Period From 1/2021 Through 6/2021

The group held conference calls on 1/8, 2/26, 4/23  and 6/25

Jan. 8: Dr. Testerman reported that in her culturing work, some of the cells lines quit growing after a period of time. She has been adding growth factor to the cultures and this helps stimulate growth. Dr. Testerman has also been looking into the effect of mitomycin C on cells and she has found variations in sensitivity among cell lines. In this work she has also been studying colorectal cell lines and comparing them to PMP cell lines in terms of sensitivity to mitomycin C. She mentioned that due to the differences she is seeing that one chemotherapeutic treatment may not be effective for every type of bacteria present in PMP. Dr. Testerman continues to study compounds that have been generated from her culture studies. Dr. Merrell mentioned that he had received email from the FDA that additional sequencing results are available. He will follow up and check whether any of Dr. Testerman’s bacteria are in the new sequences. A discussion about the two broad cellular morphologies being studied by Dr. Testerman, namely epithelial and mesenchymal took place.

Dr. Merrell said that he is finalizing the process of sending PMP specimens to Dr. Metcalf for analysis. Prior to the conference call Dr. McAvoy emailed an Excel spreadsheet with PMP diagnosis and whether a particular bacterium (acinetobacter, staphylococcus, pseudomonas, corynebacteria) was present or not in a patient’s tumor sample. The 4 bacteria were present in many of the PMP samples and except for corynebacteria they were not significantly present in the negative control specimens. Dr. Testerman discussed the information in this spreadsheet and stated that she was interested in the overlap between PMP tissue samples and negative controls. She said that she had checked the literature to see which human-associated or environmental bacteria most often contaminate tissue culture. Dr. Testerman noted that she did not find many papers. In Dr. Testerman’s experience, contaminants are often staphylococci or pseudomonas and related organisms. She also has found that corynebacterium can be a contaminant. Concerning pseudomonas which could be in PMP tumor samples, Dr. Testerman found it is surprising that she has not been able to culture pseudomonas, since it is a very easy organism to culture. Dr. Testerman also mentioned she has seen that some bacteria prefer amino acids to glucose for growth. After the call she provided the flowing discussion: “When we characterized the first new species, PMP191F, we found that it did not like glucose. We found the same for another potentially new species, PMP215. Glucose is the primary sugar used by human cells and it is a preferred carbon source for many bacteria. H. pylori has some nutrient utilization strategies that appear to be consistent with efforts to not compete with the host for critical nutrients. Starving the host is not conducive to long-term colonization. Bacteria that live in the lumen of the gut use whatever the host hasn’t already absorbed. Bacteria that live in the mucous layer in close contact with gut epithelial cells may have evolved somewhat differently. They might rely more on components of mucus or epithelial cells that have died and sloughed off. Mucus contains polysaccharides made of various sugars like galactose and fructose, so it may be more advantageous for the bacteria to use those sugars.” Dr. McAvoy raised the question about whether the lack of a pattern relating bacteria to PMP diagnosis in our microbiome results is significant. Could one draw any conclusions from the lack of a pattern? Dr. Testerman said that she thought drawing any conclusions would be difficult. She thought that to see a pattern it would be necessary to have increased quantities of bacteria in our microbiome samples. It may well be that more than one bacterium is contributing to carcinogenicity. Ms. King mentioned that in the case of DPAM patients white cells can facilitate mechanical compression of tumors. She said that Dr. Sardi has used a glucose wash in these cases. Afterwards Ms. King provided an expanded discussion of her comments. “It is 2 separate thoughts:

1)      Histopathologically, we see white blood cells and other immune/inflammatory response cells try to sequester and contain the big, largely acellular mucin pools. Patients with DPAM often succumb to mechanical issues with the organs and it is likely that a more robust immune response kind of packs down these mucin pools. It also seems that patients with a greater immune response seen pathologically actually do worse. This is the thought behind using 5-FU or xeloda, which has a major anti-inflammatory component as a systemic chemotherapy for these patients. However, there is limited data.

2)      When a complete cytoreduction is not possible for patients with big mucinous tumors, the use of HIPEC is not indicated. However, in these scenarios, Dr. Sardi commonly washes the abdomen with a glucose solution, which he believes delays progression and helps keep the mucin broken up. I will have to ask him exactly what it is/more about it, since he usually just says “we need the sugar” and the OR staff knows what he means. I will let you know once I hear from him.”

Dr. McAvoy mentioned that he was aware of studied from Japan that used sugar washes to treat PMP and he said he would see if he can locate papers on this treatment that he has.

Feb. 26: Dr. Testerman continues to work on her culturing of cell lines. She has found that mesenchymal cells tend to quit growing but that epithelial cells are fully immortal. She is testing PMP associated bacterial compounds and has found that one affects metabolism and it suppresses cell growth. Dr. Testerman does not yet know the mechanism of the suppression. She plans to continue testing other compounds. So far Dr. Testerman has not heard back from the FDA on sequencing results from her specimens that were sent to them. She anticipates that within the next couple of months she will be able to have sequencing done locally.  Another issue that Dr. Testerman is investigating is whether bacteria can metabolize any chemotherapy agent. At present she has no results but she plans to run several tests in the coming weeks.

Dr. Merrell reported that not much has happened since our last call. He has sent samples to Dr. Metcalf for her to sequence. Since there is a concern that too much host DNA affects the downstream detection of bacterial DNA, Dr. Metcalf mentioned that her technician has first initiated work with chicken tissue. This initial work will be helpful to the technician when she studies PMP samples. Dr. Metcalf plans to have her technician extract DNA from 3 of the most abundant PMP samples that Dr. Merrell sent.  She hopes that this extraction can be started next week.

Ms. King reported that collection of PMP specimens from the OR has been restarted at Mercy. These specimens can be sent out to the group. For the fecal PMP microbiome study Ms. King reported that she now has 20 complete specimens of the 24 that were originally planned. The specimens were taken pre-op, 4-6 weeks post-op, and 1 year post-op from the 20 patients. Roughly one half of the patients received antibiotics. Ms. King said that most of the specimens were from patients with lower grade tumors, DPAM patients. Because of the delay caused by COVID, it would take at least another year to get specimens from 4 additional patients. A discussion took place about whether we should send out the 20 samples now and have them analyzed to determine what they show. The consensus in the group was to submit the 20 samples now. Dr. Metcalf mentioned that she did not know what the situation is with the American Gut Project processing samples at the present time. In particular she did not know if COVID has affected their operation, but she said she would follow up and check about processing times. Ms. King said that she would check to ascertain that all the kits that she has have been properly registered with the American Gut Project. She said that due to its complexity kit registration was not a simple process. This combined with the lengthy questionnaire associated with the samples has affected getting samples from some patients.

A discussion about the sequencing work being done at the FDA took place. Dr. Merrell mentioned that the FDA had contracted out the work and that this may be a contributor to the slow rate of getting results back. A discussion also took place about using a glucose wash to treat PMP patients where surgery could not be done because of the extent of the disease. Ms. King mentioned that Mercy was trying to get approval to use a combination of drugs developed in Australia to treat inoperable PMP cases. The drugs involved are bromelain and n-acetyl cysteine.

April 23: Dr. Testerman reported that she has continued her PMP culturing but in the latest cultures no bacteria have been growing. Dr. Testerman continues to study compounds that have been purified from earlier culture work and she is testing one interesting compound on various cell lines. The compound appears to be promising in its effect on PMP cells and Dr. Testerman will study its effectiveness on colon cancer cell lines. The goal is to find a compound that is effective against cancer cells but does not affect normal cells. Dr. Testerman also is continuing her work on sequencing bacterial strains. Dr. Merrell raised a question about whether the interesting compound was synthesized or purified from a culture. Dr. Testerman said that it had been purified.

Dr. Merrell reported that not much has happened since our last call. He is waiting for Dr. Metcalf to finish her microbiome study on the PMP specimens he has sent to her. Dr. Metcalf plans to use a ZYMO kit to measure to remove host DNA in the PMP specimens. The next sequencing run at Colorado State will in early May. Dr. Merrell mentioned that he recently attended a DOD microbiome conference on bioinformatics. One topic discussed involved what to do with singleton reads in microbiome results. In particular, should singletons be considered contaminants and eliminated or kept in the results. There was a detailed and lively discussion about this subject. Dr. Testerman pointed out that singletons are significant in PMP microbiome work since the specimens tested have low microbial DNA. Dr. Metcalf sits on a committee looking into this question and she has a great deal of expertise in this area. There is currently a debate within the committee about whether it is better to look at amplicon sequence variants (ASV) or operating taxonomic units (OTU) is evaluating microbime results. Dr. Metcalf favors ASV’s since they have more resolution than OTU’s. A long discussion on singletons took place. It was pointed out that bacterial ratios can change significantly depending on whether singletons are included or not.

Prior to the conference call Ms. King and Dr. Metcalf emailed one another about the path forward on the fecal microbiome specimens that have been collected at Mercy. Ms. King raised the question about whether the specimens we now have should be sent to the American Gut Project (AGP) in San Diego for processing. At present 23 PMP patients have had 1 specimen collected preoperatively. One of these patients passed away before additional specimens could be collected. For the remaining patients 16 have had 3 specimens collected, one preoperatively, one post operatively, and a third one year after surgery. Three patients have had 2 specimens collected, pre- and post operatively. Dr. Metcalf suggested a contact at AGP that Ms. King could contact concerning submission of the specimens for analysis. In particular it is important that all of the patients’ surveys have been submitted. Dr. Metcalf also indicated that if any problem arose with contacting AGP she could use her personal contacts to help. Once specimens are submitted it could take 2-4 months to get results back depending on what is in the AGP queue. A discussion took place about whether we should continue to enroll patients in the fecal microbiome study. The group felt that continuing enrollment is a good idea and we should go forward with it. Ms. King indicated that the protocol at Mercy can be amended to add additional patients. However, the number of available fecal collection kits will be a hurdle at some point.

June 25: Dr. Testerman reported that she has continued her PMP culturing research. She has an MS student who has done the experiments and he will be writing up his results in the near future.  The student has studied the growth of cell lines. In his studies he has not changed the growth media for the cells. Dr. Testerman noted that some cells have increased sensitivity to environmental conditions compared to others. The student has also studied the sensitivity of cells to the heated chemo agent used for PMP, mitomycin C. Dr. Testerman noted that the concentrations that cells have responded to are higher than those used clinically. Dr. Testerman also reported that work on the compounds isolated from her cultures is ongoing. One compound in particular, is able to invade cells. However, its concentration is low and therefor difficult to detect. Dr. Merrell raised the question about whether the student had seen any correlation in growth rate or chemo sensitivity between DPAM and PMCA specimens. Dr. Testerman said this is not yet clear.

Dr. Metcalf asked her student, Victoria Nieciecki, to discuss her results. Victoria went over 3 Powerpoint slides that she had prepared. An important goal of the research is to develop a methodology that will allow measurement of small amounts of bacteria in samples that have significant host DNA. Three PMP samples and one chicken sample have been studied. The PMP samples were 25 mg and 100 mg and 3 different extraction methods were examined.  Interestingly, Ms. Nieciecki found that smaller samples were better for DNA extraction than larger ones. She found that bacterial amplification/detection decreases with increasing host DNA. Dr. Merrell found it interesting that for tumor microbiome work less material rather than more seems to work well. This result is opposite from our original thinking on the matter. Also, off target PCR bands can result due to host DNA. These off target bands are amplification of DNA via PCR that are not the expected size. Dr. Metcalf and Ms. Nieciecki are targeting the 16S rRNA gene but it seems that the PCR is amplifying other regions of host DNA (off-target). The Qiagen PowerSoil extraction method was repeated with test tissue inputs of 25 mg and 100 mg and successful amplification of PMP-433 was achieved. Ms.  Nieciecki  also studied the UltraClean 2-step method. The purpose was to test a non-commercial method to reduce host DNA. Successful amplification was achieved with the PMP-433 sample, and possible amplification was noted for the other 2 PMP samples. The Zymo HostZero kit was also tested and significant contamination was found with it. The contamination may have come from the Proteinase K enzyme. If a methodology can be developed to eliminate host DNA and amplify small amounts of bacterial DNA the results would be very significant for studying bacteria in tumor specimens. Dr. Metcalf noted that Ms. Nieciecki has been supported on an NIH T32 training grant that supports ~5 or so graduate students a year. Victoria applied and was awarded one, which includes up to 2 years of funding for her stipend and tuition. She wrote her application about tumor microbiomes. After Ms. Nieciecki’s presentation an extensive discussion took place. Such things as coordinating with Dr. Johnson’s results, how many PMP samples would be needed in addition to the 3 studied, and optimizing the methodology were discussed. In choosing samples to test it is important that we do not exhaust the supply of a sample.

Ms. King said that there will be one HIPEC next week and after that Dr. Sardi will be away for all of July. Dr. Merrell asked whether any new antibiotics patients were scheduled. Ms. King said the antibiotics protocol had been put on hold due to the side effects that patients had experienced. The situation with the American Gut Project and the fecal microbiome samples that Mercy submitted was discussed next. Ms. King had emailed the contact at the American Gut Project on June 7 but had not yet heard back. Dr. Metcalf said that the American Gut Project wanted all the meta data complete before doing the fecal microbiome analysis. It would be best if all our fecal specimens were processed together in one batch. Dr. Metcalf said that she would follow up with the American Gut Project, and if necessary telephone them.

Period From 7/2021 Through 12/2021

The group held a conference calls on 8/27 and 10/15 and Dec. 10

Aug. 27: Dr. Testerman reported that she has tried a new strategy in her culturing experiments. Dr. Testerman is now incubating the intact tissue samples for several days in closed tubes so that the human cells will run out of oxygen and/or nutrients and die. The bacteria should withstand that fine. Then she adds growth medium and chops up the tissue a bit and incubates longer. Dr. Testerman has not yet determined if this new technique is working. She has not studied any new cell lines. Dr. Testerman continues her collaboration on studying compounds that have resulted from her culture. Dr. Testerman currently has a student who has studied the effect of chemotherapeutic agents on cancer cell lines and who is finishing his degree. The student has studied colorectal cells and PMP cells. For the PMP cells Dr. Merrell asked whether the student had seen any pattern with patient outcome data. Dr. Testerman mentioned that they had studied only 5 PMP cell lines so a pattern would be difficult to ascertain. Dr. Merrell also mentioned that the FDA finally finished the sequencing work on the samples Dr. Testerman submitted to them. He has forwarded the results to her, and she can compare these results with some of the sequencing results determined by her collaborator.

Ms. Nieciecki, a graduate student working under Dr. Metcalf, prepared a set of slides on her microbiome research which she discussed. Ms. Nieciecki has studied two different DNA extraction methods as applied to three PMP samples (PMP 423, 433, 485). The first method is an in-house two step technique and the second uses a commercial kit, Host Zero. For the in-house two step method the host DNA is first extracted, followed by extraction of bacterial DNA. Ms. Nieciecki gave concentrations of host and bacterial DNA for both extraction methods. She suspected contamination problems for the Host Zero kit. The Host Zero sequences had a mammalian and an unassigned component that Ms. Nieciecki attributed to host DNA amplification. When these two sequences were removed the number of reads in the data dropped by ~1/2. For the in-house method Ms. Nieciecki found that there appeared to be differences in her results between extraction fractions and that further study of this point needs to be made. When the Host Zero taxonomy is compared to the in-house two step taxonomy the comparison shows that the communities found were very different between the two methods. Also, the Host Zero communities were very similar to one another for the three PMP samples, whereas that was not the case for the two step method. Ms. Nieciecki’s last 2 slides dealt with beta diversity and Rhizobiaceae contamination. She pointed out that when Pro K is removed from the extraction, Rhizobiaceae is lost. Dr. Merrell mentioned that he will send the DNA results from Dr. Johnson to Ms. Nieciecki for comparison. He will also provide the original sequencing data. Dr. Testerman addressed the fact that different fractions gave different results and she stated that in all likelihood bacteria are not evenly distributed in tumors. Also Dr. Testerman noted that we are dealing with small amounts of bacterial DNA. Both effects could explain the results obtained for different fractions.

Ms. King reported that there she has been in contact with the University of California, San Diego concerning getting the fecal microbiome samples processed. Hopefully they will be processed by the time of our next conference call.

Oct. 15: Dr. Testerman reported that she has continued working on her cell line research. Many of the cell lines grow very slowly and she has been attempting to optimize their growth via the addition of various growth factors. She reported that she does have one cell line that is growing robustly and hopes it may be beneficial. She has also made some changes to her protocol for culturing new bacteria from the PMP samples, but does not currently have any new isolates. Dr. Merrell asked about her collaborative work on characterizing the previously isolated strains and products. She said that some compounds have been tested on 20 other cancer cell lines, and that she and her collaborator plan to publish their results soon.

Ms. Nieciecki, Dr. Metcalf’s graduate student, received the prior microbiome data from Drs. Merrell and Johnson. However, she did not currently have any updates on the results she had reported to the group earlier. She asked about any deadlines for our research that were upcoming. Dr. Merrell said that there were no deadlines and our work is ongoing. When we got low reads on our microbiome samples, Dr. Metcalf volunteered to take a second look at the samples and see what she could determine from them.

Ms. King stated that Ms. Sittig has left Mercy. Among other tasks, she is now the only person who is coordinating our joint research. She is attempting to get caught up. Ms. King has been in contact with San Diego about getting them to run our fecal microbiome samples. There are apparently 2 lists for our patients and the data for ~7 out of 19 patients does not agree on each list. San Diego will not run our samples until this issue is sorted out. Some patients may have either lost their paperwork or gotten a new kit with a new barcode; this could be why there is disagreement. Ms. King will sort out what is going on. Next a long discussion took place about the genome and exome sequencing that Mercy has undertaken with a private company. Mercy had sent them specimens from DPAM, PMCA, and signet ring patients. The company has had trouble in extracting tumor DNA from normal tissue DNA in these specimens. Both Dr. Testerman and Dr. Merrell have experience in this area and it was suggested that the company be put in contact with them for their expertise on the extraction problem, since they may be able to suggest methods to overcome the problem.

Dec. 10: Dr. Testerman reported that she is in the process of shutting down her lab and she has only 1 cell line left in culture. Dr. Testerman mentioned that she finally received word from her NORD pre-proposal requesting that she submit a full proposal for her research. Dr. Testerman discussed both her in vitro culturing work and her animal research. She has had some difficulty in growing cells in vitro, but these cells clearly grow in patients. Dr. Testerman has looked at approaches to help stimulate the growth in vitro. Also, Dr. Testerman mentioned that while she would like to study peritoneal tumor growth in animals it has been difficult to detect such growth in her experiments. A good alternative option for studying PMP tumors may be to study sub-cutaneous tumor implants. She also suggested transfecting the tumor cells with CagA to see if that increased growth; Dr. Merrell reminded her that a group at Stanford had published studies using CagA expression in cell lines and that perhaps those constructs could be obtained from Stanford (see https://pubmed.ncbi.nlm.nih.gov/16258069/). Dr. Merrell asked about plans for Dr. Testerman’s collaborators to take over her research and Dr. Testerman indicated that this should be possible, and that she hopes to find a position in South Carolina so that she can continue to collaborate on the PMP research. Dr. Merrell mentioned that he does not have the resources available to keep Dr. Testerman’s cell lines going. Dr. Testerman mentioned that she has not seen a significant difference between DPAM and PMCA cell lines. Ms. King mentioned that Dr. Studeman thought that when DPAM cells were in the appendix they would not proliferate, but once they entered the peritoneal cavity they would not respond to shut off signals but would then continue to grow. The cells in the peritoneal cavity look like normal epithelial cells but they could have genetic mutations.

Ms. Nieciecki shared a Powerpoint presentation that she prepared on her microbiome work on 3 tumor specimens, PMP 423, PMP 433, and PMP 485. Ms. Nieciecki has been studying methods that facilitate the filtering of host DNA so that bacterial DNA in the tumors can be examined. She had found that the ZYMO HostZero Microbial DNA Kit contained contaminants and could not be used. As an alternative, Ms. Nieciecki has examined Tissue & Cell Kits, and PowerSoil Kits, and she presented the results of her work using both kits. After removal of host DNA, the bacterial cells that remained were lysed. Ms. Nieciecki mentioned that there was a large number of unassigned sequences resulting from both approaches that she studied. After removal of the unassigned sequences both methods had comparable reads. Ms. Nieciecki presented results on the alpha and beta diversity of her results combined with the prior results from Ryan for the 3 tumors studied. While alpha diversity is a measure of microbiome diversity applicable to a single sample, beta diversity is a measure of similarity or dissimilarity of two communities. The alpha diversity results indicated that there was no preferred DNA extraction method, while the beta diversity results indicated that specimens clustered more by tumor than by extraction method. Lastly, Ms. Nieciecki presented results for the overlap between the PowerSoil and Tissue & Cell results for the tumors she has studied. A long discussion took place during Ms. Nieciecki’s presentation. Part of the discussion focused on what additional tumor samples should be studied and another part on the preparation of a manuscript on the results that can be submitted to a journal. It was decided that a conference call among Drs. Merrell, Johnson, and Metcalf, and Ms. Nieciecki would be held on Jan. 14 to continue planning the research and discussions on the manuscript.

Ms. King mentioned that a Canadian Journal has approached Mercy to write a review article on PMP; the entire issue will be devoted to the topic of PMP. A discussion took place about what information a paper could contain. The paper would have to be submitted by March 31, 2021.

Dr. McAvoy asked about the status of the fecal microbiome specimens. Registration information for the specimens is still being sorted out. A discussion about possible funding for the microbiome research took place, and the consensus was that the microbiome research is a difficult area to get funded.