A method to induce tumor marker release

Although, Kitamura1 reported that the maximum range variation for carcinoembryonic antigen (CEA) levels in a healthy body is narrow for many years.1 We observed that serum tumor markers are not constant in any cancer case. To date, the induction of tumor marker release (TMR) has never been applied to patients during an initial examination in an outpatient clinic. Prior to patient examination, we verified the utility of this method in animal and tissue culture experiments.


Introduction
Although, Kitamura 1 reported that the maximum range variation for carcinoembryonic antigen (CEA) levels in a healthy body is narrow for many years. 1 We observed that serum tumor markers are not constant in any cancer case. To date, the induction of tumor marker release (TMR) has never been applied to patients during an initial examination in an outpatient clinic. Prior to patient examination, we verified the utility of this method in animal and tissue culture experiments.  Figure 1TMR induction was performed out on day of 16 after transplantation of MKN-45 (20million cells) in the long term. Three hours after intramuscular injection of vitamin A (2500 IU), hyperthermia treatment was performed at 40°C for 20minutes. The blood was withdrawn from the orbital vein at 0, 6, 24 and 48 hours. CEA was assayed by the CEA-EIA kit (Abbott, Co. Ltd).

Figure 2
TMR induction was performed out on day of 16 after transplantation of MKN-45 (20million cells) in the short term. Three hours after intramuscular injection of vitamin A (2500IU), hyperthermia treatment was added at 40°C for 20minutes. The blood was collected from orbital vein after 0, 1, 3, and 6 hours. CEA was assayed by the CEA-EIA kit (Abbott, Co. Ltd). In the control, the same amounts of HCO and palmitic acid were injected.   Figure 5, CEA release was induced by combined treatment with vitamin A and hyperthermia. Because mRNA inhibitors prevent this reaction, the protein synthesis observed was newly induced. There was a trend toward increased CEA release over time after stimulation by vitamin A and hyperthermia. CEA release was consistently inhibited by treatment with actinomycin D or cycloheximide. From these studies, even for small tumors, we can estimate the size of hidden tumors in the body by utilizing tumor marker parameters such as the maximum difference in a value(Δmax), the maximum value(Vmax), the average variation(Δmax/h) and the expected maximum quantity(Δmax/h xVmax). Therefore, we performed a tumor marker induction experiment with specific patients ( Table  1). The times of the final blood collection and the number of patients for each blood sampling are shown in Table 2.

Methods for inducing TMR in humans 4
A total of 138 patients presented at a holistic medical outpatient clinic and received a histopathological definitive diagnosis. The tumor size was 1 to 2 cm in diameter for cancer stage G 1 , 2 to 5 cm in diameter for cancer stage for G 2 , and 5 cm or greater in diameter for cancer stage G 3 , which was classified as advanced cancer. Stage G 1 , G 2 and G 3 numbers included 61, 37 and 40 cases, respectively. As a control, the induction of TMR was studied in 11 patients without cancer and in 4 healthy subjects. Tumor marker release was examined. Table 1 shows the types of cancer. Non-cancer patient 11   Effects of mRNA transcription or protein synthesis inhibitors on retinoic acid and hyperthermia-mediated increases in CEA content in MKN-45 cells. Cells were simultaneously exposed to 75μM retinoic acid and either actinomycin D (1ng/ml) or cyclohexamide (0.2μg/ml) followed by hyperthermia (40°C, 30min). After 6, 24 and 48h. cells and media were collected and analyzed for CEA. (□)Control: (■) retinoic acid + hyperthermia: ( ) retinoic acid + hyperthermia + actinomycinD: ( ) retinoic acid + hyperthermia + cyclohexamide.

Determination of tumor markers
CEA levels were determined by enzyme immunoassay with kits obtained from Abbott Co., Ltd. The cut-off value for CEA(Vmax) in stage G 1 and the cut-off value of Δmax (hereafter referred to as Δ in Tables 3-5) determined as the difference between the maximum and minimum values, were found to be 4.4ng/ml and 0.9ng/ml, respectively. The cut-off value of Δmax per hour (Δmax/h) and the cut-off value of (Δmax/h)xVmax were calculated to be 0.1ng/ml.h and 0.25ng 2 /ml 2 .h, respectively. Values of ferritin, alpha-fetoprotein (AFP), silalic acid and the ratio of ferritin to serum iron (FT/ Fe) were also determined using four parameters (Δmax, Vmax, Δmax/h, and Δ (Δmax/h)xVmax). The cut-off values for each stage are shown in Table 3. To quantitatively assess ferritin levels, the SPAC-Ferritin kit (The First Radioisotope Research Institute, Tokyo, Japan) and RIAgnost Ferritin kit (Hoechst, Frankfurt, FRG) were used. The AFP level was determined by RIA (double antibody method). Sialic acid levels were determined by enzyme assay with neuraminidase. Serum iron (Fe) was assayed with a Hitachi 705 autoanalyzer

Preliminary results
The cut-off value for each of the four parameters at the G 1 level was used for each tumor marker, and the number and rate of positive cases for tumor stages (G 1 , G 2 and G 3 ) are shown in Table 3. When the cut-off value of Δmax or Vmax at a G 1 level was used for CEA, the rates for positive cases in stages G 1 , G 2 and G 3 were 64, 62 and 70%, respectively. When the cut-off value of Δmax/h or (Δmax/h)xVmax was used, the rates for positive cases in stages G 1 , G 2 and G 3 were 61, 73 and 70% respectively. When any one of the four parameters were examined using the SPAC-ferritin kit to determine ferritin level, the rates of positive cases in G 1 , G 2 , and G 3 were 54, 65 and 86%, respectively. When any one of the four parameters were examined using the RIA-gnost ferritin kit to determine the ferritin level, the rates for positive cases in G 1 , G 2 and G 3 were 61, 65 and 84%, respectively. Thus, as the result of the determining the ferritin level with the above two ferritin kits, the rates for positive cases in stages G 1 , G 2 and G 3 were 57, 65 and 85%, respectively. When any one of four parameters (Δmax, Vmax, Δmax/h, or (Δmax/h) x Vmax) were used for AFP, the rates of positive cases were 26, 22, and 43% respectively. AFP was very unreliable. When any one of these parameters was used for silalic acid, the rates of positive cases in G 1 , G 2 and G 3 were 77, 82 and 89%, respectively. Because the ratio of ferritin Vmax (ng/ml) divided by serum iron (Fe, μg/dl) for the same period is considered an important tumor marker, we determined the rate of positive cases before the release induction by combing vitamin A and hyperthermia (0hr). When the SPAC-ferritin kit was used, the rates for stages G 1 , G 2 and G 3 were 29, 60 and 76%, respectively. When the RIA-gnost ferritin kit was used, they were 52, 59 and 84%, respectively. As a result of concurrent determinations with both kits, it was found that the rate of positive cases positively correlated with increased tumor stage.
The rates for five tumor markers are summarized in Table 5. When any of four parameters were used, the rates of two positive markers to four markers were 64, 70 and 93% in stages G 1 , G 2 and G 3, respectively, suggesting a linear correlation between tumor marker level and tumor size. Among any three of five positive tumor markers, any one of 4 were 53, 65 and 94% in G 1 , G 2 and G 3 , respectively, suggesting the same tendency. In this regard, none of the healthy controls or the nontumor bearing patients (except for those with elevated ferritin levels caused by liver dysfunction), showed false-positive findings for tumor markers. A cut-off value was assigned in each stage for each tumor marker (Table 3). For comparison, the rates determined using the cutoff values conventionally used before induction (at 0hr) are shown in Table 6. The positive rates per cases for any two of four markers was low, 11, 14% and 55% at level of G 1 , G 2 , and G 3, respectively, suggesting no correlation between the markers and tumor size. As shown in Table 6, when TMR was not induced, the correlations of the tumor marker parameters with a cancer stage from G 1 to G 3 were not confirmed.

Practical application of a method to induce TMR in patients with poorly defined cancer
A method for inducing TMR can facilitate the diagnosis of cancers that are difficult to diagnose morphologically or poorly differentiated cancers, as well as the diagnosis of cancers for which specific markers are unavailable. In fact, diagnosis can be made by the combined use of tumor marker combination assay (TMCA) that we developed. 5 In clinical practice, a 30-year-old woman was given a diagnosis of poorly differentiated ovarian cancer in K. Medical University, and underwent 2 surgical operations and 4 chemotherapy sessions. She was diagnosed as having "no recurrence" by Professor N. President of the Japan Society of Obstetrics and Gynecology. However, this patient consulted with another professor because of her poor condition and was referred and admitted to our hospital. The results of TMR are shown in Table 7.     Ribonuclease, 202U, NK activity (17%), T cell number (1468) Albumin: 64%, α1-globulin: 3.5%

Application results
The TMR method was applied to this postoperative undifferentiated ovarian cancer patient.
The following data were obtained after TMR induction:  Table 7. Tumor markers were evaluated by the TMCA technique, reported in the Cancer in 1994, 5 combined with the TMR induction technique. The more polished TMCA was reported in Cancer medicine. 6 Based on the results obtained with these methods, the presence of cancer in the G 2 clinical stage was estimated. Therefore, positron emission tomography (PET) and computed tomography (CT) were performed and revealed metastasis in the right axillary lymph nodes Virchow-lymph node metastasis (+). The patient was given the herbal medicine: Sun Advance® which inhibits aerobic respiration by cancer cells, 7,8 and detoxification therapy twice a week. As shown in Table 8, the patient was completely cured. As of 18 years after treatment, the patient has had no recurrence.

Conclusions
The presence and the size of hidden cancerous tumors can be estimated with tumor marker-release-related specific parameters with a combination treatment of vitamin A and hyperthermia.