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In a prospective randomized trial in patients with metastatic melanoma we compared two agents that had been used for metastatic melanoma intratumoral injections. Each patient had progressive metastatic disease no longer surgically controllable. Multiple metastases included satellitosis in the form of progressive nodules around the previously excised original melanoma site, and/or in-transit metastases in the form of observable tumor nodules progressing in a linear fashion toward a lymph node bearing area. As patients were receiving intratumoral injections, serially collected blood samples were tested for general immunologic reactivity and anti-melanoma reactivity. Specificity controls included breast cancer and lung cancer extracts. Additionally, as a measure of cell-mediated immunity, the patients were serially skin tested against antigens to measure general and melanoma-specific immunity. Depending on the patients’ clinical courses, we have divided the patients retrospectively into groups whose clinical courses were either better or worse than their cohorts, and determined the relationship between the immune testing and the clinical courses that the patients were experiencing as the serial testing was being conducted. Additionally, in a group of similar patients that were ‘cured for life’, we analyze their treatment in light of therapeutic attempts made by others to similarly haptenize melanoma antigens. We describe potential synergies between newly discovered melanoma therapies and intratumoral injection treatments and point out potential combination therapies that may offer the potential for enhancement of antitumor effects without increased systemic toxicity, a desirable goal now that combinations of recently acceptable immunotherapies have been associated with severe potential toxicities including death.

We have demonstrated in a randomized prospective study the superiority of intratumoral (intralesional) dinitrochlorobenzene (DNCB) compared to intratumoral (intralesional) bacillus Calmette-Guerin (BCG) in the treatment of progressive metastatic melanoma. The metastatic melanoma was in the form of satellitosis and/or in-transit metastases. We now demonstrate the ability of intralesional DNCB to permanently cure a selected group of patients with the same clinical criteria, and describe their clinical characteristics and treatment regimens. The described cured patients were followed for their remaining lifetimes, for up to 30 years, after being immunotherapeutically rendered free of metastatic cancer. They were each female, between the ages of 51 and 56 when intratumoral treatments were begun. Treatment was for progressive cutaneous and subcutaneous metastatic disease in the leg in two cases and for rapidly spreading scalp and forehead metastases in another. In each case the disease was not surgically controllable. Treatments were continued for 6 to 26 months. Subsequently the patients survived tumor free for either 18 years, dying at age 83; or for 24 years, dying at age 89; or for 30 years, dying at age 97. There has been a significant interest in recent years in immunotherapy for advanced cancer. The absence of systemic toxicity in the cured patients reported herein provides a basis for consideration of combining intratumoral treatments with current effective but systemically more toxic immunotherapeutic approaches to some metastatic cancers. Intratumoral treatments may be particularly applicable in cases of uncontrollable melanoma with cutaneous metastases, without evidence of distant spread, as in the current report.

e21602Background: In a previous randomized prospective study in patients receiving intralesional (IL) dinitrochorobenzene (DNCB) or IL Bacillus Calmette-Guerin (BCG) we determined that serial in vi...

59 Background: In a previous study involving in vitro serial testing in patients receiving IL BCG or IL DNCB we determined that measurements of serial in vitro phytohemagglutinin stimulation levels correlated with the patients’ clinical courses. The current evaluation attempted to determine which in vivo skin test studies were useful in this setting. Methods: All patients had locally progressive melanoma satellosis, or in-transit metastases, but no evidence of distant metastasis past regional lymph nodes. Patients had been randomized to repeatedly receive either IL BCG (9 patients), or IL DNCB (9 patients) into cutaneous or subcutaneous metastases. A "BCG worse outcome group" consisted of 6 patients in whom distant metastasis was detected within 2 to 20 months, and who died within 9 to 24 months. A "BCG better outcome group" consisted of 3 patients who were alive and in whom no distant metastases were detected after 12 to 39 months. Similarly, the 9 patients randomized to be treated with intralesional DNCB were also subsequently divided into two groups: A "DNCB worse outcome group" consisted of 6 patients in whom distant metastasis was detected within 5 to 11 months, and who died within 6 to 12 months. A "DNCB better outcome group" consisted of 3 patients who were alive and in whom no distant metastases were detected after 8 to 20 months. Following informed consent, patients were serially skin tested, as possible, with Purified Protein Derivative antigen (PPD), Mumps antigen, Streptokinase/Streptodornase (SKSD), and inactivated melanoma extracts, using coded syringes. Results: In comparing the two "better outcome" groups with the two "worse outcome" groups, patients who prior to treatment had a positive reaction to melanoma extracts appeared to fare better than those who did not. All patient groups tended to react positively to SKSD, a general sign of immune capability. Conclusions: Among the skin tests compared, testing with melanoma extract appeared to correlate best with clinical courses in patients undergoing BCG or DNCB intralesional treatments for progressive melanoma satellosis or in-transit metastases.

A review of a 14-year experience with prophylactic pigmented skin lesion removal is presented. Data obtained during a 4-year interval of this 14-year experience is analyzed specifically. During this 4-year interval, 250 patients with melanoma were seen. Of these patients, 75 with a history of stage I (localized) melanoma and three patients with stage II (history of controlled regionally metastatic melanoma) underwent removal of multiple skin lesions on a prophylactic basis. Of the removed lesions, 28% showed hyperplasia, atypia, dysplasia, or melanoma. Nine unsuspected in situ, or level I melanomas, and three unsuspected invasive melanomas were removed from these 75 melanoma patients while excising lesions prophylactically during the 4-year interval. It is estimated that four to six additional melanomas were prevented by excision of precursor lesions. During the same 4-year interval, an additional 112 of approximately 1000 patients without a previous history of melanoma underwent prophylactic lesion removals. In 31% of the 112 patients, there was a history of melanoma in a first-degree relative. In 22% of the removed lesions there was hyperplasia, atypia, or dysplasia. Three cases of melanoma in situ were detected and it is estimated that an additional three to five cases of melanoma were prevented. Atypical findings occurred in 71, or 63%, of the patients biopsied, which represented 7% of the approximately 1000 patients screened. During the 4-year interval, an average of 17.7 lesions were removed from each of the 190 melanoma and nonmelanoma patients undergoing prophylactic skin lesion excision. This was accomplished in one to four sessions per patient. This average reflects only those patients who underwent one excision or more and does not include those patients treated without operation. When including the nonoperated patients screened during this interval, the average number of lesions removed was 2.7 per patient. Death from new melanomas was prevented during the 14-year period of this study as evidenced by the fact that no patient died or developed metastatic disease from a cutaneous melanoma that was not apparent or known about at the time of first examination.

Four patients developed serious hypotension and signs of disseminated intravascular coagulation shortly after a second round of Tice bacillus Calmette-Guérin (BCG) injections into locally recurrent cutaneous melanoma satellite nodules. Each of these patients survived following intensive therapy with isoniazid, pyridoxine, steroids, pressors, antibiotics, and cardio-renal support including, in one case, three acute hemodialyses. Plasma specimens from two of the four patients caused gelation of lysate from the amebocytes of Limulus polyphemus, indicating the presence of endotoxin or an endotoxin-like substance. In vitro studies on the BCG preparations led us to conclude that this endotoxin activity in the plasma is not the result of direct injection of endotoxin with the BCG preparation, but rather from release of endotoxin from endogenous sources, such as the intestinal tract during a period of relative hypotension following an allergic reaction. Prior immunity appeared to be the consistent factor in the toxic reactions reported herein. Finally, we present recommendations for serial monitoring of these patients and discuss the use of an alternative agent for intralesional therapy.

A case of retroauricular melanoma is presented, and the literature on auricular melanoma is reviewed. A method of ear preservation in the surgical treatment of auricular melanoma is discussed.

Review of a 19 year experince in melanoma patients undergiong lymphadenectomy at the National Cancer Institute reveled that the preoperative asesment of the staus of the regional lymph nodes was accurate 91% of the tme when the surgeon felt the nodes were clinically positive, and accurate 79% of the time when the nodes were judged clinically negative. The 10-year survival in patients with one to three histologically positive nodes. or to positive nodes was 50–55%, compared to a 25% 8-year survival in patients with four or more histologically positive nods. Stepwise multivariate evaluation of prognostic factors indicated that the most important factor for predicting prognosis is the number of nodes histologically involved. Node palpability was the second most important factor becuause of its high correlation with number of nodes histologically ivolved. Node palpability was the second most important factor because of its high correlation with number of nodes histologically involved. node palpabilty was the second most important factor because of its high correlation with number of nodes histologically involved. Site of melanoma was te third most important factor, as patients with extremity (upper or lower) melanoma had a better survival (P = 0.002) than patients with axial melanoma (trunk or head and neck). Five years following lymphadenectomy there appeared to be substantial differences in survival according to differences in the level of invasion of the primary lesion, however, these differences wer not nearly as pronounced 10 years following node dissection. The division of melanoma thicknesses into 1.50 mm provided some prognostic discrimiantion at five years but again te differences were not pronounced 10 years following node dissection. The thickness measurements were esasier to determine than the level of invasion, and more reproduceable on resubmission to the same pathologist. Four patients with melanoma less than 0.76 mm had subsequent metastases, but these may repreent inadequate sampling of the primary melanoma both in our series and in the four similar patients previosusly reported with such thin metastasizing melanomas.

Eighteen patients with multiple recurrences of malignant melanoma without evident distant spread were randomly assigned to treatment with either intralesional Bacillus Calmette-Guerin (BCG) or intralesional dinitrochlorobenzene (DNCB). Both agents were able to destroy approximately 90% of the injected intradermal nodules. Intradermal disease was more easily obliterated than subcutaneous disease with intralesional treatment with either agent, and local control of satellitosis with elimination of all clinically evident tumor was achieved in the patients who had intradermal without subcutaneous satellitosis, regardless of whether the patient was receiving BCG or DNCB. The clinical courses of the treated patients were essentially the same. Although PHA reactivity was depressed, the patients in both groups were responsive to recall and melanoma skin test antigens, demonstrated leukocyte migration inhibition with melanoma antigen and were generally within normal limits when assayed for 29 degrees C E rosettes. Our study demonstrated a dramatic difference in toxicity between the two intralesional agents without a similar difference in therapeutic efficacy or immune testing.

A patient is presented who was found to have invasive squamous cell carcinoma arising in the epithelial lining of a lateral cervical cyst which had been present for more than 40 years. Radical neck dissection was performed, and metastases were found in a single cervical lymph node. A critical review of cases of branchiogenic carcinoma reported over the past quarter-century yielded no more than three others which meet the criteria for diagnosis of this entity established by Martin, Morfit, and Ehrlich in 1950.

Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected in response to tumor-associated antigens of human melanoma. The antigens were 3 M KCl-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61%) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCl extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCl extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-1 disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results indicate that the direct LMI assay may be used to measure cell immune reactivity against melanoma-associated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.

Twenty-six consecutive patients with melanoma of the lower extremities metastatic to the superficial inguinal lymph nodes were subjected to laparotomy. No patient had preoperative evidence of tumor dissemination past the superficial inguinal nodes. However three patients (12%) had metastases to the liver or para-aortic lymph nodes documented at laparotomy and were not subjected to iliac and obturator lymph node dissection. One of these patients had concomitant local recurrence of melanoma at the ankle. The other two patients had superficial inguinal lymph nodes at least 5 cm in diameter, although two other such patients with similar 5 cm lymph nodes did not have positive intra-abdominal findings. The remaining 23 of the 26 patients underwent ipsilateral iliac and obturator lymph node dissection, which proved positive in 3/23 patients (13%). Of these 23 patients undergoing iliac and obturator node dissection, 18 had clinically positive (and microscopically positive) superficial inguinal nodes prior to their dissection, while the remaining 5 patients had clinically negative (but microscopically positive) superficial inguinal nodes. The three cases of positive dissected iliac and obturator nodes occurred among the 18 patients with clinically positive superficial inguinal nodes (17%). Among the 5 patients with clinically negative, microscopically positive superficial groin nodes, there was no detectable deep inguinal nodal spread (or hepatic or para-aortic involvement).

A case of a 58–year-old woman with viscerally metastatic malignant melanoma is presented 12 years after spontaneous and complete regression of disease. Diagnosis of primary and metastatic lesions was confirmed by review of tissue sections. The presence and subsequent absence of visceral metastases were documented by open liver biopsies. Sections of metastatic lesions revealed extensive necrosis of tumor and infiltration by lymphocytes and plasma cells. Skin testing showed a strongly positive delayed hypersensitivity response to dinitro-chlorobenzene (DNCB), to a standard battery of bacterial and fungal antigens, and to two of four preparations of allogeneic melanoma antigens. Values for cell- and serum-mediated cytotoxicity against melanoma cells and the response of the patient's lymphocytes to phytohemagglutinin were slightly above the normal range. A review of the literature reveals 13 other cases of long-term spontaneous regression of melanoma. None of these, however, had biopsy evidence of visceral disease. In each of the 13 cases, regression was associated with an event that might be inferred to have altered the patient's hormonal or immune status. This patient also provides evidence of a complete, spontaneous, and long-term remission of metastatic disease associated with the spontaneous development of host immunity.

Highly purified melanoma TAA which induce melanoma-related cellmediated immune responses have been further characterized using hyperimmune TAA antisera after affinity chromatography for double immunodiffusion-immunoelectrophoresis and indirect immunofluorescence studies. An additional study of antigenic modulation was performed in 23 nonanergic and seven anergic melanoma patients, tested simultaneously with melanoma TAA prepared from primary and metastatic tumors, which had been obtained from one patient at different time periods. The results of pilot clinical trials are reported, including toxicity, timing and dosage studies in 20 patients and subsequent studies of patients with metastatic melanoma treated at three separate centers, using a single lot of purified, allogeneic melanoma TAA. The results of these latter studies in 51 patients with Stage III (distantly metastatic) melanoma and in five patients with earlier stages of disease indicate that: (1) when the interval from primary therapy to recurrence is greater than one year and when liver, bone and brain are not involved, partial or total clinical regression may be noted in up to 25% of patients with metastatic disease receiving immunochemotherapy; (2) when total regression does occur, the effect usually lasts from one to three years; (3) cytoreductive (debulking) surgery, when possible, in cutaneous, nodal retroperitoneal, and visceral regions may enhance the response to specific active immunochemotherapy, although some debulked patients had less tumor burden and this factor alone may lead to an improved prognosis in patients undergoing any subsequent treatment; (4) when circulating inhibitory factors are modified through preimmunization chemotherapy, an enhanced host response may be seen; and (5) Cancer Serum Indices (CSI) may be useful in predicting recurrence and in following tumor load and response to therapy. Information obtained from these studies suggests the need for further trials to determine the effect of immunization on patients with earlier stages of disease where recurrence rates remain high, and to evaluate the mechanisms of tumor rejection or tumor progression in the face of immune stimulation.

This is a description and evaluation of a tuition-free academic enrichment program for urban youth including a report on the use of programmed instruction to teach chemistry.

Newborn immunosuppressed rhesus monkeys were inoculated with cells containing Rauscher leukemia virus. Two animals developed a mononucleosis-like hematologic picture with monocytosis in the peripheral

Immunization with Rauscher leukemia virus resulted in high antibody levels against the virustransformed malignant cell. As expected, antibody to malignant cells followed immunization with standard virus preparations highly contaminated with cell membranes. Surprisingly, good antibody levels to malignant cells also followed immunization with highly purified virus, electron microscopically free of cell membranes. The antibody response to malignant cells paralleled the antibody response to the virus, under a variety of conditions. Immunization of mice with formalin-inactivated virus preparations, contaminated with cell membranes, resulted in protection of the mice against subsequent challenge with malignant cells. In addition, good immunity to malignant cells followed immunization with live, attenuated Rauscher virus, which was harvested from nonmalignant cells. This attenuated virus produced no malignancy after inoculation and presumably contains only viral antigens, and no malignant cell antigens. These studies strengthen the evidence that Rauscher virus is the “tumor specific transplantation antigen” on the surface of the Rauscher malignant cell. Rauscher virus is presumably integrated into the cell surface membranes which are, therefore, likely sites for transmission of cell enzymes and other proteins into the virus particle. This transfer of material from cell surface to virus particle may explain the Increasing number of enzymes, including “reverse transcriptases,” which have been isolated from RNA tumor viruses. Some such mechanism must be invoked because these viruses do not have sufficient nucleic acid to code for all the “virus-associated” enzymes.

NONSPECIFIC augmentation of tumour immunity has been achieved through the use of Bacillus Calmette-Guerin (BCG), Corynebacterium parvum, Toxoplasma gondii, Besnoitia jellisoni and other intracellular parasites1–4. Nonliving preparations, such as cell walls from BCG, also cause nonspecific augmentation of tumour immunity5. The mechanisms underlying these effects are not clear. It has been postulated that amplification of macrophage or reticuloendothelial function is responsible for the enhancement of tumour immunity by these agents. In the mouse, BCG causes a change in the rate of activation of macrophages, as well as a change in their lysosomal content and bacteriolytic power6,7. In the guinea pig, there is an initial granulomatous reaction at the BCG inoculation site, followed by a peripheral histocyte-mediated destruction of tumour cells8. We have now examined the effect of intradermal BCG on the ‘specific’ and ‘nonspecific’ responsiveness of circulating lymphocytes. We also investigated the ability of two chemical agents to produce similar or additive effects.

Anaplastic carcinoma of the thyroid is a highly malignant disease with few survivors over one year. Radical surgery and laryngectomy are usually considered contraindicated. A 50-year-old woman with a history of papillary carcinoma treated with external radiation developed anaplastic giant-cell carcinoma of the thyroid invading the soft tissues of the neck including the larynx. Since surgery was felt to represent her only chance for survival, she underwent laryngopharyngectomy, right radical-neck dissection, and pharyngeal reconstruction. Three years after the surgery was performed, she is living an active life with only minor handicaps. Nevertheless, multiple nodules were noted in her lungs on recent tomographic examination. Our experience with this woman suggests that there is a role for radical surgery and laryngectomy in selected cases of anaplastic carcinoma.

In the murine model presented for tumor-associated immune suppression, normal BALB/c mice displayed significant foodpad swelling when sensitized on the flank with 2 mg dinitrochlorobenzene (DNCB) dissolved in dimethyl sulfoxide and challenged in a footpad with 0.05 mg DNCB 10 days later. This reaction in challenged footpads seemed to be a classic delayed hypersensitivity reaction, since it took 24 hours to develop and included an extensive mononuclear infiltrate. The reaction was transmissible from sensitized to normal mice by the transfer of lymphoid cells but not to serum. When sensitized 10 days after tumor inoculation, mice bearing either an allogeneic melanoma or a syngeneic lymphoma or fibrosarcoma did not demonstrate delayed hypersensitivity to DNCB.

Prophylactic pretreatment of BALB/c mice with ip or oral vitamin A palmitate was shown to inhibit the growth and development of the S91 melanoma in the allogeneic, histoincompatible BALB/c mouse. When S91 was transplanted in the histocompatible CDF 1 system or when MCA, a nonimmunogenic sarcoma, was transplanted in BALB/c mice, vitamin A failed to protect the mice from tumor. Vitamin A was used for intralesional injection of established tumors. Established S91 tumors in BALB/c mice were successfully treated by intralesional vitamin A or BCG. Established S91 in CDF 1 or MCA in BALB/c continued to grow despite vitamin A intralesional injections, although there was a weak effect of vitamin A intralesional injection on growing MCA tumors. Dermal cutaneous metastases in melanoma patients tended to respond to intralesional vitamin A injection if the lesions were small and superficial but not if they were large and subcutaneous. Tumor destruction in this setting appeared to be due to localized tissue necrosis produced by the injection. Immunologic effects of vitamin A in the murine system are suggested but not proved by the observation that vitamin A effects are more apparent in allogeneic rather than syngeneic host-tumor combinations and by our previous observation that ALS reduces or eliminates the protection of mice pretreated with vitamin A before inoculation of allogeneic tumor.

SummaryBALB/c mice were immunized with Rauscher murine leukemia virus. Both formalin-treated virulent virus, and a live, attenuated strain of the virus were used as antigens. Immune serums and immune spleen cells harvested 64 days later caused lethal damage to the virus transformed malignant cell. The effectiveness of immune serum was removed by heat inactivation and was restored by the addition of guinea pig complement. Either X-irradiation or freezing and thawing eliminated the competence of the immunocytes. Normal spleen cells agglutinated to target cells by phytohemagglutinin were not capable of causing lethal damage to the malignant cells. Although allogeneic inhibition has been suggested as the possible mechanism underlying tumor immunity and surveillance against neoplasia in syngeneic and autochthonous systems, it does not appear to play a significant role in the immunity to transplantation of malignant cells that follows immunization with killed or attenuated murine leukemia virus.

• Since the introduction of BCG intralesional immunotherapy for melanoma, severe complications, including death, have been reported after treatment in hypersensitized individuals. This is a case report of BCG dissemination with ulceration, skin necrosis, lymphadenitis, and abscess formation following the first intralesional injection of a minimal dose of BCG in a PPD-negative patient. The case demonstrates that severe complications can occur in patients who are not hypersensitized, anergic, or debilitated or who have been treated multiple times. This patient's complication further suggests that migration of BCG after intralesional therapy may play a role in the regression of uninjected nodules seen in some patients. ( Arch Surg 113:893-896, 1978)