A case of personalized and precision medicine: Pharmacometabolomic applications to rare cancer, microbiological investigation, and therapy.

RATIONALE: Advances in metabolomics, together with consolidated genetic approaches, have opened the way for investigating the health of patients using a large number of molecules simultaneously, thus providing firm scientific evidence for personalized medicine and consequent interventions. Metabolomics is an ideal approach for investigating specific biochemical alterations occurring in rare clinical situations, such as those caused by rare associations between comorbidities and immunosuppression. METHODS: Metabolomic database matching enables clear identification of molecular factors associated with a metabolic disorder and can provide a rationale for elaborating personalized therapeutic protocols. Mass spectrometry (MS) forms the basis of metabolomics and uses mass-to-charge ratios for metabolite identification. Here, we used an MS-based approach to diagnose and develop treatment options in the clinical case of a patient afflicted with a rare disease further complicated by immunosuppression. The patient's data were analyzed using proprietary databases, and a personalized and efficient therapeutic protocol was consequently elaborated. RESULTS: The patient exhibited significant alterations in homocysteine:methionine and homocysteine:thiodiglycol acid plasma concentration ratios, and these were associated with low immune system function. This led to cysteine concentration deficiency causing extreme oxidative stress. Plasmatic thioglycolic acid concentrations were initially altered and were used for therapeutic follow-up and to evaluate cysteine levels. CONCLUSIONS: An MS-based pharmacometabolomics approach was used to define a personalized protocol in a clinical case of rare peritoneal carcinosis with confounding immunosuppression. This personalized protocol reduced both oxidative stress and resistance to antibiotics and antiviral drugs.

Parapseudoflavitalea muciniphila gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from a human peritoneal tumour and reclassification of Pseudobacter ginsenosidimutans as Pseudoflavitalea ginsenosidimutans comb. nov.

A Gram-stain-negative, microaerophilic, non-motile, rod-shaped bacterium strain designated PMP191FT, was isolated from a human peritoneal tumour. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the organism formed a lineage within the family Chitinophagaceae that was distinct from members of the genus Pseudoflavitalea (95.1-95.2 % sequence similarity) and Pseudobacter ginsenosidimutans (94.4 % sequence similarity). The average nucleotide identity values between strain PMP191FT and Pseudoflavitalea rhizosphaerae T16R-265T and Pseudobacter ginsenosidimutans Gsoil 221T was 68.9 and 62.3% respectively. The only respiratory quinone of strain PMP191FT was MK-7 and the major fatty acids were iso-C15 : 0, iso-C15 : 1 G and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids consisted of phosphatidylethanolamine and some unidentified amino and glycolipids. The G+C content of strain PMP191FT calculated from the genome sequence was 43.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strain PMP191FT represents a novel species and genus for which the name Parapseudoflavitalea muciniphila gen. nov., sp. nov. is proposed. The type strain is PMP191FT (=DSM 104999T=ATCC BAA-2857T = CCUG 72691T). The phylogenetic analyses also revealed that Pseudobacter ginsenosidimutans shared over 98 % sequence similarly to members of the genus Pseudoflavitalea. However, the average nucleotide identity value between Pseudoflavitalea rhizosphaerae T16R-265T, the type species of the genus and Pseudobacter ginsenosidimutans Gsoil 221T was 86.8 %. Therefore, we also propose that Pseudobacter ginsenosidimutans be reclassified as Pseudoflavitalea ginsenosidimutans comb. nov.

Frontline Science: Microbiota reconstitution restores intestinal integrity after cisplatin therapy.

Due to their cytotoxic activities, many anticancer drugs cause extensive damage to the intestinal mucosa and have antibiotic activities. Here, we show that cisplatin induces significant changes in the repertoire of intestinal commensal bacteria that exacerbate mucosal damage. Restoration of the microbiota through fecal-pellet gavage drives healing of cisplatin-induced intestinal damage. Bacterial translocation to the blood stream is correspondingly abrogated, resulting in a significant reduction in systemic inflammation, as evidenced by decreased serum IL-6 and reduced mobilization of granulocytes. Mechanistically, reversal of dysbiosis in response to fecal gavage results in the production of protective mucins and mobilization of CD11b+ myeloid cells to the intestinal mucosa, which promotes angiogenesis. Administration of Ruminococcus gnavus, a bacterial strain selectively depleted by cisplatin treatment, could only partially restore the integrity of the intestinal mucosa and reduce systemic inflammation, without measurable increases in the accumulation of mucin proteins. Together, our results indicate that reconstitution of the full repertoire of intestinal bacteria altered by cisplatin treatment accelerates healing of the intestinal epithelium and ameliorates systemic inflammation. Therefore, fecal microbiota transplant could paradoxically prevent life-threatening bacteremia in cancer patients treated with chemotherapy.

Antimicrobial Properties of Perfusate Fluid After Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CS-HIPEC) with Mitomycin C.

BACKGROUND: Infectious postoperative complications often delay systemic chemotherapy after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CS-HIPEC). Because the authors have empirically observed fewer incisional infectious complications than expected after CS-HIPEC with mitomycin C (MMC), they investigated the antimicrobial properties of HIPEC perfusate fluid. METHODS: This study prospectively measured in vitro bacterial growth inhibition by HIPEC perfusate (n = 18). After 10 µL of perfusate had been plated on agar plate inoculated by standard strains of either Escherichia coli (strain 25922) or Staphylococcus aureus (strain 25923), it was incubated at 37 °C for 24 h. Antimicrobial activity evidenced by a zone of complete growth inhibition was measured in millimeters. These were compared against growth inhibition produced by control groups represented by MMC solution in normal saline (MMC concentrations of 2, 4, 6, 8, and 8.75 µg/mL), 7 per group. RESULTS: Bacterial inhibition by HIPEC perfusate was stronger against E. coli than against S. aureus (13.1 ± 6.8 vs 8.3 ± 7.7 mm; p = 0.005). No E. coli inhibition was observed for MMC saline in concentrations of 2 through 8 µg/mL (p < 0.001 each), and inhibition of 4.5 ± 5.7 mm was observed for an MMC saline concentration of 8.75 µg/mL (p = 0.007). The S. aureus inhibition zones by MMC saline solutions were 2.2 ± 2.1 (p = 0.002), 5.1 ± 2.3 (p = 0.135), 7.5 ± 1.0 (p = 0.654), 9.6 ± 0.9 (p = 0.058), and 10.2 ± 0.4 mm (p = 0.021). CONCLUSION: The antimicrobial properties of HIPEC perfusate are considerable but variable between patients and stronger against E. coli than against S. aureus. Further studies of HIPEC carrier solutions and chemotherapy agents may result in reduction of surgical-site infection and thus enhanced patient recovery.

Intraperitoneal administration of tumor-targeting Salmonella typhimurium A1-R inhibits disseminated human ovarian cancer and extends survival in nude mice.

UNLABELLED: Peritoneal disseminated cancer is highly treatment resistant. We here report the efficacy of intraperitoneal (i.p.) administration of tumor-targeting Salmonella typhimurium A1-R in a nude mouse model of disseminated human ovarian cancer. The mouse model was established by intraperitoneal injection of the human ovarian cancer cell line SKOV3-GFP. Seven days after implantation, mice were treated with S. typhimurium A1-R via intravenous (i.v.) or i.p. administration at the same dose, 5 × 10(7) CFU, once per week. Both i.v. and i.p. treatments effected prolonged survival compared with the untreated control group (P=0.025 and P<0.001, respectively). However, i.p. treatment was less toxic than i.v. TREATMENT: Tumor-specific targeting of S. typhimurium A1-R was confirmed with bacterial culture from tumors and various organs and tumor or organ colony formation after i.v. or i.p. injection. Selective tumor targeting was most effective with i.p. administration. The results of the present study show S. typhimurium A1-R has promising clinical potential for disseminated ovarian cancer, especially via i.p. administration.

A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei.

BACKGROUND: Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. METHODS: To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. MAIN RESULTS: We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. CONCLUSIONS: Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.

Antibiotic treatment decreases microbial burden associated with pseudomyxoma peritonei and affects ß-catenin distribution.

PURPOSE: Pseudomyxoma peritonei is an understudied cancer in which an appendiceal neoplasm invades the peritoneum and forms tumor foci on abdominal organs. Previous studies have shown that bacteria reside within pseudomyxoma peritonei tumors and mucin. Thus, we sought to analyze the effect of antibiotics on bacterial density and ß-catenin expression within pseudomyxoma peritonei samples. EXPERIMENTAL DESIGN: The study included 48 patients: 19 with disseminated peritoneal adenomucinosis (DPAM) and 29 with peritoneal mucinous carcinomatosis (PMCA). Fourteen patients were given antibiotics (30 mg lansoprazole, 1 g amoxicillin, and 500 mg clarithromycin) twice a day for 14 days. One week after completion of therapy, surgery was conducted and specimens were harvested for pathology, bacterial culture, ISH, and immunohistochemistry. RESULTS: ISH showed the presence of bacteria in 83% of the patient samples, with a higher Helicobacter pylori density observed in PMCA versus DPAM. PMCA patients treated with antibiotics had a significantly lower bacterial density and decreased ß-catenin levels in the cytoplasm, the cell nuclei, and mucin-associated cells. Although not significant, similar trends were observed in DPAM patients. Cell membrane ß-catenin was significantly increased in both DPAM and PMCA patients receiving antibiotics. CONCLUSIONS: Bacteria play an important role in pseudomyxoma peritonei. Antibiotic treatment improved the histopathology of tissue, particularly in PMCA patients. In PMCA, antibiotics decreased bacterial density and were associated with a significant ß-catenin decrease in the cytoplasm, cell nuclei, and mucin along with a small membrane increase. These results suggest that antibiotics offer potential protection against cell detachment, cellular invasion, and metastasis.

In vivo monitoring the process of tumor growth, metastasis and bacterial infection expressing GFP via real-time optical imaging.

Noninvasive molecular fluorescence imaging in vivo which combines Optical imaging with genetic marker technology can real time monitor the development of tumor, through the use of human adenoid cystic carcinoma cell (ACC-M) and lung carcinoma cells SPC-A1 were thansfected by green fluorescent protein (GFP). This study established three types of model: Experimental metastases by tail vein injection of ACC-M-EGFP, spontaneous metastases by abdomen subcutaneous injection of SPC-A1-EGFP and subcutaneous tumor growth by subcutaneous injection of SPC-A1-EGFP. Tumor-bearing mice were viewed with whole-body fluorescent imaging system. The results showed that growth and metastasis of tumor were visualized clearly with this system. While this simple, nonintrusive technique can show in great detail the temporal behavior of the infectious process of red fluorescent protein (DsRed2)-expressing bacteria from outside intact infected animals. Therefore, this study provides a platform for monitoring tumor growth and metastasis and evaluating efficacy of antitumor drugs in vivo.

Clinicopathological features and prognostic factors of proximal gastric carcinoma in a population with high Helicobacter pylori prevalence: a single-center, large-volume study in Korea.

BACKGROUND: The incidence of gastric cancers has fallen in recent decades. However, a substantial reduction in Helicobacter pylori prevalence and a substantial increase in the incidence of proximal gastric cancer (PGC) have been observed in the West and Japan, but not in other East Asian countries. The purpose of this large-volume study was to analyze prevalence, clinicopathological features, and prognosis of PGC compared with other types of gastric cancer in Korea, where there is high incidence of H. pylori infection. METHODS: Between 2000 and 2005, a total of 3,193 patients were enrolled. We analyzed clinicopathological features and survival outcomes. RESULTS: Chronological analysis showed increasing incidence of PGC over the study period. PGC patients were younger and had higher incidence of Bormann types III and IV than did distal gastric cancer (DGC) patients. Also, PGC was associated with a significantly higher proportion of poorly differentiated type, T3 and T4 stage, and positive lymph nodes compared with DGC. Peritoneal and other distant metastases were more common in PGC group than in DGC group. The 5-year survival rate was significantly lower in PGC than in DGC group, regardless of curative resection. Also, the N0 and N1 category significantly influenced the 5-year survival rate. Tumor-node-metastasis (TNM) stage, hepatic metastasis, and curative resection were significant prognostic factors in PGC patients. CONCLUSIONS: PGC has increased in incidence with the respective decline in H. pylori prevalence in Korea. Survival was worse for patients with PGC than for those with DGC, regardless of curative respectability. PGC is often diagnosed at more advanced stage than other gastric cancers, and therefore early detection is critical for successful treatment.

Pseudomyxoma peritonei: is disease progression related to microbial agents? A study of bacteria, MUC2 AND MUC5AC expression in disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis.

BACKGROUND AND AIMS: Pseudomyxoma peritonei (PMP) is characterized by peritoneal tumors arising from a perforated appendiceal adenoma or adenocarcinoma, but associated entry of enteric bacteria in the peritoneum has not been considered as a cofactor. Because Gram-negative organisms can upregulate MUC2 mucin gene expression, we determined whether bacteria were detectable in PMP tissues. METHODS: In situ hybridization was performed on resection specimens from five control subjects with noninflamed, nonperforated, non-neoplastic appendix and 16 patients with PMP [six with disseminated peritoneal adenomucinosis (DPAM) and 10 with peritoneal mucinous carcinomatosis (PMCA)]. Specific probes were designed to recognize: (1) 16S rRNA common to multiple bacteria or specific to H. pylori; (2) H. pylori cagA virulence gene; or (3) MUC2 or MUC5AC apomucins. Specimens from one patient with PMCA were examined by ultrastructural immunohistochemistry. Bacterial density and apomucin expression were determined in four histopathological compartments (epithelia, inflammatory cells, stroma, and free mucus). RESULTS: Enteric bacteria were detected in all specimens. Bacterial density and MUC2 expression were significantly (p < 0.05) higher in PMCA than in DPAM and controls and were highest in free mucin. MUC2 was also expressed in dysplastic epithelia and in associated inflammatory cells. MUC2 expression was significantly correlated with bacterial density. CONCLUSIONS: Multiple enteric bacteria are present in PMP, and bacterial density and MUC2 expression is highest in the malignant form of PMP. Based on these observations, we propose that the bacteria observed in PMP may play a role in the mucinous ascites and perhaps promote carcinogenesis.

Suppression of peritoneal metastasis in human gastric carcinoma by enhanced immunogenicity of B7-1 transfection.

B7-1, a co-stimulatory factor, has been reported to induce cytotoxic T lymphocytes (CTL). In the present study, we transfected B7-1 genes into a gastric cancer cell line (2MD3) and analyzed the effects of B7-1 transduction on peritoneal metastasis in vitro and in vivo. We revealed that mononuclear lymphocytes show significantly stronger adherence and cytotoxicity to B7-1 transfected cells (2MD3/B7) than to their parent 2MD3 cells. We also demonstrated that mice inoculated with 2MD3/B7 cells in the peritoneal cavity have a significantly better survival rate than those inoculated with 2MD3 cells (log-rank test, p<0.01). Histologic findings showed that leukocytes intensively infiltrate the 2MD3/B7 metastatic nodules, but can scarcely be observed in the nodules associated with 2MD3 cells. These findings indicate that the B7-1 may play an important role in suppressing peritoneal metastasis by the mechanism of enhanced immunogenicity, and that B7-1 gene transduction might be effective against peritoneal metastases of gastric cancer.

Immunohistochemical demonstration of TGF-beta and decorin in paracoccidioidal granulomas.

Different patterns of granulomas have been observed in 6- to 8-week-old mice after ip inoculation with 5 x 10(6) yeast cells of Paracoccidioides brasiliensis. Transforming growth factor-beta (TGF-beta) is a cytokine that has been shown to participate in fibrosis and granuloma formation; its activities seem to be modulated by the small proteoglycan decorin. In the present study, TGF-beta and decorin expression in epiploon granulomas was assessed by immunohistochemistry in susceptible (B10.A) and resistant (A/J) mice after 15, 30, 120 and 150 days of P. brasiliensis ip infection. The epiploon was collected, fixed in Methacarn solution and embedded in paraffin, and 5-microm thick sections were used for immunohistochemical analysis employing the streptavidin-biotin-peroxidase technique. The former mouse strain developed fatal disease with many disseminated lesions increasing in size and number during the infection and the latter developed mild disease with the presence of encapsulated granulomas. In the epiploon, TGF-beta was present on macrophages, giant cells, lymphocytes and fibroblasts, and absent on neutrophils. It was also detected in areas of fibrosis and necrosis, as well as disperse in amorphous extracellular matrix, mostly in resistant mice. Decorin was present circumscribing macrophages and giant cells containing fungi, but absent on these cells. In both mouse strains, decorin was found at the periphery of the lesions, and markedly in milky spot granulomas. In resistant mice, positivity was found around fibrotic and necrotic areas of encapsulated and residual lesions containing lysed fungi. Decorin was found associated with thick fibers around encapsulated lesions. In susceptible mice, the size and number of lesions increased with the progression of the disease and were correlated with the weaker expression of decorin. We suggest an association of decorin with the fibrogenic process observed in paracoccidioidal granulomas.

Urinary undiversion for pelvic actinomycosis: a long-term follow up.

BACKGROUND: A 43-year-old woman who had been using intrauterine contraceptive devices for the past 10 years underwent an emergency operation for bowel and urinary obstruction. METHODS/RESULTS: Frozen section analysis showed undifferentiated adenocarcinoma. Incomplete tumorectomy, ileal resection, partial cystectomy, colostomy and bilateral ureterocutaneostomy were palliatively performed. Postoperatively, periodic acid-Schiff and Grocott-Gomori methenamine tests revealed Actinomyces and the final diagnosis was pelvic actinomycosis. Treatment with penicillin G administered intravenously relieved her symptoms and the lesion was dramatically improved. The patient underwent colostomy closure and urinary undiversion. CONCLUSIONS: Five years after urinary undiversion, the patient's renal function has been maintained and she can void without incontinence and dysuria.

A teratoma in a duck infected congenitally with duck hepatitis B virus.

A teratoma was diagnosed in an 8-month-old pekin duck based on the presence of tissue derived from embryonic ectoderm, mesoderm, and endoderm in the neoplasm. The neoplasm was examined for the presence of duck hepatitis B virus, because the duck was congenitally infected with the virus, a member of the hepadnavirus family that is associated with hepatic neoplasms in hepadnavirus-infected mammals. Persistent infection occurred in the liver, but no evidence of viral infection was found in the neoplasm.

C-type virus particles in urethan-induced pulmonary and renal carcinomas, in cell-graft-transmitted carcinosarcomas, and in filtrate-induced lymphomas in mice.

Repeated injections of urethan into suckling BALB/c mice induced multiple papillary adenocarcinomas in the lungs and kidneys. When the pulmonary tumors were transplanted i.p. by cell graft into 6 suckling BALB/c mice, they induced disseminated carcinosarcomas within the peritoneal cavity in all inoculated animals. Tumors resulting from the transplantation of tumor cells were used for preparation of filtered extracts. The filtrates were inoculated into 6 suckling BALB/c mice and induced generalized malignant lymphomas in all animals. The primary urethan-induced pulmonary and renal tumors, the carcinosarcomas that resulted from i.p. cell transfer, and also the generalized malignant lymphomas induced by inoculation of filtered extracts contained C-type virus particles. Theoretically, it could be assumed that both the primary urethan-induced pulmonary and renal tumors, as well as the cell-graft-induced peritoneal carcinosarcomas, contained the C-type virus particles as passengers, not necessarily related etiologically to the tumors in which they were found. It is quite likely, however, that these virus particles were etiologically related to the filtrate-induced malignant lymphomas in which they were also found.