Second Malignancy Probabilities in Patients With Breast Cancer Treated With Conventional Versus Hypofractionated External Beam Radiation Therapy in the Adjuvant Setting.

AIMS: For women with breast cancer, seminal studies have shown that adjuvant hypofractionated external beam radiation therapy (hEBRT) maintains similar outcomes and may reduce overall costs compared with conventionally fractionated external beam radiation therapy (cEBRT). However, it is unclear whether hEBRT may be associated with differential risk of development of radiation-induced second malignancies compared with cEBRT. Because the occurrence of second malignancies is small, large databases may improve our understanding of the relative risk of second malignancies between hEBRT and cEBRT. MATERIALS AND METHODS: Using the National Cancer Database, we carried out a retrospective cohort analysis of women diagnosed with non-metastatic, stage 0-III breast cancer from 2004 to 2017. All patients had a lumpectomy or mastectomy and a follow-up time of at least 60 months after diagnosis. The probability of second malignancies in women receiving adjuvant cEBRT or hEBRT was compared using multivariable logistic regression adjusting for sociodemographic, geographical, clinical and treatment factors, allowing for relative (but not absolute) comparison of second malignancy risk. Temporal sensitivity analyses stratified by year of diagnosis and length of follow-up time were also conducted. RESULTS: Of the 125 228 women in our study, 115 576 (92.3%) received cEBRT and 9652 (7.71%) received hEBRT. The median age of the cohort was 60 (interquartile range 51-68) years at diagnosis and the median follow-up time was 99.61 (interquartile range 77.5-128.49) months. Upon adjusting for sociodemographic and clinical factors, patients who received hEBRT had no difference in relative risk than patients who received cEBRT (odds ratio 0.937, 95% confidence interval 0.869-1.010, P = 0.091). In analyses stratified by year of diagnosis, and stratified by length of follow-up, there was no difference in second malignancy probability between patients who completed hEBRT and patients who completed cEBRT. CONCLUSIONS: In this analysis of over 120 000 women with non-metastatic breast cancer, hEBRT was not associated with different odds of developing second malignancies compared with cEBRT. Our findings may inform patient counselling in the choice of radiation regimens for breast cancer and further support the safety of hypofractionated regimens for breast cancer.

Skin reaction to yellow fever vaccine after immunization with rabies vaccine of chick embryo cell culture origin.

Skin reaction to yellow fever vaccine was examined after immunization with rabies vaccine. The two vaccines contained substrates from chick embryo cells (rabies vaccine) and chick whole embryo (yellow fever attenuated vaccine), as well as gelatin. A prick test with gelatin showed negative results in all vaccinees examined. An intradermal skin test revealed that the yellow fever vaccine had reacted with an anti-egg protein antibody-like substance in a case with a history of egg allergy before rabies vaccination. A case inoculated two times with the rabies vaccine revealed a positive reaction to egg-white protein as well as the yellow fever vaccine. This case had no anamnesis of egg allergy. Thus, an antibody reactive to the egg-white protein and/or the yellow fever vaccine was inducible by the rabies vaccine. The reaction of this antibody was not systemic but local at the skin test by the yellow fever vaccine. The period of the rabies vaccine sensitization reactive to the yellow fever vaccine could be estimated as longer than 14.3 +/- 9.6 days (mean +/- SD), based on a follow-up examination of the positive skin reaction in 41 of 84 cases examined. We therefore conclude that the yellow fever vaccine can be safely administered at an interval of at least four weeks after a second rabies vaccination.

Application of PCR for detection of mycoplasma DNA and pestivirus RNA in human live viral vaccines.

PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.

Evaluation of current sterility tests for human live viral vaccines.

Current sterility tests for human viral vaccines were evaluated. A total of 43 lots of bulk suspension of live viral vaccines (measles, mumps, rubella and oral poliomyelitis) produced by six manufacturers in Japan were evaluated for bacteriostatic and mycoplasmastatic activities. Some of them showed fairly high bacteriostatic and mycoplasmastatic activities, due to antibiotics added during vaccine production. It was concluded that the current sterility test for mycoplasmas is not reliable to detect viable mycoplasmas in live viral vaccines.

The views and policy of the Japanese control authorities on the three Rs.

NIH Japan has tested and regulated the three Rs of animal experiments in the development and control of biological products in a stepwise manner. (1) The number of monkeys was reduced from 108 to 72 for the neurovirulence test of OPV in each type, since paralysed monkeys inoculated intraspinally revealed a linear relationship between average scores of the lumbar lesion and cumulated paralysis occurrence ratio (%). (2) Rabbits for the pyrogen test were replaced by the endotoxin test for PPF, albumin and interferon products. The endotoxin is measured by the parallel line assay method using both turbidimetric kinetic and colorimetric methods. (3) Histopathological examination was introduced to the abnormal toxicity test as a refinement. Mean body weight loss of two guinea pigs inoculated with five ml. of an albumin product in each was far below the mean weight of pooled guinea pigs used (P < or = 0.01) and appeared repeatedly. The histopathological examination showed focal necrosis in the liver. This finding was suggestive of the presence of endotoxin in the product. The product contained 0.1 EU/ml of endotoxin. The same amount of the reference endotoxin produced a similar change in guinea pigs. The mechanism of the liver cell damage by endotoxin has been investigated by an in vitro method.

[Application of the Limulus test for practical quality control on endotoxin content in commercial human serum albumin (HSA) products. In comparison with the rabbit pyrogen test].

The bacterial endotoxin content in human serum albumin (HSA) products measured by two different Limulus amebocyte lysate (LAL) test methods, colorimetric and kinetic turbidimetric methods, were compared. So far as endotoxin-specific LAL reagents which do not show a false-positive reaction with (1-->3)-beta-D-glucan are used, a definite correlation was found between the results with the two LAL test methods. Endotoxin added to HSA products was recovered in a quantitative manner showing neither inhibition nor enhancement by HSA to the both LAL test methods. Results of the LAL tests showed a significant correlation with that of the rabbit pyrogen test. The correlation was much improved with endotoxin-added HSA. The present results indicate the practical applicability of the LAL test as an alternative method for the rabbit pyrogen test.

Thimerosal induces toxic reaction in non-sensitized animals.

The effects of injection of thimerosal solution on nonsensitized animals was investigated. Intrafootpad injection of thimerosal solution in nonsensitized mice resulted in a swelling response which peaked 1 h after injection and lasted for more than 24 h. Histopathological examination showed that there were severe edema and infiltration of polymorphonuclear neutrophils at the site of injection. An increased vascular permeability was observed after cutaneous injection of thimerosal solution on the back of nonsensitized rats. Since mercuric chloride and methyl mercury induced severer reactions, and thiosalicylic acid had no effect, mercury contained in thimerosal would have caused the reactions observed in this study. These results suggest that part of these hypersensitivity reactions against thimerosal observed among patients were possibly induced by the toxic effect of thimerosal. Therefore, thimerosal contained as a preservative in vaccine may augment the side-effects of the vaccination.

[Application of the limulus amebocyte lysate test to measurement of endotoxin in therapeutic human plasma protein fraction. Comparison with the rabbit pyrogen test].

We applied the limulus amebocyte lysate (LAL) test to the detection of bacterial endotoxins in therapeutic human plasma protein fraction (PPF) and compared the LAL-test with the rabbit pyrogen test. Two endotoxin-specific LAL-reagents were used for the colorimetric method and turbidimetric kinetic method. The amounts of added endotoxin to the PPF were correctly estimated by either method. The results of four independent assays for the 53 samples of PPF corresponded well with each other (correlation coefficient: 0.851-0.959, regression coefficient: 0.898-1.151). The amounts of endotoxin in the PPF estimated by the LAL-test significantly correlated with the rise of body temperature in rabbits (correlation coefficient: 0.547-0.642, and 0.911-0.934 for the endotoxin added samples). These results suggest that the LAL-test could be used as an alternative method for the rabbit pyrogen test to PPF.

Different susceptibilities to attenuated poliovirus of cynomolgus monkeys from the Philippines and other Southeast Asian countries.

Susceptibilities to attenuated poliovirus of cynomolgus monkeys were assessed by the intraspinal inoculation method with the same single bulk lot as the reference for each of the three poliovirus types. The lesion-inducing virus dose in the spinal cord of 50% of monkeys inoculated (LID50) varied widely from 10(6.4) to lower than 10(2.5), the difference being as much as 10,000-fold, depending on the birth place of the monkey. The birth places of the monkeys used were traced and categorized into the following three districts; the peninsular region, Indonesia and the Philippines. The LID50 value of the attenuated poliovirus in the monkeys from the Philippines were much lower than those in the monkeys from the other two districts.

Growth and spread of causative agent of Creutzfeldt-Jakob disease in intraperitoneally infected mice.

The progression of Creutzfeldt-Jakob disease (CJD) was studied in ddY mice infected intraperitoneally with the Fukuoka-1 strain, a transmissible agent isolated from a CJD patient in Japan. Infectivity became detectable simultaneously in the brain, the spleen and the liver, i.e. 19 weeks after inoculation. Infectivity titer ranged from 10(2) to 10(3) LD50/g in all three organs up to 35 weeks, in contrast to intracerebrally infected mice in which the content of infectious CJD agent is higher in the brain than in the liver and the spleen. Cuffs appeared in the brain roughly four times in the period from 3 to 37 weeks after inoculation. The observed infectivity was discussed in relation to the appearance of cuffs in the brain.

Passive transfer of experimental allergic encephalomyelitis with lymphocytes collected from brains of immunized guinea pigs.

Lymphocytes were collected from the brains of guinea pigs immunized with syngeneic spinal cord emulsified in complete Freund's adjuvant. Sixteen to 32 X 10(6) lymphocytes were inoculated into the vein of each inbred guinea pig (NIH 13). Recipient animals without clinical signs of experimental allergic encephalomyelitis (EAE) were sacrificed 20 days later. Some of the recipients had perivascular infiltrates of a large number of mononuclear cells mostly in choroid plexus and meninges of the brain and spinal cord. Demyelination, which was not so intense, was also observed in the vicinity of the perivascular infiltrates. Thus, the lymphocytes from the brain as well as lymph node and spleen were clear to have the ability to transfer EAE.

Evaluation of the neurovirulence test of oral poliovaccines in Japan during the period 1963-1982.

The present study showed that the neurovirulence test for the lesion-inducing virus dose in the spinal cord in 50% of monkeys inoculated with oral polio-vaccine (Sabin) (LID50) reflected to a large extent safety and efficacy of the vaccine upon administration to children. The degree of attenuation of the vaccine in terms of LID50 appeared to be related to the decrease in the vaccine-associated cases as well as the seroconversion rate. An exceptional case, however, was noted in which lot No. 301 was not classified into less attenuated vaccine by the neurovirulence test. This fact suggests that the method we have employed awaits further improvement.

Skin reactions to vaccinia virus infection in the rabbits immunized with vaccinia-soluble early antigen.

Rabbits were immunized with vaccinia-soluble early antigen (Es antigen) in Freund's complete adjuvant (FCA). Circulating neutralizing antibody was not induced, but CF antibody against the soluble antigens of vaccinia-infected cells was induced. By intradermal inoculation of vaccinia virus, erythema developed early at the site of inoculation and enlarged up to 3 days post infection (p.i.). No multiplication of the viruses in the erythematous areas was indicated on day 3 p.i. The erythematous areas showed histologically severe changes involving infiltration of numerous cells accompanied with necrotic and hemorrhagic lesions in a part of the dermis. Eosinophilic leukocytes were dominant and there were also some lymphocytes in the cell infiltration. The role of these kinds of cells was discussed in connection with cell-mediated immunity in the mechanism of excluding vaccinia virus from the immune rabbits.

Experimental transmission of Creutzfeldt-Jakob disease to guinea pigs.

Brain material used in this experiment was from a 47-year-old woman autopsied 5 hours after death. The brain was markedly atrophic weighing 750 g. Histopathologically, a spongy state was extensive from cortex to medulla with fibrous astrocytosis and neuronal loss. This brain tissue was aseptically removed at autopsy and a 20% emulsion was prepared. Intracranially 0.1 ml and intraperitoneally 0.2 ml of the emulsion were inoculated into each guinea pig. Five and three of them were killed 10 and 22 months later, respectively, nevertheless showing no clinical signs. The brains from these guinea pigs did not reveal any changes as seen in the human autopsy brain both macroscopically and microscopically. However, all the brains indicated ultrastructural changes characterized by vacuolation with curled membranes in dendrites and free membranes with cellular debris in extracellular space. These changes were not found in guinea pigs inoculated with material from normal guinea pig brain or uninoculated guinea pigs, but similar to those observed in the human autopsy brain used as inoculum. Therefore, it could be considered that Creutzfeldt-Jakob disease was transmitted from man to guinea pig to present an early stage of the lesion. Several organs were observed ultrastructurally after whole bleeding in monkeys. Curled membranes were generated only on the vacuoles of dendrite from 2 1/2 hours after bleeding and not in those of other organs. This may explain at least a part of the pathogenesis of spongiform changes restricted in the central nervous system.

Late effects of whole or partial body x-irradiation on mice: life shortening.

Life table data were obtained on 2400 ddY/SLC female mice exposed at 10 weeks of age to a single dose of X-rays. Doses given to the whole body were 95, 190, 380 and 570 rad, and to head, trunk or lower body, 190, 380, 570 and 760 rad. The mean survival time of unirradiated controls was 73 weeks. The mean survival times after whole body exposure decreased linearly with increasing dose. The estimate of life shortening per 100 rad was 7.2 per cent for whole body exposure. The life shortening after head or lower body exposure to a dose of 190 rad was 6.2 and 7.1 weeks respectively, and almost no further life shortening up to 760 rad. After trunk irradiation with 190 rad, life shortening was 10.4 weeks and beyond that dose life shortening was 1.1 per cent per 100 rad.

Retrovirus particles in spontaneously occurring and radiation-induced tumors in ddY mice.

Among spontaneously occurring tumors, pulmonary tumor, malignant lymphoma, mammary tumor, and ovarian tumor were the major ones in 232 ddY female mice. The former three tumors appeared significantly earlier and the latter one increased in incidence in 229 mice given 600 R whole or 800 R partial body (tunk) x-irradiation at the age of 10 weeks. Five tumors were examined electronmicroscopically from each tumor type of both the spontaneously occurring and radiation-induced tumors. C type virus particles were present only in the malignant lymphoma and B particles in the mammary, ovarian and pulmonary tumors, which are all of epithelial character. Thus, as far as we were concerned in this mouse strain, B particles were produced preferentially in epithelial cells and C particles in non-epithelial cells. The retrovirus particles were found in the same frequency, namely, 10 of 20 tumors examined in both the tumor groups. From our results, the intervention of virus in radiation carcinogenesis still remains in question.

Experimental infection of young rabbits with rabbit parvovirus.

A newly isolated parvovirus of rabbits (F-7-9 strain) was inoculated into young female rabbits by the oral or intravenous route. Virus was recovered from the feces 3 to 13 days after inoculation. Viremia was demonstrated in orally inoculated rabbits. The virus was also isolated from tissue extracts of liver, pancreas, spleen, small intestine, appendix and mesenteric lymph node on days 10 and/or 14, and from the small intestine on day 30 after inoculation. Hemagglutination inhibiting antibody appeared in the serum after 8 days and neutralizing antibody was detected later. An 11S neutralizing antibody was demonstrated in fecal extracts between 15 and 30 days after inoculation. Infected rabbits showed very mild clinical signs of listlessness and inappetence lasting for a few days. Histologically, mild to moderate catarrhal enteritis was observed in the small intestine.