Latest Research 2019

Period From 7/2019 Through 12/2019

The group held a conference call on 8/2, 9/27 and Dec. 6. The next conference call will be held on Jan. 24, 2020.

Aug. 2: Dr. Testerman reported that another animal in her study was found dead in the cage. She believes that it had not been dead very long and when it was examined, it had visible ascites, what appeared to be mucinous tumor, a tumor in the liver and the liver was discolored. They recovered the ascites and trypan blue staining suggested that some of the cells were alive, so these were injected into another animal. The interesting thing about this animal was that it was originally injected with a PMP cell line that was derived from a patient (PMP457, DPAM patient). Cells from the patient tumor were cultured in vitro for a period of time to create the cell line and then these were what were injected into the animal approximately 1 year ago.

A very thin animal was also euthanized, but there was no obvious tumor. Additionally, a new bacterial isolate from a recent PMP tumor sample was cultured. This new isolate is filamentous and looks a lot like PMP215, though she believes it is different species. 16S sequencing will be performed next week for identification.

She submitted an abstract to the NORD conference. Mercy mentioned that they also submitted several to the same conference. Among these, Ms. King mentioned that one of their abstracts is focused on their recent analysis that indicates that the number of epithelial cells found in the mucus may negatively correlate with tumor progression; the more cells, the faster the tumor will progress.

Dr. Testerman said that she met someone from GE that has the ability to stain tissue slices with up to 60 antibodies; this is a new machine that they want to test. They have agreed to look at a few of the PMP samples and she is picking particular antibodies that will target pathways of interest (differentiation, cell signaling, Muc2, etc.)

Finally, she said that she is planning on two upcoming papers; a mouse model paper and an in vitro tissue culture model paper. Additionally, the original 191F paper is almost ready to be resubmitted.

Dr. Merrell mentioned that he had set up a collaboration for the sequencing of these samples. The plan is to sequence 96 samples (including sequencing controls) to start. If these work well, the number of samples will be expanded in subsequent rounds of sequencing. It was suggested by Dr. McAvoy that the ovarian cancer samples be included in this round. Mercy suggested that we not include the tumors from the interesting familial case. The hope is to start prepping samples in about 2 weeks.

 Dr. Testerman’s PMP strains were cultured and sent to the FDA for sequencing.

Ms. King reported that one new patient was added to the microbiome study and one new patient was added to the antibiotic study since that last conference call. Current numbers for microbiome are 10 patients on antibiotics, 10 not on antibiotics and 4 withdrew; the goal is 24 patients total.  Current totals for the antibiotic study are 20 enrolled and 9 withdrew. One new control specimen was sent to Dr. Merrell.

 Ms. King mentioned that they also submitted the familial case to the NORD meeting as an abstract. They are developing an extensive data collection on this mother and daughter; germ line sequencing, appendiceal tumor sequencing, fecal microbiome, the tumor microbiome, etc.

The one follow up item from the last conference call involved Dr. Testerman sending a spreadsheet to Mercy on the samples that she has been processing and injecting into animals. She did this and Mercy is working on completing their portion of the spreadsheet.

Sept. 27: Dr. Testerman reported that she has not cultured any new isolates. Also, her funding for the mice has been exhausted, so any new animal research will await additional funding. As a result, Dr. Testerman has been euthanizing mice and, in some cases, she has seen abnormalities that appear to be tumors; she is waiting on results from pathology. Dr. Testerman has been freezing material for future animal research, which she feels will be easy to restart when more money for animals is available. In the case of some mice, Dr. Testerman has been able to re-isolate bacteria injected into mice. She has re-isolated PMP215, which is a waxy, branched bacterium. Interestingly, unlike other bacteria this bacterium decreases host cell respiration, and Dr. Testerman has been able to quantitate the decrease. PMP215 is similar to other bacteria that have been found in the PMP microbiome. Dr. Testerman has also re-isolated a Streptococcus species as well. A discussion about the bacteria sent to the FDA for sequencing took place. PMP215 was one of the species that the FDA will sequence. The specimens were sent to the FDA in late June and hopefully results should be available shortly. Ms. Sittig raised the point about making sure the material Dr. Testerman has implanted is actually positive for cancer. Species have been harvested at the time of surgery and sent to Dr. Testerman for implantation. Pathology results are obtained sometime later. Ms. Sittig said she would provide pathology information for the specimens sent to Dr. Testerman.

Dr. Merrell discussed his plan to sequence 96 microbiome samples. His Postdoc, Dr. Blum has selected the 96 samples as follows:

35 DPAM subjects (24 female, 11 male)

35 PMCA (mucinous adenocarcinoma) subjects (21 female, 14 male)

6 other cancer subjects (4 ovarian, 1 mucinous ovarian, 1 uterine carcinosarcoma)

6 PMP CONTROL samples (peritoneal tissue)

Controls: 8 no-input controls; 6 positive controls (dilution series of mock community)

 

The no-input controls are used to detect reagent contamination. The specimens to be analyzed were selected based on suggestions that were received from the group. In choosing samples, priority was given to specimens that had a majority of tumor since these produce more DNA than mucinous specimens. The size of the microbiome specimens is 250 mg, and if there is not enough tumor then the specimen has mucin added to it. The DNA preparation for PCR should be completed today. Carrying out the PCR reactions should take 1 to 2 weeks. The samples produced by PCR will be sent to a collaborator at BDRD, which is a military facility in Frederick Maryland. Analyzing the microbiome of the 96 specimens should take about 1 month. Once the results are available Dr. Merrell said he would email them to the group. Dr. Merrell briefly discussed our recent antibiotics PMP paper and noted its publication. Dr. McAvoy checked on Researchgate.net and found that the paper had 27 readers.

Ms. Sittig reported that there were no new fecal microbiome patients enrolled since the last call. Ms. Sittig noted that we need 5 additional patients to make the 24 that are sought. We currently have 10 abx and 9 non-abx patients in the study. Ms. Sittig mentioned that 1 new surgical abx patient was enrolled giving a total of 21 patients in the surgical study. The surgical abx study is close to 3 years old and it will need to be renewed with Mercy’s IRB. Ms. Sittig did not feel that there would be a problem with the renewal. Ms. Sittig said she would email data on the 21 patients, such as diagnosis, status, etc. to the group before the next conference call.

Dec. 5: Prior to the conference call Dr. Testerman emailed copies of pathology slides from her mouse studies. Dr. Testerman had discussed the slides yesterday with a pathologist at her institution. A detailed discussion of what Dr. Testerman’s slides showed took place. Dr. Testerman mentioned that even when a slide did not contain tumor in many cases it was not totally normal. One of her slides contained signet ring tumor from a specimen from a patient with signet ring disease. In one of the mice the liver showed a great deal of tumor and bleeding. This result could explain the occurrence of bloody ascites in some mice. Other mice exhibited large pancreases, which led to a question about whether PMP patients might be hyper-glycemic as a result of the disease. Dr. Gushchin stated that hyperglycemia is not seen clinically with PMP patients. Another mouse exhibited tumor on the outside of the spleen. An enlarged spleen indicates irritation. Dr. Testerman mentioned that she has had success using peritoneal lavage to get solutions from which cells can be cultured. Even when mice show no signs of tumor per se but they simply look sickly and are losing weight, peritoneal lavage has allowed Dr. Testerman to culture cells. Dr. Testerman mentioned that her slides have contained a mixture of tumor types, with both low and highly malignant tumors showing up in the same mouse. Dr. Testerman’s results correlate with disease type, either DPAM or PMCA. Dr. Studeman will compare Dr. Testerman’s slides with slides from the patients from whom Dr. Testerman’s specimens were obtained. Dr. Testerman has tried using antibiotics on mice to see if they help with disease progression but whether the antibiotics have helped remains to be evaluated. Dr. McAvoy suggested that Dr. Testerman probably has enough material to write a couple of papers, which could be co-authored with people from Mercy Medical. Dr. Gushchin asked what hypothesis would be tested in each paper. Dr. Testerman said that one paper could focus on her animal model and compare her mouse results with patient tumor pathology. A second paper could focus on new methods of culturing cells in vitro. Dr. Testerman has been successful in culturing cells from both DPAM and PMCA patients and her culturing results could be correlated to a patient’s clinical outcome. Ms. King mentioned that for DPAM some patients can do poorly and it would be interesting if culturing results could shed some light on why. Lastly a discussion about organoids took place.

Dr. Merrell updated the microbiome research. The 96 specimens that his postdoc Faith chose were successfully amplified. These specimens were sent to the sequencing facility and Dr. Merrell expects to have results back before the holidays. At that time he will be able to compare the PMP microbiomes with those of controls. Dr. Merrell mentioned that Faith will be leaving on Jan. 3 to take up an IRTA Fellowship at NIH. He will need to identify someone to continue the work that Faith has been doing. Dr. Merrell also updated the status of the PMP isolates that Dr. Testerman had sent him to be sequenced at the FDA. Roughly 1/3 of the isolates have been sent on for sequencing and results should be available reasonably soon from the FDA. Included in the specimens sent on were bacteria from PMP patients 215 and 238. Dr. Testerman mentioned that the PMP215 results would be particularly interesting because of the type of bacteria present.

Ms. King reported on the various studies underway at Mercy. After the call she emailed the information below about these studies. For the fecal microbiome project 24 people were enrolled but 5 were off study. Five more patients are needed to get to the goal of 24. Of the 19 patients enrolled, 10 have taken antibiotics and 9 are non-antibiotic patients. For the PMP antibiotics study one new patient was enrolled but that patient withdrew because of going to another institution. Ms. King also provide an overview of all the PMP patients at Mercy. Since 9/27/2019 14 new patients were enrolled. Specimens have been collected on 12 of them. Overall 261 specimens have been collected from 335 enrolled patients. Control specimens from 25 patients have been collected. Recently the sample size for collecting PMP specimens was increased from 350 to 1000 through Mercy’s IRB.

Prior to the conference call Ms. King mailed out two very detailed documents. Both were discussed during the conference call. The Antibiotics Study Update was a 17 page Research Summary giving results from 11/4/2014 to 10/18/2019 for our PMP antibiotics study. Patients in the study take pre and post-operative antibiotics in conjunction with cytoreductive surgery and HIPEC. The following information, taken from Ms. King’s report, was discussed. Of the 217 patients screened for inclusion in the antibiotics study, only 30 were enrolled and currently 19 remain in the study, but results for 18 were included since the last patient has not yet started preop antibiotics. Numerous reasons exist for patients failing the screening including such things as allergies to antibiotics, advanced disease, patients declining enrollment, not enough time before surgery, negative malignancy, among others. Patients who went off the abx protocol did so for reasons such as surgery was aborted, patient went to another institution, aborted HIPEC, or palliative HIPEC. For the patients remaining in the abx study 89% completed the preop antibiotics regimen, but only 50% the postop regimen. Patient refusal is the primary reason for the postop percent being low. The cytoreductive surgery often leads to patients struggling with diarrhea, nausea, and vomiting, and patients do not want to add antibiotics. Of the 18 antibiotics patients 12 were DPAM, 3 PMCA, 2 PMCA signet cell, and 1 PMCA goblet cell. Of the 18 patients 13 had a CC (completeness of cytoreduction) score of 0 and 5 a score of 1. At present all 18 patients are surviving and only 1 has experienced disease recurrence. Significant additional data is given in Ms. King’s report. The second document discussed was a tumor tracking pathology specimen excel spreadsheet. The spreadsheet gives details on the pathology reports from PMP patients at Mercy and it also details the specimens that were sent to Dr. Testerman for her culturing and animal studies. This information will be very useful in comparing Dr. Testerman’s results with what was found clinically. The group complimented Ms. King on the thoroughness of her work on the two reports.