- Email: [email protected]
Intralesional injection of herpes simplex virus 1716 in metastatic melanoma
RESEARCH LETTERS
Research letters
Intralesional injection of herpes simplex virus 1716 in metastatic melanoma Rona M MacKie, Barry Stewart, S Moira Brown We have previously shown that avirulent but replicationcompetent herpes simplex virus (HSV) 1716 causes cell death in human melanoma cell lines in vitro and selectively replicates in melanoma tissue in nude mice. We now present a pilot study of intratumoral injection of HSV1716 into subcutaneous nodules of metastatic melanoma in five patients with stage 4 melanoma. Two patients each received one injection, two received two injections, and one received four injections of 103 plaque-forming units HSV1716. In one patient, flattening of previously palpable tumour nodules was seen 21 days after two direct injections of HSV1716, and in injected nodules from all three patients who received two or more injections there was microscopic evidence of tumour necrosis. Immunohistochemical staining of injected nodules revealed evidence of virus replication confined to tumour cells. These findings suggest that HSV1716 is non-toxic and could be of therapeutic benefit in patients with metastatic melanoma.
Melanocytes from which malignant melanoma develops are derived in fetal life from the neural crest; neurotropic viruses therefore localise in melanoma cells. Herpes simplex virus (HSV) is neurotropic and its genome contains a gene, RL1, whose protein product ICP34.5 is a specific virulence determinant. The mutant virus HSV1716,1 which is deficient in ICP34.5, has a median lethal dose (LD50) of greater than 106 plaque-forming units (pfu), compared with 2 pfu for the parental wild-type strain 17. Despite this reduced lethality, it replicates as efficiently as wild-type virus in actively dividing cells.2 In tumour lines and in a range of cancer-model systems, HSV1716 has led to tumour killing and improved survival times of tumour-bearing animals.3 Specifically, HSV1716 replicates preferentially in human melanoma cell lines in vitro, causing cell lysis and death. Injection of HSV1716 into intracranial murine melanoma in C57 Black mice led to an increase in time to development of neurological symptoms, tumour regression, and long-term survival of some animals. In human melanoma cell lines injected into SCID mice and subsequently directly injected with HSV1716, virus replication was confined exclusively to melanoma tissue and tumour regression was achieved.4 Taken together, these studies indicated that HSV1716 is non-toxic and of therapeutic benefit. To assess the therapeutic potential of HSV1716 in human disease, we did a preliminary study of toxicity and possible efficacy in five patients with metastatic melanoma. All patients gave written informed consent, and the study was approved by the UK Gene Therapy Advisory Committee, the UK Medicines Control Agency, and the local ethics committee. This study complements our recently reported trial of HSV1716 in patients with recurrent glioma.5 All five patients had pathologically proven stage 4 metastatic melanoma which was not surgically resectable, including softtissue nodules accessible to direct intratumoral injection at sites other than on the face. For each patient, a test nodule was injected with 103 pfu HSV1716 (supplied by Q1 Biotech, Glasgow, UK) in 1 mL diluent, and a second nodule was
THE LANCET • Vol 357 • February 17, 2001
injected with 1 mL sterile saline. All patients were monitored for systemic adverse effects and the injected nodules were examined for signs of inflammation. The first two patients had the injected nodules excised 14 days after injection for pathological and immunohistochemical examination. Patients 3, 4, and 5, had a second 1 mL injection of 103 pfu HSV1716 on day 14 and the nodule excised on day 21. The fifth patient had a second set of two injections of 103 pfu HSV 2 weeks apart injected into a different nodule 8 weeks after the first series of injections (four injections in total). Fresh frozen sections from excised nodules were stained with ZIF11 antibody (supplied by H Marsden), which is specific for the HSV 65 kD DNA-binding protein. All patients tolerated HSV1716 well. All were seropositive for HSV before virus injection, and their IgG and IgM titres did not substantially alter during the trial. There was no virus
Figure 1: Sections from nodules of secondary melanoma excised from patient 5 (stained with haematoxylin and eosin) A= uninjected nodule. B: nodule excised 21 days after two injections each of 103 pfu HSV1716 on days 1 and 14. A focus of necrotic tumour cells associated with abundant free melanin pigment is present.
525
For personal use only. Reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
shedding nor reactivation of endogenous latent HSV in any patient. No clinical change was seen in the HSV-injected nodule in four of the patients. However, in patient 5, who had two virus injections in each of two nodules, there was a flattening of both injected nodules. No patient showed evidence of regression of tumour mass at any other adjacent or distant site of tumour. Pathological examination of the excised nodules showed necrotic melanoma cells in three of the virus-injected excised nodules from the three patients who had two injections on days 1 and 14 and excision on day 21. Nodules excised from patients 3 and 4 both showed this necrosis adjacent to areas of apparently viable tumour cells, but no morphological evidence of damage to adjacent normal tissues. The nodule from patient 5 showed the most striking tumour cell necrosis, with adjacent foci of free melanin (figure 1). No cell death was seen in the saline-injected nodules. Immunohistochemical examination of the excised HSV1716-injected nodules showed evidence of viral replication within the limits of the tumour mass (figure 2). In all five patients, the HSV 65 kD DNA-binding protein was seen in the melanoma cells from the virus-treated nodules, with no antigen staining in the adjacent normal connective tissue. The basal layer of the epidermis was also unstained, suggesting that, in contrast to melanoma cells, normal melanocytes were antigen-free. Until now, use of ICP34.5-deficient HSV has been described only in glioma where tumours are not readily accessible after virus injection and hence there has been no opportunity to obtain clear evidence of virus replication. We showed that at least at an input dose of 103 pfu, HSV1716 is non-toxic to human beings when directly injected into secondary melanoma nodules. As expected with a low input dose of 103 pfu HSV1716, the cell death in the melanoma nodules was localised to the region adjacent to the HSV1716 injection site. Immunohistochemical staining of sections from virus-injected and saline-injected nodules showed that HSV antigen was confined to the melanoma cells in virus-injected nodules only with no evidence of virus antigen in normal cells, and specifically none in the basal layer of the epidermis. This finding is encouraging in light of reports from animal studies that the metabolically active cells of this layer such as basal layer keratinocytes, and also normal melanocytes might support HSV1716 replication. In the cascade of HSV protein expression, the 65 kD DNA-binding protein is made early in the infectious cycle and its presence in the biopsy specimens is evidence of de-novo protein synthesis and hence virus replication. The extent of antigen presentation in the melanoma nodules supports the assumption of virus-induced tumour necrosis.
The fact that each of the patients was seropositive for HSV before HSV1716 injection indicates that the ability of the virus to replicate is not blocked by previous exposure to HSV. That the nodules that flattened had more than one virus injection also indicates that the second dose of virus was capable of replication and was not blocked by an immune response. This pilot study shows that HSV1716 replicates in melanoma cells, causes tumour-cell necrosis, and is not toxic. The results are sufficiently encouraging to continue with higher doses of HSV1716 in patients with metastatic melanoma. This study was supported by the Scottish Executive, Department of Health, Chief Scientist Office. 1
2
3
4
5
Maclean AR, Fareed MU, Robertson L, Harland J, Brown SM. Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence related sequences in Glasgow strain 17+ between immediate early gene 1 and the ‘a’ sequence. J Gen Virol 1991; 72: 631–39. Brown SM, Harland J, Maclean AR, Podlech J, Clements JB. Cell type and cell state determine differentiated in vitro growth of nonneurovirulant ICP34.5-negative herpes simplex virus. J Gen Virol 1994; 75: 2367–77. Kesari S, Randazzo BP, Valyi-Nagy T, et al. A mutant herpes simplex virus replicates in brain tumours but not in neurons derived from a human embryonal carcinoma cell line. Lab Invest 1995; 73: 636–48. Randazzo BP, Bhat MG, Kesari S, Fraser NW, Brown SM. Treatment of experimental subcutaneous human melanoma with a replication restricted herpes simplex virus mutant. J Invest Dermatol 108: 933–37. Rampling R, Cruikshank G, Papanatassiou V, et al. Toxicity evaluation of replication competent herpes simplex virus ICP34.5 null mutant 1716 in patients with recurrent glioma. Gene Ther 2000; 7: 859–66.
Department of Dermatology, University of Glasgow, Glasgow G12 8QQ, UK (R M MacKie FRCP); and Glasgow University, Neurovirology Research Laboratories, Institute of Neurological Sciences, Southern General Hospital, Glasgow (B Stewart PhD, S M Brown PhD) Correspondence to: Prof Rona MacKie (e-mail: [email protected])
Controlled oxygen therapy and carbon dioxide retention during exacerbations of chronic obstructive pulmonary disease Edward D Moloney, John L Kiely, Walter T McNicholas
Figure 2: Immunohistochemical staining of frozen sections taken from post-injection nodule excised from patient 1
Hypoxaemic patients with exacerbations of chronic obstructive pulmonary disease (COPD) are at some risk of carbon dioxide (CO2) retention during oxygen therapy. We quantified the risk of CO2 retention with oxygen therapy in COPD in 24 consecutive patients presenting to the accident and emergency department with acute exacerbations associated with hypercapnic respiratory failure (partial arterial pressure of oxygen [PaO2] <8 kPa and partial pressure of CO2 [PaCO2] ⭓6·5 kPa). Only three patients developed clinically important CO2 retention (defined as a rise in PaCO2 >1 kPa) with controlled oxygen therapy (24–40% by Venturi mask to maintain the oxygen saturation at 91–92%). These patients presented with more severe hypercapnia, but all three required only low-flow oxygen (24–28%). These findings suggest only a small risk of aggravating hypercapnia with controlled oxygen supplementation.
Red reaction product is seen only on tumour cells.
Hypoxaemic
526
patients
with
exacerbations
of
chronic
THE LANCET • Vol 357 • February 17, 2001
For personal use only. Reproduce with permission from The Lancet Publishing Group.
Research letters
Intralesional injection of herpes simplex virus 1716 in metastatic melanoma Rona M MacKie, Barry Stewart, S Moira Brown We have previously shown that avirulent but replicationcompetent herpes simplex virus (HSV) 1716 causes cell death in human melanoma cell lines in vitro and selectively replicates in melanoma tissue in nude mice. We now present a pilot study of intratumoral injection of HSV1716 into subcutaneous nodules of metastatic melanoma in five patients with stage 4 melanoma. Two patients each received one injection, two received two injections, and one received four injections of 103 plaque-forming units HSV1716. In one patient, flattening of previously palpable tumour nodules was seen 21 days after two direct injections of HSV1716, and in injected nodules from all three patients who received two or more injections there was microscopic evidence of tumour necrosis. Immunohistochemical staining of injected nodules revealed evidence of virus replication confined to tumour cells. These findings suggest that HSV1716 is non-toxic and could be of therapeutic benefit in patients with metastatic melanoma.
Melanocytes from which malignant melanoma develops are derived in fetal life from the neural crest; neurotropic viruses therefore localise in melanoma cells. Herpes simplex virus (HSV) is neurotropic and its genome contains a gene, RL1, whose protein product ICP34.5 is a specific virulence determinant. The mutant virus HSV1716,1 which is deficient in ICP34.5, has a median lethal dose (LD50) of greater than 106 plaque-forming units (pfu), compared with 2 pfu for the parental wild-type strain 17. Despite this reduced lethality, it replicates as efficiently as wild-type virus in actively dividing cells.2 In tumour lines and in a range of cancer-model systems, HSV1716 has led to tumour killing and improved survival times of tumour-bearing animals.3 Specifically, HSV1716 replicates preferentially in human melanoma cell lines in vitro, causing cell lysis and death. Injection of HSV1716 into intracranial murine melanoma in C57 Black mice led to an increase in time to development of neurological symptoms, tumour regression, and long-term survival of some animals. In human melanoma cell lines injected into SCID mice and subsequently directly injected with HSV1716, virus replication was confined exclusively to melanoma tissue and tumour regression was achieved.4 Taken together, these studies indicated that HSV1716 is non-toxic and of therapeutic benefit. To assess the therapeutic potential of HSV1716 in human disease, we did a preliminary study of toxicity and possible efficacy in five patients with metastatic melanoma. All patients gave written informed consent, and the study was approved by the UK Gene Therapy Advisory Committee, the UK Medicines Control Agency, and the local ethics committee. This study complements our recently reported trial of HSV1716 in patients with recurrent glioma.5 All five patients had pathologically proven stage 4 metastatic melanoma which was not surgically resectable, including softtissue nodules accessible to direct intratumoral injection at sites other than on the face. For each patient, a test nodule was injected with 103 pfu HSV1716 (supplied by Q1 Biotech, Glasgow, UK) in 1 mL diluent, and a second nodule was
THE LANCET • Vol 357 • February 17, 2001
injected with 1 mL sterile saline. All patients were monitored for systemic adverse effects and the injected nodules were examined for signs of inflammation. The first two patients had the injected nodules excised 14 days after injection for pathological and immunohistochemical examination. Patients 3, 4, and 5, had a second 1 mL injection of 103 pfu HSV1716 on day 14 and the nodule excised on day 21. The fifth patient had a second set of two injections of 103 pfu HSV 2 weeks apart injected into a different nodule 8 weeks after the first series of injections (four injections in total). Fresh frozen sections from excised nodules were stained with ZIF11 antibody (supplied by H Marsden), which is specific for the HSV 65 kD DNA-binding protein. All patients tolerated HSV1716 well. All were seropositive for HSV before virus injection, and their IgG and IgM titres did not substantially alter during the trial. There was no virus
Figure 1: Sections from nodules of secondary melanoma excised from patient 5 (stained with haematoxylin and eosin) A= uninjected nodule. B: nodule excised 21 days after two injections each of 103 pfu HSV1716 on days 1 and 14. A focus of necrotic tumour cells associated with abundant free melanin pigment is present.
525
For personal use only. Reproduce with permission from The Lancet Publishing Group.
RESEARCH LETTERS
shedding nor reactivation of endogenous latent HSV in any patient. No clinical change was seen in the HSV-injected nodule in four of the patients. However, in patient 5, who had two virus injections in each of two nodules, there was a flattening of both injected nodules. No patient showed evidence of regression of tumour mass at any other adjacent or distant site of tumour. Pathological examination of the excised nodules showed necrotic melanoma cells in three of the virus-injected excised nodules from the three patients who had two injections on days 1 and 14 and excision on day 21. Nodules excised from patients 3 and 4 both showed this necrosis adjacent to areas of apparently viable tumour cells, but no morphological evidence of damage to adjacent normal tissues. The nodule from patient 5 showed the most striking tumour cell necrosis, with adjacent foci of free melanin (figure 1). No cell death was seen in the saline-injected nodules. Immunohistochemical examination of the excised HSV1716-injected nodules showed evidence of viral replication within the limits of the tumour mass (figure 2). In all five patients, the HSV 65 kD DNA-binding protein was seen in the melanoma cells from the virus-treated nodules, with no antigen staining in the adjacent normal connective tissue. The basal layer of the epidermis was also unstained, suggesting that, in contrast to melanoma cells, normal melanocytes were antigen-free. Until now, use of ICP34.5-deficient HSV has been described only in glioma where tumours are not readily accessible after virus injection and hence there has been no opportunity to obtain clear evidence of virus replication. We showed that at least at an input dose of 103 pfu, HSV1716 is non-toxic to human beings when directly injected into secondary melanoma nodules. As expected with a low input dose of 103 pfu HSV1716, the cell death in the melanoma nodules was localised to the region adjacent to the HSV1716 injection site. Immunohistochemical staining of sections from virus-injected and saline-injected nodules showed that HSV antigen was confined to the melanoma cells in virus-injected nodules only with no evidence of virus antigen in normal cells, and specifically none in the basal layer of the epidermis. This finding is encouraging in light of reports from animal studies that the metabolically active cells of this layer such as basal layer keratinocytes, and also normal melanocytes might support HSV1716 replication. In the cascade of HSV protein expression, the 65 kD DNA-binding protein is made early in the infectious cycle and its presence in the biopsy specimens is evidence of de-novo protein synthesis and hence virus replication. The extent of antigen presentation in the melanoma nodules supports the assumption of virus-induced tumour necrosis.
The fact that each of the patients was seropositive for HSV before HSV1716 injection indicates that the ability of the virus to replicate is not blocked by previous exposure to HSV. That the nodules that flattened had more than one virus injection also indicates that the second dose of virus was capable of replication and was not blocked by an immune response. This pilot study shows that HSV1716 replicates in melanoma cells, causes tumour-cell necrosis, and is not toxic. The results are sufficiently encouraging to continue with higher doses of HSV1716 in patients with metastatic melanoma. This study was supported by the Scottish Executive, Department of Health, Chief Scientist Office. 1
2
3
4
5
Maclean AR, Fareed MU, Robertson L, Harland J, Brown SM. Herpes simplex virus type 1 deletion variants 1714 and 1716 pinpoint neurovirulence related sequences in Glasgow strain 17+ between immediate early gene 1 and the ‘a’ sequence. J Gen Virol 1991; 72: 631–39. Brown SM, Harland J, Maclean AR, Podlech J, Clements JB. Cell type and cell state determine differentiated in vitro growth of nonneurovirulant ICP34.5-negative herpes simplex virus. J Gen Virol 1994; 75: 2367–77. Kesari S, Randazzo BP, Valyi-Nagy T, et al. A mutant herpes simplex virus replicates in brain tumours but not in neurons derived from a human embryonal carcinoma cell line. Lab Invest 1995; 73: 636–48. Randazzo BP, Bhat MG, Kesari S, Fraser NW, Brown SM. Treatment of experimental subcutaneous human melanoma with a replication restricted herpes simplex virus mutant. J Invest Dermatol 108: 933–37. Rampling R, Cruikshank G, Papanatassiou V, et al. Toxicity evaluation of replication competent herpes simplex virus ICP34.5 null mutant 1716 in patients with recurrent glioma. Gene Ther 2000; 7: 859–66.
Department of Dermatology, University of Glasgow, Glasgow G12 8QQ, UK (R M MacKie FRCP); and Glasgow University, Neurovirology Research Laboratories, Institute of Neurological Sciences, Southern General Hospital, Glasgow (B Stewart PhD, S M Brown PhD) Correspondence to: Prof Rona MacKie (e-mail: [email protected])
Controlled oxygen therapy and carbon dioxide retention during exacerbations of chronic obstructive pulmonary disease Edward D Moloney, John L Kiely, Walter T McNicholas
Figure 2: Immunohistochemical staining of frozen sections taken from post-injection nodule excised from patient 1
Hypoxaemic patients with exacerbations of chronic obstructive pulmonary disease (COPD) are at some risk of carbon dioxide (CO2) retention during oxygen therapy. We quantified the risk of CO2 retention with oxygen therapy in COPD in 24 consecutive patients presenting to the accident and emergency department with acute exacerbations associated with hypercapnic respiratory failure (partial arterial pressure of oxygen [PaO2] <8 kPa and partial pressure of CO2 [PaCO2] ⭓6·5 kPa). Only three patients developed clinically important CO2 retention (defined as a rise in PaCO2 >1 kPa) with controlled oxygen therapy (24–40% by Venturi mask to maintain the oxygen saturation at 91–92%). These patients presented with more severe hypercapnia, but all three required only low-flow oxygen (24–28%). These findings suggest only a small risk of aggravating hypercapnia with controlled oxygen supplementation.
Red reaction product is seen only on tumour cells.
Hypoxaemic
526
patients
with
exacerbations
of
chronic
THE LANCET • Vol 357 • February 17, 2001
For personal use only. Reproduce with permission from The Lancet Publishing Group.