The group held a conference calls on March 1, April 26, and June 14. The next conference call is scheduled for August 16, 2024.
March 1: Ms. Nieciecki presented several Powerpoint slides that covered new microbiome results that she has recently gotten. Ms. Nieciecki mentioned that the meta data provide by Mercy had been very useful for her analysis. One slide discussed the core PMP microbiome at the genus level that she has found. Included in the core genus microbiome were: Staphylococcus, Pseudomonas, Acinetobacter, Streptococcus, and Corynebacterium. Ms. Nieciecki found that the bacteria in PMP tumors clustered into two groups, independent of metadata. One cluster was higher in Bacteroides, and the other cluster was higher in Staphylococcus, Pseudomonas, and Acinetobacter. Finally, Ms. Nieciecki’s results showed that patients who had received prior chemotherapy had a less diverse tumor microbiome than those who had not received chemotherapy. Ms. Nieciecki did not have information on whether the patients had received antibiotics. She will incorporate her new results into the paper she is working on. A lengthy discussion of Ms. Nieciecki’s results took place. Dr. Merrell suggested that that it might be good to look at patient overall survival.
Dr. McAvoy mentioned the recent paper that he had found on DNA testing of 15 PMP patients.The paper title is:Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms. https://pubmed.ncbi.nlm.nih.gov/38262376/. This paper is the first report of comprehensive DNA methylation profiles of PMP patients in the world. The paper indicated that genes associated with neuronal development and synaptic signaling may be involved in the development of PMP. Dr. Metcalf suggested that the neurons and synapses probably were in the peritoneal region and not the brain. Their change could lead to a suppressed immune system. After the conference call Dr. McAvoy found that the supplementary data from the paper had been published, but not the paper itself.
April 26: Dr. Li presented several Powerpoint slides that covered his research on compounds that he has isolated from PMP cell line cultures. The slides showed the apoptosis activity that two of the compounds have against PMP cell lines. Their activity is comparable to that of mitomycin C which is used in heated chemotherapy treatment for PMP. The compounds also have activity against other types of cancer cell lines including colon cancer and breast cancer. The compounds did not show activity against normal cell lines. Additional compound analogs have been isolated by Dr. Li and they will be studied by him. Dr. Merrell raised the issue of performing Synergy Assays to see if the novel compounds show synergistic interaction with mitomycin C and other cancer-related drugs. An extended discussion took place about Dr. Li’s presentation and the culturing of PMP cells from tumors.
Dr. Metcalf discussed the manuscript on our latest microbiome research. The paper will be submitted and she recommended Cell, Host, and Microbe as the journal for submission. Dr. Metcalf felt that our results have broader implications than for PMP itself and that Cell, Host, and Microbe would reach a broader audience of readers. A discussion then took place about the paper that Dr. McAvoy had emailed to the group prior to the conference call. This paper, which is different from the paper mentioned in the March 1 summary above, discussed microbiome findings on mucin taken from PMP patients. The most prevalent species that was found was Pseudomonas plecoglossicida (~20%). Several questions were raised by the group about this finding. After the conference call Dr. Merrell clarified that the author’s approach sequenced on the order of 450-500 base pairs from the 16S gene, which has about 1600 base pairs. To get fully accurate species identification, you typically need to sequence the full-length gene. Our group was trying to point out that getting a species level identification is very difficult with the type of approach discussed in the paper. Thus, the author’s report of a specific bacterium that has not been found by others in the human microbiome can be questioned. In addition, the quality of the journal in which the paper was published was questioned. As a result of the discussion, the group felt that the results in the paper were questionable. Dr. Li raised the issue of sequencing the entire genome of bacteria obtained from PMP tumors. Ms. Nieciecki pointed out that typically the samples she has analyzed have had low biomass. For whole genome sequencing to be successful, a tumor specimen with a lot of biomass should be studied. Ms. Nieciecki mentioned that one of her samples did contain significant biomass and Dr. Li might want to consider it.
June 14: Dr. Li discussed two updates on his research. In the first he talked about his work on isolating metabolites from cultures developed from PMP cell lines. In his purification process he noted that there were additional peaks, suggesting that there were additional metabolites that could be purified. In addition to the two compounds which he discussed in the last conference call, he has now isolated a third compound. Like the first two compounds this new isolate has cytotoxic activity against PMP cells but not against normal cells. The three compounds also have activity against other cancer cell lines, but their activity seems to be strongest against PMP cells. The second update involved whole genome sequencing, which Dr. Li is investigating for use with the bacteria he is studying. Dr. Li has been in contact with a company that does whole genome sequencing. He discussed a paper that appeared in the April 25 issue of Cell (“A pan-cancer analysis of the microbiome in metastatic cancer”). The brief description given in the paper is as follows: “Characterization of microbiome genomes at the species level in over 4,000 metastatic tumor biopsies identifies the distribution and diversity features of tumor-resident bacterial DNA at a pancancer scale, highlighting the associations between microbial community dynamics and tumor immunity and immunotherapy efficacy.” A discussion of this paper took place. Ms. Nieciecki pointed out that contamination is an important issue in whole genome analyses. It is important that human DNA not be included in the samples analyzed. The Cell paper does discuss the contamination issue. Dr. Metcalf mentioned that a 2020 paper that she was co-author on was recently retracted due to inclusion of human reads classified as bacteria. Dr. Li mentioned that the compounds showing PMP activity that he has isolated are not produced by humans, so that they must have originated from bacteria.
The group held a conference call on August 16, October 4, and December 6. The next conference call is scheduled for Jan. 17, 2025.
August 16: Dr. Li presented an overview of his research on isolating compounds that have shown anti-cancer properties for PMP cell lines. His goal is to develop chemotherapeutic molecules that kill cancer cells by stimulating T-cells in the immune system. The compounds he has found can lead to endoplasmic reticulum (ER) stress in cancer cells leading to apoptosis. The PMP cell lines on which he is testing his compounds were developed by Dr. Testerman.
Dr. Metcalf mentioned that she and Victoria have submitted their paper on the PMP microbiome research to the mSystems journal. Victoria will finish her Ph. D. studies next spring and Dr. Metcalf does not have any new PMP projects planned.
Ricki is new to our group and he went over his technical background. He has 15 years of experience in software engineering. He has also started 3 software companies starting in 2007. Ricki’s interest in PMP research started when his wife Bina was diagnosed with the disease in 2021. She was operated on and her disease recurred in 2023. Ricki gave a very detailed presentation on his research on PMP which involves docking simulations. Docking is a simulation process that attempts to predict the binding mode of interactions as well as the binding affinity of a molecule inside the active site of a known 3D structure of the target protein. Ricki’s presentation can be accessed here. A long discussion followed Ricki’s presentation. Two possible interactions with Dr. Li were mentioned. Ricki has been looking at repurposing NIH approved drugs for use with PMP. His top candidate is mebendazole. It was suggested that Dr. Li could try this drug on the PMP cell lines to assess its effect. Secondly, the question was raised about whether Ricki could simulate the compounds that Dr. Li has found in his studies. If the structure of the compounds can be determined then such simulation seems possible. After the call Dr. Sardi responded that in the past mebendazole was used at Mercy to treat colon cancer. He also said that it is very safe. There was also the possibility to study the use of Ivermectin which is another drug that Ricki found to be potentially effective. It was also suggested that it could be useful to contact NORD and ACPMP to let them know about Ricki’s research.
October 4. Li presented an overview of his research on isolating compounds that have shown anti-cancer properties for PMP cell lines. His goal is to develop chemotherapeutic molecules that kill cancer cells by stimulating T-cells in the immune system. The compounds he has found can lead to endoplasmic reticulum (ER) stress in cancer cells leading to immunogenic cell death. The PMP cell lines on which he is testing his compounds were developed by Dr. Testerman. Dr. Li presented several slides that gave results from his latest research. In the first slide he showed IC50, CRT, and ATP values for treating 3 cancer cell lines (breast, colon, and leukemia) in addition to results for treating the PMP cell line with the compounds he has been studying. CRT and ATP are two hall markers of ICD (immunogenic cell death). So active secretion of these markers (meaning both becoming high) means induction of ICD. IC50 is which is a measure of how much of a drug or compound is needed to inhibit a biological process by half. He compared the results from his compounds with positive controls, mitocycin C for IC50 and doxorubicin for CRT and ATP. The cancer cells were treated with 10 mM of compound for CRT and APT measurements. The table below gives the results for PMP.
| Compound | IC50 (mM) | APT (mM) | CRT (mM) |
| Positive control | 8.7 | 14.8 | 95.9 |
| 1 | 8.9 | 20.2 | 91.1 |
| 2 | 12.9 | 22.6 | 90.0 |
| 4 | 10.8 | 25.4 | 94.3 |
Compounds 1,2, and 4 not only showed cytotoxicity, but they also induced immunogenicity to the PMP cell line. Compound 1 produced the same effects on the other cancer cell lines studied. Next Dr. Li discussed ICD for compound 4. He pointed out that ER stress and ROS (Reactive oxygen species) are linked to ICD. Further he pointed to a paper that stated that both ER stress and ROS should occur in parallel to induce ICD. His third slide showed that compound 4 can target ER stress, generate ROS, and induce ICD. Dr. Li’s fourth slide gave results calculated by Mr. Dahl for initial screening of binding affinities of Dr. Li’s compounds across multiple KRAS variants. Dr. Li’s fifth slide gave very preliminary results for CCK-8 assay results for Mebendazole. The CCk-8 assay measures cell viability by correlating the production of colored formazan dye to the amount of living cells in a culture. The 2 results shown in slide 5 had very different IC50’s and Dr. Li will rerun this assay. Dr. Li’s results are very encouraging.
Dr. Metcalf discussed the paper that she and Victoria have submitted to mSystems journal. Since the paper has been under review for two months, Dr. Metcalf contacted the journal editor to find out where things stand. She found out that one review has been obtained and the editor is waiting for a second review before making a decision. Dr. Metcalf then mentioned that she plans to study the issue of whether surgical contamination could be an important contributor to the study of tumor microbiomes. She will be participating in a study of liver cancer in dogs and attempting to measure a high-resolution microbiome of the cancer. Dr. Metcalf also expressed the opinion that rather than studying PMP tumors per se it would be better to study the microbiome in the appendix. The PMP tumors appear downstream from what takes place in the appendix, so whether a particular bacterium contributes/causes PMP would be better studied in the appendix. During the discussion Dr. Merrell asked about getting updated survival information on the PMP surgical patients at Mercy that were treated with antibiotics. Ms. King gave an overview of some of the compliance issues associated with these patients. She said she would be able to get the updated survival information and get it to the group.
December 6: Dr. Li had two items to report. He mentioned that this past week the material transfer agreement to send the PMP cells that he has been studying to Bina Therapeutics was approved. A study will be carried out by Archie Svetlov (working in collaboration with Ricki Dhal) who secured research access to GMP facilities to evaluate a new formulation of mebendazole (discussed below) on these cell lines. Dr. Li has also started research to improve the immunogenic cell death potential of the compounds he has been studying. His modified compounds appear to increase reactive oxygen species produced by the compounds. Dr. Li will continue this avenue of his research. After Dr. Li’s presentation Mr. Dhal raised the question of whether a transdermal patch might be a useful vehicle for administering drugs for PMP. This led to a discussion of how to best give drugs to treat PMP. Dr. Mc Avoy mentioned the idea of using the same approach that is used for peritoneal dialysis, since this would help circulate a drug through the peritoneal cavity. Dr. Terterman discussed the idea of using an ultrasound targeted needle to delivery drug intraperitoneally directly toward tumors. She also discussed the idea of using laparoscopic delivery of a drug.
Dr. Metcalf discussed the reviews that she and Victoria received for their paper submitted to the mSystems journal. Referee 1 raised a number of questions that should be easy to address. This referee did not appear to be a microbiome person, The second referee was very satisfied with the paper and did not have any major concerns. Dr. Metcalf discussed the fact that better surgical controls are needed for the microbiome research. She and Victoria will work on answering the referees and they hope to complete their reply by the end of the week of Dec. 9. The paper should be published in January 2025.
Mr. Dhal used a Powerpoint presentation to discuss his research discovery and development of a candidate drug for PMP. He gave a timeline of his efforts so far and it included his contacting regulatory and funding agencies in the UK. Ricki also discussed his plans for the next 2 months. The discussion below is taken from Ricki’s Powerpoint presentation. Ricki has formed Bina Therapeutics with a scientific co-founder (Archie Svetlov) and collaborators from the UK and USA. He hopes to complete preclinical work in January and to look into selecting a contract development and manufacturing drug organization then. The compound they have designed has a unique formulation to improve drug delivery in PMP. Their approach enhances drug sustainability and therapeutic effects, with studies showing superior efficacy against mucinous adenocarcinoma cell lines like LS174T. Below is a table from Ricki’s presentation comparing the new drug to mitomycin.
| Compound | LS174T |
| Lead Compound | 1.03mM – 4.07mM |
| Mitomycin C | 8.8mM – 11.6mM |
Ricki discussed the advantages of using benzimidazoles for treating PMP and other cancers. Ricki also discussed the additional studies that he plans to have completed by the end of January 2025