pembrolizumab (antiPD-1) with T-VEC in patients with unresectable stage IIIB-IV melanoma led to 48% ORR and 14% complete response (CR) as well as an acceptable toxicity profile.

Pembrolizumab in combination with CAVATAK is currently being tested in a phase I/III expansion trial in patients with advanced melanoma (NCT02565992) and in a phase I trial including other advanced tumor types (NCT02043665).

In situ vaccines present several obvious advantages over traditional vaccines. First, they present an &ldquo;off the shelf&rdquo; approach to personalized vaccination, obviating the need for identification and synthesis of patient-specific antigenic targets. Second, such vaccination has a potential to generate an immune response against a broader antigen repertoire, thus decreasing the possibility of emergence of resistant clones that lose antigen expression. Third, the intratumoral delivery route allows for the use of agents that would otherwise be toxic if given systemically. An obvious limitation of in situ vaccination is a potential challenge with tumor accessibility for intratumoral administration, especially with repetitive dosing protocols. With advances in modern interventional radiology, endoscopy, and surgical techniques, however, such studies are becoming more feasible. Fong and colleagues have demon- strated that oncolytic herpes virus can be delivered to liver metastases via hepatic arterial infusion.

Infusion through intraperitoneal or intrapleural catheters can be used for patients with peritoneal and pleural disease, respectively. Direct intratumoral injection through catheter implantation has been shown to be possible in glioma.

With improved understanding of tumor cells and the immune microenviroment came the recognition that therapies targeting tumor and immune cells directly within the patient can, in essence, generate cancer vaccines in vivo. Such approaches encompass a range of experimental and local therapeutic techniques, some of which have traditionally not been thought of as immune therapies. With provision of appropriate local immunostimulatory signals, such in situ vaccination approaches can enhance tumor recognition by the innate and the adaptive immune systems and present an opportunity to generate an immune response against a broad repertoire of TAs. When combined with systemic immunomodulatory antibody therapy, in situ vaccination presents a powerful strategy to generate a strong systemic anti-tumor immune response and to extend the benefit of immunotherapy to a broader range of patients and cancer types.

Butter eld LH. Cancer vaccines. BMJ. 2015;350:h988. doi: 10.1136/bmj.h988.

Katsnelson A. Mutations as munitions: neoantigen vaccines get a closer look as cancer treatment. Nat Med. 2016;22(2):122-124. doi: 10.1038/nm0216-122.

Gaipl US, Multho G, Scheithauer H, et al. Kill and spread the word: stimulation of antitumor immune responses in the context of radiotherapy. Immunotherapy. 2014;6(5):597-610. doi: 10.2217/imt.14.38.

Pierce RH, Campbell JS, Pai SI, et al. In-situ tumor vaccination: bringing the ght to the tumor. Hum Vaccin Immunother. 2015;11(8):1901-1909. doi: 10.1080/21645515.2015.1049779.

van den Bijgaart RJ, Eikelenboom DC, Hoogenboom M, et al. Thermal and mechanical high-intensity focused ultrasound: perspectives on tumor ablation, immune e ects and combination strategies. Cancer Immunol Immunother. 2017;66(2):247-258. doi: 10.1007/s00262-016-1891-9.

Gage AA, Baust J. Mechanisms of tissue injury in cryosurgery. Cryobiology. 1998;37(3):171-186.

A locoregional approach to cancer vaccines, when combined with immunmodulatory antibody therapy, could generate a significant anti-tumor response.

2017 April | The Journal of Targeted Therapies in Cancer

Hammers Recommends Revisiting Vaccines in Renal Cell Carcinoma

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A Phase II Trial of Cetuximab and Docetaxel With Radiation for Head and Neck Cancer

Locoregional Therapeutics: Using One's Own Tumor as a Vaccine