Samer Singh ^1* Rakesh K. Singh ²

• ¹ Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
• ² Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India

by subsequent Mycobacterium spp. exposure seem protectively associated with a significant reduction in BCNS cancer incidences in less than 5-year-olds (0-4Y-olds). A role for early childhood Mycobacterium spp. exposure in competent immune system development and reduction in early childhood BCNS cancer incidences may need to be explored further to establish a protective role for certain microbial exposure in cancer incidence reduction. Epidemiological data suggests that among early childhood vaccines, the neonatal BCG vaccination followed by "boosting" may reduce early childhood BCNS cancer incidences. Childhood exposure to common microbes, pathogens, and childhood vaccines is supposed to provide appropriate immune training and surveillance capability that generates a cross-protective immunity against number of pathogens to which children get exposed later in life [1][2][3]. It is associated with better immune function, characterized by reduced allergies to different allergens, and reduced disease occurrence, including cancer [1,[3][4][5][6][7][8][9]. Primary brain tumors and associated central nervous system (BCNS) cancers are the most common solid tumors in children (0-14 years old) and adolescents (15-19 years old), accounting for the highest number of solid cancer-related deaths [10,11]. The incidence of childhood BCNS cancers is highest in children less than 5year-olds (i.e., 0-4Y-olds) that progressively reduces until adolescence before again starting to steadily rise with age during adulthood [10,11]. The BCNS cancers in children are inherently different from those in adults. Those found in children are predominantly malignant gliomas, embryonal tumors, and germ cell tumors, with a minority of nonmalignant tumors primarily comprising pituitary tumors [11][12][13]. A large number of environmental and genetic risk factors have been studied for their association with BCNS cancer incidences [11,12,14]. Based on epidemiological studies, exposure to ionizing radiation (increased incidence) and inherited single-gene syndromes (~4% of childhood cases) are the only validated primary risk factors for BCNS cancer incidence [11,12,14]. Structural birth defects (non-chromosomal) have been associated with approximately 7% of childhood BCNS cancers [11,15,16]. Higher incidence rates have also been observed in the population's higher socioeconomic position (SEP) sub-groups [17][18][19][20]. The "hygiene hypothesis" has been supposed to provide an explanation in the form of a change in SEP-associated unknown risk factor(s) that could alter the exposure of the immune system to common allergens and pathogens, resulting in aberrant immune system development or immune training and an associated risk of childhood cancer [9,11,12,21]. For the majority of early childhood BCNS cancers, the precise nature or identity of risk factors that could be promoting or protecting 0-4Y-old age group children against BCNS cancer incidence remains unknown, while those identified or suspected in a minority of cases lack any interventional value [11,12].Childhood vaccines play an important role in protecting children from major infections and diseases by augmenting the developing immune system [1]. As the first year of life is important in shaping and training the developing immune system, the recommended immunization schedule [22], starting from birth to the first year of life, generally includes vaccine shots against the following common diseases: tuberculosis (Bacillus Calmette-Guérin: BCG), polio (polio vaccine: POL), Hemophilus influenza B (HIB), hepatitis B (HEPB), pneumococcal illness (Pneumococcal conjugate vaccine: PCV), measles infections (measles containing vaccine: MCV), diphtheria, tetanus, and pertussis (DTP). These childhood vaccines are supposed to enhance the trained innate immunity, and humoral-and cell-mediated immunity in children [1][2][3]23,24]. Types of these vaccines range from subunit vaccines, heat-inactivated to live-attenuated vaccines, each activating and training a specific combination of the effector arms of the developing immune system [1]. The timing and number of shots vary for each, beginning soon after birth to throughout the entire first year of life, and many times boosters are given later in life based on past experiences with vaccines and perceived threats to children's lives [22]. The first doses of the BCG, HEPB, and OPV vaccines are administered at birth. Two doses of MCV (MCV2), two additional doses of HEPB (HEPB3) and POL (POL3), and three doses each of PCV (PCV3), DPT (DPT3), and HIB (HIB3) are administered at different time intervals during the first year of life to generate, strengthen, and sustain immunity against target pathogens, as well as general immunity during the most susceptible period. It may be pertinent to state that the previously recommended booster doses of BCG have been discontinued worldwide due to a lowering of threat perception resulting from a general increase in hygiene and the lowered tuberculosis incidence and associated risk of exposure to the environmental Mycobacterium tuberculosis complex. In many countries of the industrialized worldwhich also happen to be the countries with the highest childhood cancer incidence rates-BCG vaccination has been completely discontinued, including the neonatal dose, for the perceived lack of benefit for these countries with their elimination of tuberculosis.The nonspecific positive effect of the BCG vaccine on the survival of children is well recognized [References in [25][26][27]. It is presumed to be through providing needed immune training to enhance general immune surveillance and strengthening the "trained immunity" of the naïve developing immune system of neonates by functional reprogramming of the immune cells involved in innate immune response [23,[28][29][30][31] that ultimately seems to cross-protect them against various pathogens and diseases [1,6,24,[25][26][27]32,33].A number of epidemiological studies around 1970 found that neonatal BCG vaccination was associated with a reduced incidence of leukemia, the most frequent childhood cancer, in up to 6-year-old children [6,[34][35][36][37]. The proposed mechanisms for the observed reduced cancer incidence in these children have envisaged a role for the neonatal BCG vaccinations or infections in priming and training the developing immune system to efficiently eliminate pre-existing pre-cancerous clones or cells-remnants of embryonic development [6,36]. Based on the observed need for a second hit in the genetically predisposed models of the disease and the investigation of familial cases of leukemia that are often supposed to get triggered by infections, Greaves hypothesized the need for infections that could lead to overt cancer growth following a second genetic hit [38]. However, many retrospective studies failed to see the protective impact of childhood vaccination and early infections on childhood cancer incidences, with some even suggesting and arguing for an increase in cancer incidence on vaccination [References in 6,39-42]. For a brief account of other theories proposed for childhood leukemia incidence as a result of differential pathogen exposure and immune training, refer to the review by Hauer et al. [43]. Unlike the studies that demonstrate a reduction in the incidence of childhood cancers (leukemia and BCNS cancer) with neonatal BCG vaccination, those witnessing no effect or arguing for an increase in cancer incidences had children vaccinated at times other than the neonatal period [REFs in 37,39-42].Furthermore, their follow-up cancer incidence periods varied, ranging from beyond early childhood to old ages, when the origin and nature of cancers markedly differ from those of early childhood cancers [6,11,12,36,38,43,44]. Some studies even seem to equate a lack of curative potential in age groups other than early childhood with a lack of preventive potential in early childhood. However, for the lack of any clear-cut epidemiological evidence and any apparent way forward for their validation, the role of any childhood vaccination, including BCG, for childhood cancer prevention has remained highly debated and controversial [6,32,37,39,40,42,44,45].Previously, based on our observation that the incidence of BCNS cancer in up to 5-yearold children in similar HDI countries worldwide was significantly inversely correlated with tuberculosis incidence rates in these countries, we have suggested that exposure to Mycobacterium spp. could be potentially protectively linked to BCNS cancer incidence in young children [45]. In this opinion article, we further develop our assertion by presenting an epidemiological analysis of early childhood BCNS cancer incidence in the WHO European region (ER), which has more similar underlying socioeconomic conditions, genetic makeup, comparable health and medical infrastructure access, and disease reporting as compared to the rest of the world. However, there is a diversity in universal vaccination programs/policies that range from never-ever to all eligible for Early childhood vaccinations and incidence of BCNS cancer: The tuberculin immunoreactivity (TI), as measured by TST and interferon gamma release assays (IGRAs), is employed to indirectly measure the elicited or persisting cellmediated immune response resulting from exposure to Mycobacterium spp. (BCG or environmental) antigens, as well as to ascertain BCG vaccine efficacy [49,50,57]. In the absence of a clinically active tuberculosis disease, it is also referred to as LTBI by the WHO for tuberculosis management purposes, due to these individuals supposedly "being at risk of developing TB" in their lifetime from reactivation or fresh infections [49,50,51,59]. Intriguingly, the prevalence of TI/LTBI in the European Region countries [48], which provides a measure of the cell-mediated trained-immunity persistence in the populations resulting from their exposure to Mycobacterium spp., including that from the BCG vaccine-is found to be significantly negatively correlated with BCNS cancer incidence rates in 0-4Y-olds [Pearson's correlation coefficient, r(47): -0. (co-vaccination) or their combinations also does not seem to affect BCNS cancer incidence reduction in less than 5-year-old children, in addition to TI in the neonatally BCG-vaccinated populations. Interestingly, when all countries are considered, completely disregarding their neonatal BCG vaccination policy and coverage, the countries with medium-to-high BCNS cancer incidence rates (i.e., ASR 2-4 and >4 per 100,000) exclusively belong to low TI/TST-positive populations (<15%), whereas the low BCNS cancer incidence countries (i.e., ASR >0-2 per 100,000) are primarily BCGvaccinating countries (8/9) with high prevailing TI/TST-positive populations (7/9 with >15 to 20%). Overall, early childhood BCNS cancer incidence in countries that use neonatal BCG vaccination is strongly but negatively linked to TI/TST positivity in the community. These findings suggest that neonatal BCG vaccination plays a protective role in countries where the immune system of any child is more likely to be boosted by exposure to environmental Mycobacterium spp. Together, these results imply that environmental Mycobacterium spp. exposure-associated boosting in children primed with neonatal BCG vaccination may play an important role in lowering the incidence of BCNS cancer in early childhood (less than 5 years of age). Regarding the impact of potential confounders on our observations and outcomes of any prospective study, it must be remembered that many studies in the past have positively associated the high BCNS incidence rates with socioeconomic position (SEP), nonchromosomal structural defects, a high birth rate, syndromes (e.g., neurofibromatosis types I and II, Li Fraumeni syndrome, tuberous sclerosis), and polymorphisms in a number of genes and infections [reviewed in 11,12]. Infection of Toxoplasma gondii, a protozoan, has also been associated with an increased risk of glioma incidence (a subtype of BCNS cancers). Human leukocyte antigen (HLA) alleles, composition of immune cells, and the genomic architecture of T-, NK-, and myeloid cells have been associated with glioma risk. However, the role of most viral infections studied has been inconsistent in glioma-the malignant BCNS cancer-and responsible for the majority of deaths. Only allergies, atopic conditions, and infection with Varicella Zoster Virus (VZV), a herpes virus that causes chickenpox and shingles, have been consistently associated with a reduced risk of glioma. The existence of a potential protective association between environmental Mycobacterium tuberculosis complex spp. exposure and BCNS cancer incidence has been suggested by us based on the observed lower incidence rates in high tuberculosis reporting countries [45]. These may very well remain underlying confounders for swaying the outcome of any exploratory study and hence the conclusions, including those of the current article. Their control, as possible, would be desired in future studies.From a mechanistic point of view, the observed negative correlation of Mycobacterium spp. exposure with the BCNS cancer incidence in 0-4Y-olds when compared with general vaccination could be related to their ability to epigenetically reprogram innate immune cells possibly making them better at recognizing and eliminating abnormal transformed cells [28][29][30][31][32][33]44,45]. No matter how significant and strong any statistical correlation could appear between potential variables, it can never be considered to have a causeand-effect relationship. Endeavors may be made to identify and validate such protective variables that may have a potential cause-and-effect relationship [51]. The channelization of resources to evaluate and explore the preventive and protective potential of childhood vaccinations, especially BCG vaccination and boosting events (BCG and environmental Mycobacterium spp.), may be warranted to ascertain a potential cause-and-effect relationship, if any. It should also be remembered that there are certain inherent lineage-specific and preparation-specific differences with regard to content and immunogenicity in BCG sub-strains [60,61] that are historically differentially employed in the Eastern and Western European region countries for children's vaccination [47], along with differences in circulating Mycobacterium spp. [62-64] and their inherent immune activation potential [65-67] on exposure. The covaccination during the period may also have an impact [27,68]. While designing and performing any such exploratory study, more emphasis should be placed on identifying variables potentially causatively associated with direct reduction in early childhood BCNS cancer incidence (having cause-and-effect relationships) with some interventional value rather than just being an explanatory variable (e.g., income, GDP, socioeconomic position in society, gene polymorphism number of children, etc. [11,12]). Additional study design considerations may be needed to control the effect of possible confounders that have been found associated previously with BCNS cancer incidences and could be supposedly overrepresented in any specific study populations (e.g., syndromes, gene polymorphisms, mutations in genes associated with immune system functioning, etc.; Supplementary Materials and Methods: For the current Opinion article, the associative analysis was performed on the European Region countries' data using routine statistical methodology as described previously [51,69]