Bacteria in neoplastic diseases: A brief note on Wilms tumor

Table 1 Selected studies that utilized the bacterial system in targeted therapeutic strategies against cancer

Ref.	Authors and brief study outline	Salient findings
Carroll et al[8]	This study had a few separate components, which included the intra-tumoral injection of killed mycobacteria (complete Freund’s adjuvant/CFA) in in vivo models, natural tumors in dogs (mastocytoma/mast cell tumor) and horses (melanoma), and patients with different advanced cancers from Switzerland and Australia (administered into primary or metastatic tumors). In female DBA/2 J mice, for tumor development, the following cells were injected: P815 (mouse mastocytoma) cells and 4T1 (mouse mammary adenocarcinoma) cells orthotopically, and CT26 (mouse colon carcinoma) cells subcutaneously	The efficacy of intra-tumoral CFA injection was observed in orthotopically injected P815 and 4T1 tumors but not in the non-orthotopically injected CT26 tumors. Out of 14 dogs, 3 dogs presented complete regression of their tumor and substantially extended survival in 2 cases. So, in dogs, 21% showed a complete response and an overall response for 35%. Among horses (n = 11), 3 horses (27%) exhibited the sign of clinical response (mass remission and reduction in size). CFA administration induced extensive infiltration of immune cells into the tumors in comparison to control (saline). Out of 8 patients, 5 showed substantial immune cell infiltration in tumor tissue along with extensive necrosis
Park et al[9]	This was a clinical study conducted in Korea; 19 patients with HPV-16 single infection and histologically confirmed CIN3 were included between 2014-April and 2016-March, and they were administered orally Lactobacillus casei that expressed HPV-16 E7 antigen on the surface. Phase 1 was a safety test, and phase 2a was an efficacy test, which finally included 8 women who were administered this bacterial agent (1000 mg) in weeks 1, 2, 4, and 8 (5 times a week)	There was a steady increase of HPV-16 E7-specific IgG level in plasma. Histological responses were assessed 7 weeks after the final medication. Clinical efficacy was observed in 6 patients (out of 8, 75%); complete remission to normal was seen in 3 women, and in the other 3 women, regression to CIN1 was noticed. There were no seriously observed adverse effects at 1000 mg administered dosage of this oral vaccine
Augustin et al[10]	This was an in vivo study that utilized BALB-neuT mice and a virulence-attenuated strain of Salmonella typhimurium for the colonization of tumors and the delivery of immunomodulators. The plasmids in the bacteria were designed to express IL-15 superagonist IL-15Ra-IL-15 (having anticancer efficiency) as well as anti-CTLA-4 and anti-PD-L1 (immune checkpoint inhibitors). BALB-neuT transgenic mouse model overexpresses HER2 and spontaneously develops mammary carcinoma. The investigators also used cannabidiol, a vascular disrupting agent, to create intra-tumoral necrotic spaces	In transgenic mice, the combination treatment regimen showed a statistically significant 64% slower tumor growth and a 25% increase in mean survival time in comparison to without treatment control mice. Furthermore, the experiments were completed with minimal toxicity, as assessed by less than 7% weight loss and a restoration of normal weight gain in 3 days after administration of the bacteria intravenously
Manuel et al[11]	In this in vivo study, KPC-luc pancreatic cancer cells were orthotopically implanted into the pancreas of C57BL/6 mice and treated with the combination of PEGPH20 and shIDO-ST. PEGPH20 diminishes hyaluronic acid in the tumor extracellular matrix, causing stromal remodeling, vascular reduction, and improved drug delivery, while shIDO-ST is a Salmonella typhimurium-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase	The study noticed extended survival and frequent total regression of tumors. Along with this type of response to the treatment, there was also migration and accumulation of activated neutrophils into tumors. Furthermore, CD8+ T cells were recorded to be associated with the late control of tumors. Following treatment, supportive care for animals was not required, and maintenance of normal body weight was noted
Hsieh et al[12]	The aim of this in vivo study was to determine the efficacy of Escherichia coli engineered to overexpress β-glucuronidase [E. coli (lux/βG)] in anticancer activity. Irinotecan (CPT-11) is used in chemotherapy for colon cancer and hydrolyzed into SN-38 (active form). SN-38 is rapidly metabolized to inactive glucuronide conjugate SN-38G. However, β-glucuronidase can enhance anti-tumor effectiveness by converting SN-38G into SN-38. NOD/SCID mice were inoculated subcutaneously with different colon cancer cells (HCT116, LS174T, and CT26). A single intravenous injection of E. coli was administered when the tumor size reached about 150 mm3	E. coli (lux/βG) could hydrolyze SN-38G to SN-38 and selectively accumulate in tumors. However, E. coli (lux/βG) did not efficiently enhance SN-38 anticancer activity in tumor xenografts in comparison to non-engineered E. coli. On the other hand, the incubation of SN-38G with E. coli (lux/βG) increased the inhibition of HCT116 cell growth by 135-fold. In addition, E. coli (lux/βG) can produce cytotoxic concentrations of SN-38 from SN-38G
Yamatsugu et al[13]	In this study (both in vitro and in vivo), antibody mimetic-drug conjugates based on high-affinity binding of mutated streptavidin and modified iminobiotin were used to target HER2-positive KPL-4 breast cancer cells and in KPL-4 xenograft model (BALB/cSlc-nu/nu nude mice). Antibody mimetics (i.e., non-immunoglobulin miniproteins or affibody molecules) were fused to a streptavidin variant, which was subsequently expressed in E. coli inclusion bodies	By utilizing bis-iminobiotin conjugated with photo-activating silicon phthalocyanine, the drug conjugates developed against HER2 successfully killed cancer cells. Moreover, the HER2-targeting antibody mimetic-drug conjugates were demonstrated to be useful for the in vivo xenograft model
Shahid et al[14]	This in vitro study utilized genetically engineered E. coli (E. coli BL21) that secretes a pore-forming toxin HlyE, and binds to HER2 receptors on JIMT-1 breast cancer cells by anti-HER2Rs15d nanobody. JIMT-1 cells are hormone receptor negative (both estrogen and progesterone receptors), amplified HER2 positive, and insensitive to trastuzumab	The results showed that the nanobody effectively bound to HER2-positive JIMT-1 cells in vitro, and E. coli BL21 killed the target cells with HlyE toxin, which was secreted by using the YebF secretion system (a type VI secretion system)

CFA: A potent immune stimulant that contains mineral oil, surfactant, and heat-killed Mycobacteria; CIN: Cervical intraepithelial neoplasia; HPV: Human papillomavirus; IL: Interleukin; Luc: Luciferase (firefly); Lux: Luciferase (bacterial); PEGPH20: PEGylated human recombinant PH20 hyaluronidase; E. coli: Escherichia coli; CTLA-4: Cytotoxic T lymphocyte-associated antigen-4; PD-L1: Programmed cell death ligand 1.

Table 2 Salient findings of recent studies that analyzed a considerable number of patients with Wilms tumor for prognostic characteristics

Authors and brief study outline	Statistical significance of the results	Study outcomes
The study by Neuzil et al in 2020[47], tried to characterize health differences among Tennessee (United States) children treated for renal cancers that included Wilms tumor, renal cell carcinoma, and sarcoma	In TCR, for both race groups (black and white), Wilms tumor represented 80% of recorded malignancies. However, in the subgroup analysis of Wilms tumor cases, there was an over-representation of black children (n = 33, 26%) in relation to the state census for this specific race (16.8%). Compared to other renal malignancies, Wilms tumor exhibited the youngest median age at presentation (36 months/IQR: 12.0-48.0). Regarding overall survival, there was no significant difference between black and white patients with Wilms tumor	Wilms tumor remained the most common cancer of the kidney, and renal cell carcinoma was the least common. When combining all renal malignancies, specifically renal cell carcinoma and sarcoma, black children in the state of Tennessee presented with more advanced disease and experienced worse survival
The authors used the TCR (between 2004 and 2015) for patients aged 18 years or younger diagnosed with any form of renal malignancies (n = 160; Wilms tumor cases - 129, i.e., 81%). To further investigate treatment and outcomes, they conducted a retrospective cohort study of pediatric renal cancer patients from their Vanderbilt University Registry (ICR, n = 121; Wilms tumor cases - 100, i.e., 83%)	With reference to Wilms tumor, univariate logistic regression analysis for predicting mortality from pediatric renal malignancies showed a significant difference in case of renal cell carcinoma (OR = 5.75, 95%CI: 1.25-26.5, P value: 0.025) in TCR and (OR = 45.00, 95%CI: 3.77-536.92, P value: 0.003) in ICR, as well as for sarcoma (OR = 11.5, 95%CI: 4.00-33.07, P value < 0.001) in TCR. However, in multivariate analysis, only sarcoma showed a statistical significance (OR = 17.86, 95%CI: 4.68-68.2, P value < 0.001) in TCR	The survival disparities appeared to be reduced when treated at a comprehensive pediatric cancer center, which could alleviate the inequalities. Among both TCR and ICR, metastasis at diagnosis was independently predictive of worse prognosis
	Black patients were 26% of all patients in TCR (in ICR - 21%), presented more frequently with metastasis than white patients (37% vs 16%, P value: 0.021), and demonstrated worse overall survival (73% vs 89%, P value: 0.018). In ICR, similar survival among race groups (92% vs 93%, P value: 0.868) was recorded	The study highlighted the disparities in health outcomes due to socio-economic factors among pediatric renal cancer patients in the state of Tennessee
The study by Ekuk et al[48] in 2023 determined the one-year overall survival of Wilms tumor cases and their predictors among children diagnosed in the Pediatric Oncology and Surgical Units of MRRH, western Uganda	The one-year overall survival rate was found to be 59.3% (95%CI: 40.7-73.3)	Overall survival of Wilms tumor at MRRH was found to be 59.3%, and prognostic factors documented were unfavorable histology and tumor size greater than 15 cm
In this study, the treatment charts and files of children diagnosed and managed for Wilms tumor (n = 41) were retrospectively reviewed for the period from January 2017 to January 2021. The clinical details of children with histologically confirmed diagnoses were examined for demographic information, clinical and histological characteristics, and treatment modalities	Tumor size greater than 15 cm was identified as a significant predictor of poor survival, with a P value of 0.021. Tumor size above 15 cm was found to increase the risk of death from Wilms tumor by 6 times (95%CI: 1.32-34.95)
	Unfavorable Wilms tumor type (anaplasia) was another significant predictor of poor survival, with a P value of 0.012	The study highlighted the importance of tumor size and histology in predicting the survival outcomes for children with Wilms tumor
	Unfavorable histology type was found to increase the risk of death from Wilms tumor by 5.1 times (95%CI: 1.77-92.50)

TCR: Tennessee Cancer Registry; ICR: Institutional Cancer Registry; IQR: Interquartile range; OR: Odds ratio; CI: Confidence interval; MRRH: Mbarara Regional Referral Hospital.