The US Food and Drug Administration has been rapidly approving new checkpoint inhibitors and targeted therapies for melanoma and other tumors. Recently, it approved the first intralesional therapy, talimogene laherparepvec (T-VEC), for the treatment of metastatic melanoma lesions in the skin and lymph nodes. Several other intralesional therapies (PV-10, interleukin-12 electroporation, coxsackievirus A21 [CVA21]) are entering later-stage testing. Locally injected agents have clearly shown their ability to produce local responses that can be durable. The possibility that they also stimulate a regional and even systemic immune response is exciting, as this potential effect may have utility in combination regimens; such regimens are an area of active research. Favorable responses with minimal toxicities in monotherapy trials have led to the first melanoma studies of T-VEC in combination with the cytotoxic T-lymphocyte–associated antigen 4 inhibitor ipilimumab and, separately, with the programmed death 1–blocking antibody pembrolizumab. Studies of PV-10 with pembrolizumab and of CVA21 with pembrolizumab are also being initiated. Preliminary analyses of the results of the first combination trials, which show higher response rates than with either agent alone, offer some optimism that these locoregional therapies will find application-as treatment for patients who cannot tolerate systemic immunotherapies, to alleviate locoregional morbidity, and perhaps even to “prime” the immune system.
Background
Melanoma accounts for about 4.5% of new cancers (21.6% per 100,000) and 1.7% of all cancer deaths in the United States. The incidence of melanoma has been gradually increasing for several decades, with the annual rise averaging 1.4% per year over the last 10 years. It is estimated that the number of deaths from melanoma approached 10,000 in 2015.[1]
After frustrating decades of little progress, the landscape of melanoma therapy was dramatically altered with the first report in 2010 of significantly improved overall survival with the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) inhibitor ipilimumab, compared with a vaccine, and subsequently, compared with the then “standard” chemotherapeutic agent dacarbazine (DTIC). Following the US Food and Drug Administration (FDA) approval of ipilimumab the following year, approvals of other targeted therapies (against BRAF and MEK), and most recently, approvals and extended indications for other checkpoint inhibitors, especially the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors, have constituted a revolution characterized by higher response rates and the potential for extended survival. Clearly, we are now in an era where new treatments are showing positive results and are becoming available at an unprecedented rate.
Why an Interest in Locoregional Therapies?
Direct treatment of cutaneous melanoma lesions is on a separate track from that of systemic therapies, with its rapid advances. However, interest in strategies focused on the direct treatment of cutaneous melanoma lesions has its own pedigree. Melanoma is unique in its propensity to produce cutaneous, subcutaneous, and nodal metastases. This unique pattern of metastatic spread affords us an opportunity to access the tumor and manipulate it for potential therapeutic benefit. Furthermore, while it is true that metastatic melanoma is a systemic disease-and also that mortality is not caused by dermal or subcutaneous lesions but rather by metastases to the liver, brain, lung, and other sites-locoregional disease is often disfiguring and painful, and locoregional therapy may provide clinical benefit.
A particular challenge is posed by the in-transit metastases that develop in 6% to 10% of patients with primary melanomas > 1.0 mm in Breslow thickness.[2] In some patients, in-transit metastatic disease may remain indolent and regionally confined for years or decades. In these patients, clinical results with systemic therapy have been less than optimal, and locoregional therapeutic options, such as limb perfusion and intralesional treatments, have thus been explored.
Intralesional therapy for melanoma has a long history. The unique relationship of melanoma to the immune system has stimulated research in this area for many years. Murine experiments showed heightened host immune responses against transplanted experimental tumors after inoculation with bacillus Calmette-Guérin (BCG).[3,4] Dummer et al demonstrated BCG-induced increases in melan-A–specific cytotoxic T cells.[5] Morton et al, in 1974, reported 7 years’ experience with 151 malignant melanoma patients who received BCG immunotherapy alone or as an adjunct to surgery.[6] In lesions limited to skin, regression was 90% in injected lesions and 17% in uninjected lesions, with one-fourth of patients remaining disease-free for 1 to 6 years.
Enthusiasm for intralesional BCG therapy waned following reports of anaphylactic reactions; disseminated BCG mortality[7]; and, in our own research, the occurrence of punctate abscesses in more than two-thirds of treated patients, as well as failure to impact outcomes.[8] Other agents of mostly historical interest include intralesional interleukin (IL), interferon (IFN), and granulocyte-macrophage colony-stimulating factor (GM-CSF), which have not shown consistent or durable efficacy and have not been rigorously tested in randomized trials. Here we review agents that have been evaluated or are currently undergoing evaluation in the modern era of melanoma therapeutics.
Velimogene Aliplasmid
Velimogene aliplasmid (Allovectin-7), a plasmid/lipid complex with human leukocyte antigen (HLA)-B7 and β2 microglobulin DNA encoding sequences, was granted orphan drug designation by the FDA back in 1999. HLA-B7 and β2 microglobulin are components of major histocompatibility complex class I (MHC-I), expression of which is often altered during cancer progression, allowing tumor cells to evade the immune system.[9] A role in augmenting the immune system’s ability to recognize and target melanoma cells was hypothesized because velimogene aliplasmid increases HLA-B7 cytotoxic T-cell frequency fivefold, upregulates/restores MHC-I molecules, and induces a proinflammatory response.
The agent showed promise in a phase II trial, which was conducted in 133 patients with stage IIIB/C and IV M1a/b injectable cutaneous, subcutaneous, or nodal melanoma lesions.[10] Complete responses (CRs) and partial responses (PRs) were reported in 3.2% and 8.7% of patients, respectively, with stable disease (SD) in 25% of the two patient cohorts combined. Time to death was significantly longer in responders than in nonresponders (P = .036). Of the patients with stage IV M1a/b disease, 21% (9/42) had responses in noninjected target lesions. However, the subsequent phase III Allovectin Immunotherapy for Metastatic Melanoma (AIMM) trial failed to show benefit.[11] Among 390 patients with stage IIIB/IV M1a/b melanoma randomized 2:1 to velimogene aliplasmid or to intravenous DTIC or oral temozolomide (TMZ), the primary endpoint of response rate at ≥ 24 weeks was lower in the velimogene aliplasmid group at 4.6%, compared with 12.3% for DTIC/TMZ (P = .010). While the duration of response in the velimogene aliplasmid responders was marginally longer than with DTIC/TMZ (P = .066), overall survival was shorter (median, 18.8 months [95% CI, 16.6–21.3 months] vs 24.1 months [95% CI, 17.1–27.9 months]; P = .491). The velimogene aliplasmid program was discontinued.
Electroporation With Plasmid IL-12
With intratumoral electroporation, tumor cell pores are opened when a mild electrical current is introduced directly through an electrode. The intention is to thereby facilitate greater influx of a cytotoxic agent over a longer period of time than would occur with systemic administration of the same agent. Achieving high levels of IL-12 protein expression stimulates a local proinflammatory process, which leads to a targeted immune response; enhances the immune capacity of natural killer (NK) and T cells; and upregulates IFNγ, as well as antigen presentation and processing. With reduced systemic drug concentrations, side effects are minimized. This feature is of particular value with respect to IL-12, a cytokine that is toxic when given systemically.
Interim phase II results[12] of first treatment with electroporation of IL-12 in 28 patients with advanced melanoma, after 24 weeks of treatment, reported at the American Society of Clinical Oncology 2014 Annual Meeting, revealed a 32.2% objective response rate (ORR; the primary endpoint), with a CR in 10.7%. Among lesion responses (n = 85), the CR rate was 45%, and the PR rate was 8%, with SD in 31%. Responses were observed in untreated lesions in 59.1% of evaluable patients (13/22).
The electroporation treatment produced no toxicity and no serious adverse events, aside from injection site pain (69.0%) and inflammation (20.7%). Grade 3 pain was reported in 1 patient.
An exploratory analysis showed that intratumoral NK cells doubled from pretreatment through day 11, and doubled again by day 39, with an increase in the frequency of activated circulating NK cells. In the phase II trial, a maximum of four treatment cycles at 12-week intervals were allowed. A planned expansion protocol in melanoma patients will evaluate increased treatment frequency.
T-VEC
In October 2015-around the same time that the FDA was granting several approvals and indication extensions to checkpoint inhibitors-the agency approved the oncolytic virus (herpes simplex virus 1)–derived therapy talimogene laherparepvec (T-VEC) for the treatment of melanoma lesions in the skin and lymph nodes. T-VEC encodes GM-CSF, and is thought to replicate in tumor cells, lysing cells in injected tumors. Antigen-presenting cells then take up the lysed cells. Local expression of GM-CSF may also evoke an enhanced adaptive antimelanoma response (Figure).
T-VEC was compared with GM-CSF in the phase III OPTiM study in 436 stage IIIB/C and IV melanoma patients who had injectable and unresectable disease.[13] Durable response (complete or partial), the primary endpoint, was defined as a response lasting continuously for at least 6 months and begun within 12 months of initiation of therapy. Patients were randomized 2:1 (295:141) to intralesional T-VEC (initially, ≤ 4 mL × 106 pfu/mL; then after 3 weeks, ≤ 4 mL × 108 pfu/mL every 2 weeks) or subcutaneous GM-CSF (125 µg/m2 daily × 14 days every 28 days).
The durable response rate (DRR) in the intention-to-treat analysis was 2.1% in the GM-CSF arm and 16.3% in the T-VEC arm, a treatment difference of 14.1% (95% CI, 8.2%–19.2%; P < .0001). The ORR in the 141 patients who received GM-CSF was 5.7% (95% CI, 1.9%–9.5%). It was 26.4% in the 295 patients who received T-VEC (95% CI, 21.4%–31.5%; P < .0001), for a treatment difference of 20.8% (95% CI, 14.4%–27.1%; P < .0001). In the T-VEC responders, the CR rate was 41% (10.8% among all patients who received T-VEC; 0.7% for all patients who received GM-CSF). PR rates were 15.6% and 5.0%, respectively, for the entire T-VEC and GM-CSF arms.
In the stage IIIB/C melanoma patients with no disease spread to distant organs, the DRR was 33% for T-VEC compared with 0% for GM-CSF. Differences in DRRs between groups were smaller for stage IV patients (11% for T-VEC, 7% for GM-CSF).
With regard to median overall survival (OS), a secondary endpoint, T-VEC had a 4.4-month advantage over GM-CSF (23.3 months vs 18.9 months [hazard ratio (HR), 0.79 (95% CI, 0.62–1.00); P = .051]); this approached but did not achieve statistical significance.
The OPTiM investigators, in an analysis of lesion-level responses, reported that among 2,116 lesions in patients treated with T-VEC, tumor area decreases of ≥ 50% occurred in 64% of injected lesions, in 34% of uninjected nonvisceral lesions, and in 15% of uninjected visceral lesions.[14]
The most common adverse event was fatigue (occurring in 50.3% of patients in the T-VEC arm, and in 36.2% of those in the GM-CSF arm), with chills (48.6% in the T-VEC arm; 8.7% in the GM-CSF arm) and pyrexia (42.8% in the T-VEC arm; 8.7% in the GM-CSF arm) the next most common. The only grade 3/4 event that occurred in ≥ 2% of patients was cellulitis (seen in 2.1% of the patients who received T-VEC).
PV-10
PV-10 (rose bengal disodium 10%), a small-molecule fluorescein derivative, while excluded from normal cells, transits the plasmalemma of cancer cells and accumulates in lysosomes, triggering lysosomal release and complete autolysis within 30 to 60 minutes.[15] “Bystander” effects observed in uninjected tumors are believed to ensue as a consequence of acute exposure of antigenic tumor fragments to antigen-presenting cells. The double benefit is immediately reduced tumor burden and immunologic activation.
Our 80-subject, multicenter, international phase II study[16] enrolled patients with measurable stage III/IV melanoma. PV-10 injections were given in up to 10 target lesions of ≥ 0.2 cm in diameter. Also, investigators observed 1 to 2 biopsy-confirmed bystander lesions in each patient. These were typically small or difficult to access, and included visceral lesions.
The CR rate in target lesions was 24% in preliminary reporting. The disease control rate, which combined the CR rate with the PR rate of 25% and the SD rate of 22%, was 71%. CRs were also observed in 24% of untreated bystander lesions, and regression in target lesions correlated strongly with bystander lesion responses. The ORR for bystander lesions was 37%, and the locoregional control rate was 55%.
By the final analysis of this study, completed in 2012, ORRs had risen to 58% and 40% in target and bystander lesions, respectively, with locoregional disease control at 80% and 60% for the target and bystander lesion groups, respectively. Also, regression or stasis was noted in a few patients in distant visceral lesions.
An analysis of progression-free survival (PFS) in the first 40 patients revealed that those who achieved CRs had significantly longer PFS than those with SD or progressive disease (11.1 months vs 2.8 and 2.7 months, respectively). Also, disease stage and prior treatment appeared to have no effect on ORRs in injected lesions. In a later analysis,[17] the ORR in 28 patients who had all lesions injected was 71%, and the mean PFS was 9.8 months.
Target lesion responses with PV-10 in the phase II trial were consistently more robust in patients with stage III melanoma than in patients with stage IV disease; similarly, response duration was significantly longer in patients with stage III melanoma than in those with stage IV melanoma-9.6 months vs 3.1 months (P < .001).[18] Clearly, the higher tumor burden at baseline in stage IV patients adversely affected responses. Progression of non-study lesions, which precluded repeat treatment, had the same effect.
The choice to exclude all but stage IIIB/C patients in the ongoing phase III trial of PV-10[19] was influenced by these findings. This international multicenter, open-label, randomized controlled trial will include 225 patients with locally advanced cutaneous melanoma. The trial is enrolling patients who are BRAF V600 wild-type and in whom ipilimumab has failed or who are not otherwise candidates for ipilimumab or another immune checkpoint inhibitor. It is comparing single-agent intralesional PV-10 (every 4 weeks) vs systemic chemotherapy (every 4 weeks) with investigator’s choice of either DTIC or TMZ, in a 2:1 randomization.
Coxsackievirus A21
Surface intercellular adhesion molecule-1 (ICAM-1) is upregulated in melanoma and some other cancers (eg, prostate, bladder, breast, non–small-cell lung). In preclinical research, tumor cells lysed by coxsackievirus A21 (CVA21), a naturally occurring “common cold” ICAM-1–targeted RNA virus, induced a secondary systemic host-generated antitumor immune response.
In the phase II CALM trial (ClinicalTrials.gov identifier: NCT01227551),[20] 57 patients with stage IIIC (42.1%) or stage IV (57.9%) melanoma and at least 1 injectable dermal, cutaneous, subcutaneous, or lymph node lesion received 10 series of multi-intratumoral CVA21 injections. The primary endpoint was immune-related (ir)PFS-ie, the proportion of patients at 6 months with CR, PR, or SD (immune-related Response Evaluation Criteria in Solid Tumors [irRECIST] 1.1). The secondary endpoint was ORR (CR + PR).
Analysis of CALM revealed an irPFS rate of 38.6% (22/57) and an ORR of 28.1% (16/57), with 8 CRs and 8 PRs. Median OS was 26 months (95% CI, 16.7 months–not reached), and the 1-year survival rate was 75.4% (43/57). Tumor responses were observed in injected lesions, in noninjected cutaneous lesions, and in noninjected visceral lesions. The fact that responses occurred in the presence of high levels of anti-CVA21 antibody and in the absence of circulating infectious CVA21 supports an immune-mediated response as opposed to one caused by the virus entering the tumor.
Multi-dose intralesional CVA21 was well tolerated; no grade 3 or grade 4 treatment-related adverse events were observed.
To further examine systemic antitumor responses, a CALM study extension[21] evaluated sequential tumor biopsies of both injected and noninjected lesions in a cohort of 13 patients. Levels of viral replication and evidence of viral-induced immune activation within the tumor microenvironment were monitored. Serial serum samples were monitored for viral loads, anti-CVA21 neutralizing antibody, and levels of immune-inflammatory cytokines. Increases in immune cell infiltrates were observed in 4/4 patients in whom single- or double-line immune checkpoint blockade had failed. These results, Andtbacka et al concluded, offer a solid rationale for investigating CVA21 given sequentially or concurrently with T-cell checkpoint antibodies.
Early Combination Results Suggest Higher Response Rates
TO PUT THAT INTO CONTEXT
[[{"type":"media","view_mode":"media_crop","fid":"48694","attributes":{"alt":"","class":"media-image","id":"media_crop_6857593592561","media_crop_h":"0","media_crop_image_style":"-1","media_crop_instance":"5844","media_crop_rotate":"0","media_crop_scale_h":"0","media_crop_scale_w":"0","media_crop_w":"0","media_crop_x":"0","media_crop_y":"0","style":"height: 161px; width: 144px;","title":" ","typeof":"foaf:Image"}}]]
Robert H. I. Andtbacka, MD, CM
University of Utah & Huntsman Cancer Institute, Salt Lake City, UtahIntralesional Therapies: An Old Treatment Is RebornIn the late 1800s, William Coley described the use of an intralesional bacteria-free extract, known as “Coley toxins,” in the treatment of sarcomas. Coley’s data showed an over 60% remission rate of sarcomas. Intralesional therapies are especially well suited to the treatment of melanoma, in particular in-transit and lymph node metastases. Intralesional bacillus Calmette-Guérin and interleukin-2 have both shown good responses in injected lesions; however, they have had minimal to no effect on noninjected lesions, likely due to inadequate activation of the adaptive immune system. Yet over the past 15 years, an improved understanding of immune activation has led to the development of intralesional therapies with enhanced immune activation and improved effects in noninjected metastases. More than 20 different intralesional therapies, ranging from viruses and cytokines to glycolipids, plasmids, antibodies, and small molecules, have been tested in clinical trials for metastatic melanoma alone.What Is the Current Role of Intralesional Therapies in Melanoma?To date, the only approved intralesional therapy for metastatic melanoma is talimogene laherparepvec (T-VEC), which has been shown to have a durable response not only in injected dermal, subcutaneous, and lymph node metastases, but also in noninjected regional metastases. It also has some effect on visceral noninjected lesions, although this appears less robust. These treatment effects, coupled with its limited toxicity, make T-VEC monotherapy a great addition to the treatment options available to patients with nonvisceral metastases and for patients who, because of comorbidities, may not be candidates for other melanoma therapies. This is likely to be the role for other intralesional monotherapies as well.What Is Likely to Be the Future Role of Intralesional Therapies?Early results from combination studies of intralesional therapies with other immunotherapies have indicated no increased toxicity and possibly an additive improvement in response rates above that of single-agent use; these results will likely cement the role of intralesional therapies as important immunomodulatory agents in combination therapies for metastatic melanoma. These agents may also have a future role in neoadjuvant treatment for resectable metastatic melanoma. To date, most of the intralesional agents have been administered in palpable dermal, subcutaneous, and lymph node metastases. There are ongoing studies with injection of T-VEC into visceral lesions, and also of intravenous administration of intralesional therapies, such as coxsackievirus A21. If these new routes of administration are found safe and responses are seen, it will certainly expand the role of these agents, not only in metastatic melanoma, but also in many other cancers. Indeed, intralesional therapies have the potential, in the near future, to transform the way we treat metastatic cancer.Financial Disclosure: Dr. Andtbacka has received honoraria and/or served on advisory boards for Amgen, Merck, and Provectus; his institution has received research support from Amgen, Provectus, Takara, and Viralytics.
It has been generally surmised that intralesional therapies will be combined with the successful systemic immunotherapies-the checkpoint inhibitors or targeted therapies already approved or in development. It has been suggested that the intralesional viral vector and chemoablative activity of the intralesional agents just discussed ruptures the tumor, causing the release and presentation of intact antigens. The observed effect, an influx of T cells to the tumor, is distinct from the programmed cell death produced by the checkpoint inhibitors.[22] The potential of synergies arising from these different mechanisms of action, the safety and lack of dose-limiting toxicities of the intralesional therapies, and the nonoverlapping toxicity profiles of the contemplated combinations, offer an attractive rationale. In addition, the relatively lower toxicity of the PD-1/PD-L1 agents compared with ipilimumab intensifies interest in the ongoing trials of their combination with intralesional therapies.
Early data from a small phase Ib/II study[23] of ipilimumab combined with T-VEC, which demonstrated that the combination offers much higher complete and overall response rates than have been seen with either agent alone, appear to confirm that there are beneficial synergies between these two therapeutic modalities. In 18 patients with stage IIIB/IV M1c melanoma, the investigator-assessed CR rate was 33%, with PR in 22%, SD in 17%, and an overall response rate of 56% (95% CI, 31%–79%). There were no dose-limiting toxicities, and most toxicities appeared to be ipilimumab-related. In a pattern suggestive of T-VEC responses, activated CD8+ T-cell increases were higher in patients with disease control, according to flow cytometry. The trial’s randomized phase II segment is ongoing.
The phase II part of a phase Ib/II study (ClinicalTrials.gov identifier: NCT02263508; MASTERKEY-265)[24] assessing the safety and efficacy of T-VEC in combination with the PD-1–blocking antibody pembrolizumab in previously untreated, unresected stage IIIB/IV melanoma is evaluating confirmed ORR by irRECIST at week 24. T-VEC plus pembrolizumab is being compared against pembrolizumab alone. In early results, of 16 evaluable patients, 9 had objective responses (56.3%); the disease control rate was 68.8%. Although all patients had treatment-related serious adverse events, no grade 4 events or dose-limiting toxicities were reported (Tables 1 and 2).
A phase III study comparing T-VEC plus pembrolizumab vs pembrolizumab alone in 660 patients with treatment-naive, stage III/IV melanoma with injectable lesions is being planned.[25]
The Melanoma Intra-Tumoral CAVATAK and Ipilimumab (MITCI) study[26] of CVA21 in combination with systemic administration of ipilimumab in patients with unresectable melanoma is being conducted at three US sites. Preliminary reports indicate that to date the CVA21 + ipilimumab combination has led to no serious adverse events. Early signs of antitumor activity at 14 weeks after initiation of treatment have been observed in metastatic visceral and nonvisceral lesions, according to a case report.
Similarly, a combination phase Ib/II trial of PV-10 and pembrolizumab[27] has been initiated in patients with stage IV melanoma. The phase Ib portion will include 24 patients. The phase II part, with an expanded cohort of up to 120 patients in a 1:1 randomization to pembrolizumab with or without PV-10, will assess clinical benefit (PFS).
Likely Future and Unresolved Questions
If the ongoing and planned clinical trials confirm initial trends observed in early studies, combinations of systemic immunotherapies with intralesional agents may find a place in the ever-expanding therapeutic toolbox available to clinicians who treat patients with melanoma. As long as a lesion amenable to intralesional injection is available, it may allow clinicians to exploit the potential synergy and nonoverlapping toxicities of these approaches.
Another interesting area for ongoing and future research is in the neoadjuvant setting. Would it be justifiable to consider T-VEC, PV-10, or CVA21 as an upfront strategy in patients with surgically resectable disease before surgery, with the intent of making the tumor our ally? If ablating the patient’s autologous tumor can stimulate a tumor-specific immune response that persists after the tumor is removed, then creating an ally is what we would be doing. However, would the benefits be substantial enough to warrant incurring the risk inherent in delaying surgery? A phase II multicenter, randomized, open-label trial of T-VEC[28] in patients with resectable stage IIIB, IIIC, or IV M1a melanoma testing that hypothesis is in the enrollment phase. About 50 international sites will ultimately participate. Neoadjuvant T-VEC followed by surgery will be compared with surgery alone. Recurrence-free survival from the time of randomization to the date of the first local or distant recurrence of melanoma or death due to any cause is the primary endpoint. Biomarker analyses will look for correlations between baseline CD8+ T-cell density in injected lesions and outcomes.
Through the emergence of new immunotherapies, treatment of melanoma is undergoing a long-awaited revolution. Ongoing research will clarify the outlines of the place that intralesional therapies will occupy in the therapeutic armamentarium in the years ahead.
Financial Disclosure:Dr. Agarwala has acted as an ad hoc consultant to Amgen, Merck, and Provectus.
Acknowledgement:The author gratefully acknowledges medical writing assistance from Walter Alexander.
References:
1. National Institutes of Health. SEER stat fact sheets: melanoma of the skin. http://seer.cancer.gov/statfacts/html/melan.html. Accessed April 1, 2016.
2. Thompson JF. Local and regional therapies for melanoma: many arrows in the quiver. J Surg Oncol. 2014;109:295.
3. Goldie JH. Scientific basis for adjuvant and primary (neoadjuvant) chemotherapy. Semin Oncol. 1987;14:1-7.
4. Weiss DW. MER and other mycobacterial fractions in the immunotherapy of cancer. Med Clin North Am. 1976;60:473-97.
5. Dummer R, Rochlitz C, Velu T, et al. Intralesional adenovirus-mediated interleukin-2 gene transfer for advanced solid tumors and melanoma. Mol Ther. 2008;16:985-94.
6. Morton DL, Eilber FR, Holmes EC, et al. BCG immunotherapy of malignant melanoma: summary of a seven-year experience. Ann Surg. 1974;180:635-43.
7. de la Monte SM, Hutchins GM. Fatal disseminated bacillus Calmette-Guerin infection and arrested growth of cutaneous malignant melanoma following intralesional immunotherapy. Am J Dermatopathol. 1986;8:331-5.
8. Agarwala SS, Neuberg D, Park Y, Kirkwood JM. Mature results of a phase III randomized trial of bacillus Calmette-Guerin (BCG) versus observation and BCG plus dacarbazine versus BCG in the adjuvant therapy of American Joint Committee on Cancer stage I-III melanoma (E1673). Cancer. 2004;100:1692-8.
9. Garrido C, Paco L, Romero I, et al. MHC class I molecules act as tumor suppressor genes regulating the cell cycle gene expression, invasion and intrinsic tumorigenicity of melanoma cells. Carcinogenesis. 2012;33:687-93.
10. Bedikian AY, Richards J, Kharkevitch D, et al. A phase 2 study of high-dose allovectin-7 in patients with advanced metastatic melanoma. Melanoma Res. 2010;20:218-26.
11. Andtbacka RHI, Gonzalez R, Wloch MK, et al. A phase 3 clinical trial to evaluate the safety and efficacy of treatment with 2 mg intralesional Allovectin-7® compared to dacarbazine (DTIC) or temozolomide (TMZ) in subjects with recurrent metastatic melanoma. 10th International Meeting of the Society for Melanoma Research; 2013 Nov 18; Philadelphia, PA.
12. Daud A, Algazi P, Ashworth MT, et al. Systemic antitumor effect and clinical response in a phase 2 trial of intratumoral electroporation of plasmid interleukin-12 in patients with advanced melanoma. American Society of Clinical Oncology 2014 Annual Meeting; 2014 May 30–Jun 3; Chicago, IL. Abstr 9025.
13. Kaufman HL, Andtbacka RHI, Collichio FA, et al. Primary overall survival from OPTiM, a randomized phase III trial of talimogene laherparepvec (T-VEC) versus subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment of unresected stage IIIB/C and IV melanoma. American Society of Clinical Oncology 2014 Annual Meeting; 2014 May 30–Jun 3; Chicago, IL. Abstr 9008a.
14. Andtbacka RH, Ross MI, Delman K, et al. Responses of injected and uninjected lesions to intralesional talimogene laherparepvec (T-VEC) in the OPTiM study and the contribution of surgery to response. Society of Surgical Oncology 67th Annual Meeting; 2015 Mar 12–15; Phoenix, AZ. Abstr 52.2015.
15. Wachter EA, Dees C, Harkins J, et al. Functional imaging of photosensitizers using multiphoton microscopy. Proc. SPIE. 2002;4620:143.
16. Agarwala SS, Thompson JF, Smithers BM, et al. Immuno-hemoablation of metastatic melanoma with intralesional Rose Bengal (PV-10). European Society for Medical Oncology 2012 Annual Meeting; 2012 Sep 28–Oct 2; Vienna, Austria. Abstr 1137P.
17. Agarwala SS, Thompson JF, Smithers BM, et al. Efficacy of intralesional Rose Bengal in patients receiving injection of all existing melanoma in phase II study PV-10-MM-02. American Society of Clinical Oncology 2014 Annual Meeting; 2014 May 30–Jun 3; Chicago, IL. Abstr 9027.
18. Agarwala S. PV-10. 2nd European Post-Chicago Melanoma Meeting; 2012 Jun 21–22; Munich, Germany. https://www.pvct.com/news/PV-10_Second-PostASCO-22Jun2012.pdf. Accessed April 1, 2016.
19. US National Institutes of Health. PV-10 vs chemotherapy or oncolytic viral therapy for treatment of locally advanced cutaneous melanoma. https://clinicaltrials.gov/ct2/show/NCT02288897. Accessed April 1, 2016.
20. Andtbacka RHI, Curti B, Kaufman H, et al. CALM study: secondary endpoints of a phase II study of a novel oncolytic immunotherapeutic agent, coxsackievirus A21, delivered intratumorally in patients with advanced malignant melanoma. Ann Oncol. 2014;25(suppl 4):iv374-iv393.
21. Andtbacka RHI, Curti BD, Hallmeyer S, et al. Phase II CALM extension study: Coxsackievirus A21 delivered intratumorally to patients with advanced melanoma induces immune-cell infiltration in the tumor microenvironment. J Immunother Cancer. 2015;3(suppl 2):P343.
22. Promising strategy for advanced melanoma: intralesional therapies plus systemic immunotherapies. Cancer Watch. 2014;23:186-7.
23. Puzanov I, Milhem MM, Andtbacka RHI, et al. Primary analysis of a phase 1b multicenter trial to evaluate safety and efficacy of talimogene laherparepvec (T-VEC) and ipilimumab (ipi) in previously untreated, unresected stage IIIB-IV melanoma. J Clin Oncol. 2014;32(suppl 5s):abstr 9029.
24. Ribas A, Puzanov I, Gajewski T, et al. A multicenter, open-label trial of talimogene laherparepvec (T-VEC) plus pembrolizumab vs pembrolizumab monotherapy in previously untreated, unresected, stage IIIB-IV melanoma. J Clin Oncol. 2015;33(suppl):abstr TPS9081.
25. Fuerst ML. T-VEC plus pembrolizumab combo shows activity in advanced melanoma. http://www.cancernetwork.com/smr-2015/t-vec-plus-pembrolizumab-combo-shows-activity-advanced-melanoma. Accessed April 1, 2016.
26. Curti B, Richards J, Faries M, et al. Phase 1b study of a novel immunotherapy combination therapy of intralesional Coxsackievirus A21 and systemic ipilimumab in patients with advanced melanoma. American Association for Cancer Research 2016 Annual Meeting; 2016 Apr 16–20; New Orleans, LA. Abstr CT021.
27. Provectus Biopharmaceuticals, Inc. Provectus Biopharmaceuticals confirms first patients dosed in trials of PV-10 for melanoma. https://www.pvct.com/pressrelease.html?article=20160112.1. Accessed April 1, 2016.
28. US National Institutes of Health. Efficacy and safety of talimogene laherparepvec neoadjuvant treatment plus surgery versus surgery alone for melanoma. https://clinicaltrials.gov/ct2/show/NCT02211131. Accessed April 1, 2016.
