Daniel R. Vlock

Phase Ib trial of the effect of peritumoral and intranodal injections of interleukin-2 in patients with advanced squamous cell carcinoma of the head and neck : an eastern cooperative oncology group trial

Thirty-six patients with unresectable squamous cell carcinoma of the head and neck were entered into a phase Ib trial evaluating the toxicity, maximally tolerated dose (MTD), and immunomodulating effects of locally administered interleukin-2 (IL-2). Patients received daily IL-2 injected perilesionally in divided doses in each of four quadrants and bilaterally into the superior jugular lymph nodes. The dose of IL-2 began at 200 U/day and was escalated to 4 x 10(6) U/day in groups of six patients. Overall, regionally administered IL-2 was well tolerated. The most frequently encountered toxicities were fever, hepatotoxicity, and hypotension. Dose-limiting toxicity was encountered at 4 x 10(6) U. Of the 36 patients treated, 2 partial responses were noted at 2,000 and 4 x 10(6) U. We conclude that regionally administered IL-2 is well tolerated in patients with head and neck cancer and that the MTD is 2 x 10(6) U/day, similar to what has been reported with systemically administered IL-2. Although the overall response rate was low, it may be improved with prolonged administration of IL-2 or by combining it with other biologic or cytotoxic agents.

Phase II trial of interferon-alpha in locally recurrent or metastatic squamous cell carcinoma of the head and neck: immunological and clinical correlates.

The objective of this study was to study the antitumor, host toxicity, and immunomodulatory effects of recombinant interferon-alpha 2b (IFN) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Seventy-one patients with recurrent or metastatic SCCHN were entered into a phase II noncomparative randomized trial of IFN at two dosage schedules. Eligible patients with histologically proven SCCHN were randomized to receive low-dose IFN, 6 x 10(6) U/m2 daily x 3 every 4 weeks or high-dose IFN, 12 x 10(6) U/m2, 3 x/week. Pretreatment levels of natural killer (NK) activity, CD3, CD4, CD5, CD8, CD16, CD19, CD56, DR, and the CD4/CD8 ratio were evaluated for any relationship with survival. The toxicity encountered in patients receiving low-dose IFN was for the most part mild to moderate. With high-dose IFN, toxicity was greater with significantly more episodes of grade 3 and 4 toxicity encountered. Dosage reduction was required in the majority of patients receiving high-dose IFN. Of the four lethal complications, only one was thought to be possibly associated with therapy. Of the 32 evaluable patients receiving low-dose IFN, there were 1 complete response, 1 stable disease, 24 patients with progressive disease, and 6 unevaluable. Of the 29 evaluable patients taking high-dose IFN, there were 2 complete responses, 7 with stable disease, 16 with progressive disease, and 4 patients were unevaluable. Median survival in the two arms was similar (6.2 months). Because it was postulated that a more prolonged exposure to IFN might be needed for it to be effective, patients receiving > or = 6 weeks of therapy were evaluated. Median survival in that subset was 10 and 12 months for patients receiving low- and high-dose IFN, respectively. None of the immune parameters tested was a significant predictor of survival when evaluated in all cases entered into study regardless of therapy duration. No difference in baseline NK activity was noted between patients who received < 6 or > or = 6 weeks of IFN (p = 0.90). However, among the 35 patients who received > or = 6 weeks of therapy, a high baseline NK activity was a significant predictor of the duration of survival (p = 0.04). IFN was well tolerated in patients with recurrent or metastatic SCCHN. The higher incidence of toxicity encountered in the high-dose arm could be ameliorated by reducing the dose 50%. In patients receiving 6 or more weeks of therapy, elevated baseline NK activity was associated with increases in survival, suggesting that IFN may play an immunomodulatory role. Although the overall response rates were low, disease stabilization was noted, suggesting an antiproliferative, noncytotoxic role of IFN in this group of heavily pretreated patients.

Tomographic gallium-67 citrate scanning: useful new surveillance for metastatic melanoma.

Conventional gallium scans are not useful to evaluate patients with metastatic melanoma. We evaluated a new method of tomographic gallium imaging. One hundred fourteen tomographic scans were obtained in a prospective surveillance study of 67 patients over a 3-year period. Scans were evaluated and compared to findings of independent clinical evaluations. Sensitivity of gallium identification of tumor involving peripheral lymph nodes and soft tissues, abdomen, mediastinum, and osseous sites was 68% to 100%; overall sensitivity of this technique is 82% with specificity of 99% in 570 organ system assessments. Analysis of discordant findings when a site was clinically occult but gallium-positive showed gallium uptake to be true-positive in six of seven lymphatic sites, three of three lung and mediastinal sites, six of six abdominal sites, but in no brain or bone sites. Gallium lesions identified by computed tomographic scans proved to be false-positive at one lymphatic and one bone site, and false-negative at four otherwise clinically evident lymph node and soft tissue sites, seven pulmonary sites, and four brain sites. Gallium tomographic scanning provides a composite assessment of melanoma and may eliminate the need for other studies.

Synergistic enhancement by interleukin-1 α of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice

Administration of interleukin-1 α (IL-1 α) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 α itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 αs ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 α on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 α and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 α and cDDP being analyzed through median dose-effect. In vitro, IL-1 α had no enhancing effect on the cDDP-mediated tumorcell kill. For examination of the in vivo efficacy of this regimen. RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 α and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 α /cDDP was dose-dependent, with significant enhancement by IL-1 α being observed (P<0.001), even at the lowest doses tested (2 mg/kg and 6 μg/kg for cDDP and IL-1 α, respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 α significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P<0.001). These results demonstrate that IL-1 α synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 α and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.

Tomographic gallium-67 citrate scanning. Useful new surveillance for metastatic melanoma

Editors Comment: This is the second in a series of papers being exchanged by ANNALS OF INTERNAL MEDICINE and ANNALS OF SURGERY for republication with the view that specially selected subjects will be of considerable interest to the readers of these journals and are exchanged accordingly.Conventional gallium scans are not useful to evaluate patients with metastatic melanoma. We evaluated a new method of tomographic gallium imaging. One hundred fourteen tomographic scans were obtained in a prospective surveillance study of 67 patients over a 3-year period. Scans were evaluated and compared to findings of independent clinical evaluations. Sensitivity of gallium identification of tumor involving peripheral lymph nodes and soft tissues, abdomen, mediastinum, and osseous sites was 68% to 100%; overall sensitivity of this technique is 82% with specificity of 99% in 570 organ system assessments. Analysis of discordant findings when a site was clinically occult but gallium-positive showed gallium uptake to be true-positive in six of seven lymphatic sites, three of three lung and mediastinal sites, six of six abdominal sites, but in no brain or bone sites. Gallium lesions identified by computed tomographic scans proved to be false-positive at one lymphatic and one bone site, and false-negative at four otherwise clinically evident lymph node and soft tissue sites, seven pulmonary sites, and four brain sites. Gallium tomographic scanning provides a composite assessment of melanoma and may eliminate the need for other studies.

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

SummaryIn previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.

Isolation and purification of a squamous cell carcinoma of the head and neck-associated antigen identified by autologous antibody

We have previously shown that detection of autologous antibody activity to squamous cell carcinoma of the head and neck may be augmented by dissociation of immune complexes. Western blot analysis with autologous antibody has identified a 60 kDa squamous cell carcinoma of the head and neck-associated antigen in spent media and immune complex-dissociated serum ultrafiltrate not recognized by normal human sera. Antigen-containing fractions of spent media were eluted from anion exchange columns immediately after serum albumin indicating that the antigen has an acidic pI < 4. Preparative purification of the squamous cell carcinoma antigen was accomplished by anion exchange of concentrated spent media (protein concentration 300 mg/ml) followed by lectin affinity chromatography with a Triticum vulgaris column. A single 60 kDa band was detected by silver stain and Western blot in antigen-containing fractions eluted following lectin affinity chromatography and SDS-PAGE. Final concentration of the antigen was determined to be 1 microgram/ml of protein with relative activity increased 1600 x over unfractionated spent media. We conclude that a squamous cell carcinoma of the head and neck-associated antigen, detected by autologous antibody, is an acidic 60 kDa glycoprotein.

Purification and partial characterization of a shed 66 kDa melanoma-associated antigen identified by autologous antibody

We have previously reported the isolation of a 66 kDa melanoma-associated antigen, identified by autologous antibody, in serum and unfractionated spent tissue culture media by Western blot analysis. The antigen, detected by autologous serum S150, was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma and head and neck carcinoma cell lines. S150 did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, autologous cultured lymphocytes or fetal calf serum. To further characterize the antigen, spent tissue culture media, obtained from autologous melanoma cell line, Y-Mel 84:420, was separated by an isoelectric focusing column. Unabsorbed control serum S150 was noted to have a maximum titer of 1:2040 against autologous melanoma cells as measured by protein A hemadsorption. Following isoelectric focusing the greatest decrease in autologous antibody titer (30-fold) occurred with fractions having a pI between 2 and 3. Further resolution of the antigen was accomplished with high-pressure ion-exchange chromatography. One of these fractions showed a significantly higher concentration of antigen and was distinctly resolved from bulk serum albumin. Subsequent Western blot analysis, with autologous antibody, of the isolated antigen-containing fraction, confirmed the presence of a single 66 kDa band. Exposure of the antigen, purified by high-pressure ion-exchange chromatography, to neuraminidase ablated recognition by autologous antibody and suggests that sialic acid is present on the protein and may be part of the antigenic epitope. Binding of antigen, obtained following DEAE anion exchange chromatography, was noted to lectins derived from Triticum vulgaris, Dolichos biflorus and Lycopersicon esculentum. Preparative purification of the antigen was accomplished by anion exchange followed by lectin affinity chromatography with a Dolichos biflorus column. Antigen obtained following lectin affinity chromatography subjected to SDS-PAGE and silver stain revealed a single band at 66 kDa. We conclude that a melanoma-associated antigen detected by autologous antibody in spent tissue culture media is an unusually acidic glycoprotein (pI 2-3).

Biologic response modulation by tumor necrosis factor alpha (TNFα) in a phase Ib trial in cancer patients

During a phase I study of recombinant human tumor necrosis factor (TNF) in cancer patients, serial immune studies were performed and analyzed for effects of TNF. The TNF (specific activity 9.6 X 106 U/mg protein, <5.0 endotoxin units/mg protein) was given over 2 h intravenously on days 1, 8–12, 29–33, 50–54, and 71–75 at doses of 40, 80, 160, 200, and 240 μg/m2. Immunologic testing was performed before therapy three times and subsequently on days 2, 8, 10, 12, 29, 33, 50, 54, 71, 75, and off-study two times. Immune parameters evaluated included cytotoxicity [natural killer (NK), spontaneous lymphokine activated killer cells (LAK), LAK, and monocyte], cytokine production [spontaneous and stimulated interferon (IFN)-γ and interleukin (IL)-2], superoxide production [resting and stimulated polymorphonuclear leukocytes (PMN) and mononuclear cells (MNC)], and phenotype of peripheral blood lymphocyte subsets (CD3, CD4, CD8, CD16, CD56, CD19). Data were analyzed for long-term effects, the effect after 1 day of treatment (day 1), and for weekly effect (change from day 1 to day 5 of a given treatment week). Significant decreases were seen in the spontaneous cytotoxicity of peripheral blood NK cells and IL-2-inducible LAK cells, whereas increases in spontaneous peripheral blood LAK activity were seen with TNF treatment. Consistent increases in superoxide production of resting PMN and MNC were demonstrated, with late increases in superoxide production by opsonized, zymosan-treated PMN. No spontaneous IFN-γ or IL-2 were noted in sera with treatment, but production of IL-2 by MNCs rose with TNF treatment. During 5 days of TNF treatment, the percentages of circulating CD8+ and CD56+ cells decreased, whereas that of CD4+ and CD19+ cells increased significantly and consistently, as determined by a multivariate analysis. Significant changes in several independently measured parameters were observed, including a dose-related diminished production of IFN-γ by MNC stimulated by phytohemagglutinin and increased in vitro-generated LAK activity. Because there was no clinical response in this trial, no association of immunologic change with clinical response can be made. No biologically optimal dose of TNF was evident. The data suggest that TNF may act as a trigger cytokine, initiating a broad immune/inflammatory response.