Period From 7/2018 Through 12/2018

The group held a conference call on 8/24, 10/05, and 11/16.

Aug. 24: Dr. Testerman reported that her animal studies are going well. She has seen tumors growing in mice and will have slides made from them and sent to Dr. Studeman for pathology analysis. One tumor has been implanted from one mouse to another and it has grown well. A pathology report on this tumor indicates that it is DPAM. This tumor goes to organs and when implanted forms new tumor fast. Dr. Testerman plans to perform sequencing analysis of new bacterial isolates. Dr. Testerman has done some immunohistochemistry (ISH) staining for such things as CA 19.9, MUC2, etc. and this staining has been positive. A discussion took place about whether ISH is done routinely at Mercy on PMP specimens. Dr. Nieroda said that such staining is not routinely done. In June Dr. Testerman re-submitted her R21 NIH proposal and she expects this proposal to be reviewed by November.

Dr. Merrell reported that his postdoc, Faith, is continuing to optimize conditions for the microbiome analysis. One approach that was tried was to concentrate material from the purification step to determine if that would be helpful. Dr. Merrell said that the concentration did not help when the PCR reaction was carried out and in one case it inhibited results. The problem appears to be that there is little specific bacterial DNA in a sea of human DNA in the specimens to be tested. Faith has found a couple of enzymatic treatments online that might be useful for the microbiome work. She plans to try these on some samples. Dr. Merrell said that it is likely that for the final analysis the Qiagen kit will be used since it appears to be the best so far. Overall progress has been slow and additional tweaking needs to be done to generate results. Dr. Testerman asked whether Dr. Merrell has tried using nested primers to improve results, but he replied that he has not.

 Ms. Sittig  reported that 26 patients total have been enrolled in the Abx protocol.  One new patient, PMP25, had  a positive breath test for H. pylori. Samples from this patient were sent to Dr. Merrell and Dr. Testerman. Mercy reported a total of 20 patients enrolled in the microbiome study with 2 added since the last conference call. This includes 8 concurrently on the antibiotics protocol, 9 not on antibiotics, and 3 who were removed/withdrawn from the study

Dr. Merrell reported that he has made good progress on a draft of the opinion article. He still needs updated statistics on the original study patients and he has emailed Mercy to request this data. Ms. Sittig said that she has collected this data and will check it over and email it to Dr. Merrell in the next day or so. Dr. Merrell will finalize the draft and it should be available for the group to review before the next conference call.

One of the follow up items involved Mercy sending pictures from surgery to Dr. Testerman. It was agreed that Dr. Testerman will develop a list of patients for which she would like pictures and email the list to Mercy. A second item involved checking patient charts to see if there was a pattern in their glucose levels. Since it is difficult to do this checking retrospectively it was agreed that these levels would be checked for new patients in the future.

Oct. 5. Dr. Testerman reported that she continues to get tumors growing in mice. She focused on results from tumors from 1 patient who has had 2 surgeries. Tumors from this patient’s first surgery were injected into a mouse and the tumors grew aggressively, especially as they were passaged to additional animals. After only 2 weeks, several small tumors had grown in the mouse. Tumors were attached to the liver and bloody ascites was seen. Dr. Testerman is considering doing some genome sequencing of these tumors. Dr. Merrell may have specimens from the original patient that can be used for the sequencing. Dr. Merrell suggested that it would be useful to compare the sequence results from both the patient and the mouse. Dr. Testerman mentioned that cells from her PMP191F culture appear to slow cell growth and that it could be interesting to inject them into a mouse with a tumor to see what happens to the tumor. Dr. Testerman mentioned that she has sent out cultured bacteria for sequencing, but that 1 strain (a gram negative cocci) did not have enough DNA for sequencing, which she found unusual.  A discussion about genomic testing at Mercy then took place. Ms. King mentioned that genomic testing is very expensive, ~$5k per test. Mercy received a follow up from a genomic company yesterday expressing interest in collaborating. Dr. Merrell noted that there is a large sequencing facility at USUHS and if necessary he can approach them about collaborating with Mercy, potentially at a reasonable cost. Dr. Testerman mentioned that the University of South Carolina also has a sequencing facility.

Dr. Merrell reported that his postdoc, Faith, is continuing to troubleshoot amplification and DNA prep for the microbiome work. So far the results of the troubleshooting show that there is not enough bacterial DNA; both the specimen and PCR controls often give the same results in terms of amplified bands. Dr. Merrell questioned whether it will be possible to move on. One possible issue is that the kit reagents they are using have a low level of DNA contamination. If so, when controls are tested this contaminating DNA is amplified. Dr. Merrell has arranged with a colleague to carry out sequencing to determine if chip contamination is an issue. Next week PMP specimens and controls will be sent to the colleague to be sequenced. Dr. Merrell expects that answers about possible contamination will be gotten in the next 2 to 3 weeks, after which a plan to go forward can be made.      

Ms. King reported that 1 new patient has been added to the antibiotics protocol. This same patient has been enrolled in the fecal microbiome protocol. Two additional patients have been screened for addition to the antibiotics protocol. At present 27 patients total have been enrolled in the antibiotics protocol, including 12 DPAM and 8 PMCA. There is the need for control specimens from 3 additional patients. However, due to Mercy changing over to an electronic records system for patient information harvesting of these control specimens has been delayed.

Prior to the conference call Dr. Merrell emailed the group a copy of the opinion article that he redrafted from the prior patient update article that was written. A discussion about the article took place. One issue discussed at length involved whether antibiotics patients had complied with taking 2 courses of antibiotics. In earlier publications our group had mentioned that 17 out of 21 did complete 2 courses. However, a check of records at Mercy indicates that the number 17 is too high, and something close to 11 is a better estimate. Due to digestive issues, patients in some cases either do not complete the second course or fail to begin it. Most patients did take the first course. Dr. McAvoy suggested that we add a couple of sentences to the opinion article to point out this information. Dr. Testerman suggested that lengthening the time after surgery for the second antibiotics course might help with compliance. Dr. Merrell has received feedback from almost all group members, which he is incorporating into the opinion article. A discussion took place about which journal to submit the article to. It was the consensus that the European Journal of Surgical Oncology would be a good choice. This journal is indexed on PUBMED. Dr. Merrell will check the format that this journal requires for opinion articles. He will circulate a draft to the group in the near future.

Nov. 16. Dr. Testerman reported that she continues to sort through issues with the mouse tumors. She has done ISH staining for T cells which were mostly negative, but occasionally positive. The staining for positive controls has been positive, but not as positive as she would like. When she implanted the tumor from the patient with colon cancer it produced muscinous tumors with ascites. Some of the SCID mice Dr. Testerman have been using developed lymphoma, but they should not have done so. These mice should only have a low level of lymphoma development and develop this disease only when they are old. Dr. Testerman is checking with NIH for new mice strains where this problem may not arise. In her in vitro work, the cells that are produced are definitely epithelial. A discussion took place about either taking pictures of the lymphomas that the mice develop and sending them to Mercy for evaluation, or sending tumor slides to Mercy

Dr. Merrell reported that he had contacted a collaborator to do some sequencing related to his microbiome work. Dr. Blum prepared DNA from samples, controls, and even empty wells that contained no tissue. Three different microbiome kits were used for the DNA preparation. The amplified 16S products were sent to the collaborator for sequencing. Dr. Merrell has gotten the data files back from his collaborator, but before they can be analyzed bar codes need to be removed from the data. If the sequencing shows significant differences between samples and controls plus empty wells, then the microbiome work can move forward. Dr. Merrell’s collaborator noted that some wells had very few reads and hopefully this indicates that the work can progress. Since the last conference call Dr. Metcalf sent out an email indicating that she could have her graduate student help with the microbiome work. 

Ms. Sittig reported that 2 new patients have been added to the abx protocol. At present, 28 patients total have been enrolled. Ms. King reported that 2 new patients were added to the fecal microbiome study. The total number of patients stands at 22 out of the 27 which was the goal. There are 9 abx patients and 10 non-abx patients. Ms. King will try to contact Dr. Metcalf to coordinate the analysis of the fecal microbiome specimens.

Dr. Merrell thanked the group for the fast response to his requests on the opinion article. Dr. Blum uploaded the version of the article that Mercy had edited to conform to the word length required by the journal, the European Journal of Surgical Oncology. The paper will be submitted later today.

 The date for the next conference call was set as Jan. 11, 2019 at noon.

Period From 1/2018 Through 6/2018

The group held conference calls on 2/9,  3/23, 5/11, and 6/29.

Feb. 9. Dr. Merrell’s postdoc, Faith, continues to work on DNA prep for the microbiome study. Faith has ordered different DNA kits and will test them for consistency of results. Dr. Testerman sent Dr. Merrell information on using higher concentrations of primers as a means to improve the DNA amplification results. When Faith tried using the higher primer concentrations, the specimens that were positive with normal amounts of primer gave improved, more robust results. However, specimens that were negative remained negative. The negative specimens may contain too few bacteria or inhibitors may be present in these samples. Dr. Merrell will try the new kits with increased primers as a means to determine if good results can be achieved. Dr. Testerman made a suggestion about using nested primers for DNA amplification. She said she had seen this approach in a paper which she will try to find and send to Dr. Merrell.

Ms. Sittig reported that since the last conference call 2 new antibiotics patients have been enrolled. There is still the need to get 3-4 control specimens. For the fecal microbiome study 2 new patients were enrolled this year, giving a total of 7 antibiotics and 7 non- antibiotics patients in the study.

Ms. King discussed results on comparing survival and disease free survival between antibiotic-treated patients and non-antibiotic-treated patients. She had emailed an expanded powerpoint presentation giving results. The results were limited to lymph negative PMP patients who had completeness of cytoreduction (CC) scores of 0 or 1 and who did not have signet ring disease. This is the group that appears to benefit most from antibiotic treatment. The expanded powerpoint presentation gave plots of overall survival and progression free survival. In both cases statistically significant results were not achieved. While there is a large number of non-antibiotic-treated patients the antibiotic-treated group is small and it contains 2 patients who were treated since the beginning of 2016. Drawing statistical conclusions will require having additional antibiotic-treated patients.

Ms. King discussed a review of appendiceal cancer that is being developed at Mercy. After the call she emailed the following information about this review. “One of our research fellows is working on a thorough appendiceal review paper. There are some interesting findings in the surgical complications results. The analysis showed that at 10 years, the progression free survival for grade III/IV complications was longer (7.4 years) than those with only grade I/II complications (3.5 years). Dr. Sardi and our team have discussed that this significant difference may be due to the fact that grade III/IV complications likely require antibiotic therapy (due to UTI, pneumonia, abscess, etc.) and probably had more antibiotics postoperatively than those with grade I/II complications. We use the Clavien-Dindo Classification of Surgical Complications, but basically grade I/II complications are those that resolve on their own and do not need intervention or if they do require treatment, it is limited to things that are very commonly used like antiemetics, diuretics, blood transfusions, electrolytes, etc. Grade III/IV complications require pharmacologic (except for those drugs outlined in grade I complications), surgical, or endoscopic intervention.”

March 23. Dr. Testerman reported that she found a very large, fluid-filled cyst in one mouse that had been injected with tumor material.  Cells from both the cyst fluid and peritoneal fluid were isolated and they initially grew very fast and had an epithelial appearance.  The cells have since stopped growing and Dr. Testerman is trying to rescue them by co-culturing with bacteria.  Another mouse was found dead; however it had mucinous ascites filling the peritoneal cavity.  Dr. Testerman is trying to recover cells, since it didn’t appear that the mouse had been dead for very long. She has isolated a couple of bacterial species.  One of them won’t grow on plates and is growing very poorly in broth.  Dr. Testerman is testing different conditions to see if growth can be improved.  Dr. Testerman’s  graduate student is preparing to do more cell motility testing.  The student recently did a gene expression microarray of conditionally-immortalized colonocytes with 2 different species of bacteria.  The student found upregulated genes in cell cycle control and p53 pathways.

Dr. Merrell discussed his suggestion about how to follow up with the earlier paper on PMP and antibiotics. He mentioned that there was a question raised about whether we had enough patient results to draw meaningful statistical conclusion. As an alternative to the paper, a letter to the editor was also considered earlier. Dr. Merrell checked with the journal we were targeting and found that they do accept opinion articles. He has started to work on an opinion article about using antibiotics in conjunction with cytoreductive surgery and HIPEC to treat PMP. Ms. King had previously discussed an appendiceal cancer survey paper that is being developed at Mercy. Interestingly, when the Mercy records were examined it appeared that patients who had more complications fared better long term than those with fewer complications. One hypothesis was that the patients with more complications may have received more antibiotics.  A discussion about how this result could be fitted into the opinion article took place. Both the opinion article and the survey will be written up over the next several months. It might be possible to reference the Mercy survey paper in the opinion article.

May 11.  Dr. Testerman reported that in a recent attempt to culture PMP tumor cells that she tried adding insulin. When she did, she saw very strong cell growth. It is not clear why insulin would produce this result. Dr. Testerman is now experimenting with using insulin to help promote cell growth in several PMP tumor specimens. Dr. Testerman described the cells that she has grown as fibroblasts that have epithelial characteristics and that they seem mucinous. She plans to stain the cells for MUC2 and to run a number of additional tests on them. Dr. Testerman also mentioned that she has cultured another Gram negative bacterium that she believes to be E coli, which is the second to be cultured. Dr. Testerman has also cultured an interesting coccoid bacterium, but this bacterium is not easily grown. One of the mice that had tumor plus bacteria implanted in it developed a gigantic ovarian cyst. A second mouse similarly implanted had a gigantic spleen. Spleen specimens will be sent to a pathologist for evaluation. In the future Dr. Testerman would like to run experiments using only tumor and no added bacteria. Dr. McAvoy raised the question about whether insulin levels could be checked in PMP patients prior to surgery. Dr. Nieroda said that insulin would not be measured but that glucose levels would be available. If glucose levels are low then this could indicate that insulin levels were high. The database at Mercy will be checked to determine what the glucose levels of PMP patients have been.

Dr. Merrell mentioned that his postdoc, Faith, had checked the Qiagen DNA kit for use in the microbiome study. Faith used the same specimens that were tried in earlier kit studies. The results with the Qiagen kit were not much better than those produced by the earlier kits. One problem may be that the Qiagen kit only allows for a very small specimen sample. Between now and the next conference call Faith will be testing other kits that have been obtained. Dr. Testerman had earlier suggested increasing the quantity of primers and when Faith tested this suggestion it worked well. Dr. Merrell also reminded the Mercy group that if they sent a proposal concerning patient genomic sequencing, he would discuss this with the USUHS sequencing facility to determine if this would be feasible on site.

Ms. King reported that since the last conference call, no new abx patients have had surgery. Since March 5, several non-abx patients have been operated on. Most of the patients that are being seen at Mercy are patients with recurrent disease. As of the present conference call, 10 out of 12 non-abx fecal microbiome specimens have been harvested. When 2 more are obtained the entire batch can be sent in for processing. Since the last call, no completely benign potential control patients have been treated. Mercy is currently looking at its database to determine parameters that are linked to some patients doing very well after surgery while other do poorly. An abstract has been submitted on this study and it will be circulated to the group.

Dr. Testerman reported that she will be submitting a paper on naming bacteria PMP191F that she cultured to the International Journal of Systematic and Evolutionary Microbiology.

June 29: Dr. Testerman reported that she has gotten a few more tumors to grow. One of the tumors was huge, and it looked like tumors that she has seen from patients. Another tumor was seen on a mouse spleen. This tumor was harvested and inserted into another mouse. Dr. Testerman has cultured additional PMP cell lines and carried out antibody analysis on some of the lines.  Dr. Testerman will order additional mice and she is ready to carry out systematic animal studies. In her initial work she will implant tumor material from a single patient and she will focus on PMCA first. Dr. Testerman raised the question about whether notes were usually written down during PMP surgery. Dr. Testerman noted the large variability in tumor specimens. Dr. Nieroda mentioned that pictures were taken for some patients and these might be better than notes describing the tumors. It was agreed that Mercy would send pictures to Dr. Testerman as well as pathology reports. In turn Dr. Testerman can take pictures of mouse tumors and send them to Mercy. Dr. Testerman will resubmit her R21 proposal today.

After the conference call Dr. Merrell’s postdoc, Dr. Faith Blum provided the following summary of her microbiome work. “We used the same set of samples for all extractions (see exception below): 6 PMP samples (3 mucin and 3 tumor), S. aureus (10⁷, 10⁵, and 10³ CFU), H. pylori (10⁷, 10⁵, and 10³ CFU), and an empty tube. Bacterial DNA extraction was assayed by PCR using 16S primers (V4 region). The Qiagen DNeasy Blood & Tissue kit extracted bacterial DNA from S. aureus and H. pylori (10⁷ and 10⁵ CFU only), but not from the PMP samples. The Qiagen QIAamp Fast DNA Tissue kit (using VortexGenie for homogenization) extracted bacterial DNA from S. aureus and H. pylori (10⁷ and 10⁵ CFU only). The empty tube and all PMP samples generated similar bands, suggesting that there is some contaminating DNA in the kit, and that we didn’t extract DNA above this background from the PMP samples. The MP Biomedicals FastDNA kit did not extract DNA from any sample (and/or there was interference with downstream PCR amplification). The Qiagen QIAamp Fast DNA Tissue kit (using bead beater for homogenization) was tested with only one PMP sample, H. pylori (10⁷ CFU), and an empty tube. PCR generated a stronger band from the PMP sample than the empty tube, suggesting that this protocol will work for a larger set of samples. Amplification from the H. pylori sample was strong. Our next step is to repeat the QIAamp Fast DNA extraction (with bead beater) with more PMP samples and several empty tubes.”

Ms. Sittig  reported that 25 patients total have been enrolled in the antibiotics protocol.  The last patient to be enrolled had a positive breath test for H. pylori. Samples from this patient were sent to Dr. Merrell and Dr. Testerman this week and pathology is pending. Eighteen patients have been enrolled in the fecal microbiome study. In the next few weeks, Mercy will have the full samples (3 from each patient) on half of the cohort (12 patients).  Mercy will coordinate with Dr. Metcalf on getting those samples sent to UCSD and processed.

One of the follow up items involved sending an abstract that Mercy has submitted to an upcoming conference. This abstract was emailed to the group just after the May 11 conference call. The second item involved checking PMP patients’ glucose levels to determine if they were low, indicating that patient insulin levels were high. This item still needs to be done.

Dr. Metcalf mentioned an interesting paper that she had seen entitled: “Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response”. After the conference call Dr. Metcalf emailed a copy of the paper to the group.

The date for the next conference call was set as Aug. 24.