Period From 1/2017 Through 6/2017

The group held conference calls on 1/20, 3/10, 4/28 and  6/9.

Jan. 20. Prior to the January conference call Dr. Metcalf sent out an email giving the fecal microbiome results for a mother/daughter pair of PMP patients. The microbiomes of both the mother and daughter fall on the fringes of the results for the US population. The daughter has a very low diversity of microbes (far fewer types than most people), which is common for people with a disease of some type. The results for the mother/daughter pair are the only results so far for our PMP study, and it will take some time to accumulate additional results. It should be possible to do the fecal analysis in two batches. This approach will help eliminate small differences due to run to run effects. It will be interesting to compare the fecal results for patients before and after cytoreductive surgery. Dr. Metcalf said that surgery tends to lead to a crash in gut bacterial diversity. In addition to the fecal results for patients we will also have results from the tumors from the same patients to be analyzed in Dr. Merrell’s microbiome research. A comparison of these results could prove to be very interesting.

Ms. Sittig reported that there are now 18 patients that have been enrolled in the antibiotics protocol. Of the 18, 9 have completed two antibiotics courses. Five patients did not complete an antibiotics course due to various reasons. No new control tissue has been harvested but Dr. Teresa P. Diaz-Montes, from Mercy, has agreed to participate and contribute specimens.

March 10. During the March conference call we found out that our NIH proposal would not be funded. Dr. Merrell emailed the NIH review summary to the group prior to the conference call. He mentioned that 2 of the 3 referees were incredibly critical and they did not think we had enough data about the connection of micro-organisms to PMP and that everything that was proposed was correlative in nature. One referee questioned whether bacteria could be initiative and Dr. Testerman commented that the referee was wrong in this assessment. Dr. McAvoy mentioned that there was only 1 carry over member in the NIH study group and that he tried to find a reference to a paper by Sew given by referee 2 but was unable to get it after searching the web for 20-30 minutes. It was decided to schedule a follow up conference call among Drs. Merrell, Testerman, Metcalf, and McAvoy to discuss a path forward on generating funding.

Dr. Testerman reported that recently she was successful in culturing a new bacterium. It was a waxy bacterium that she thinks may be an Actinobacterium, which is represented in our microbiome results. Dr. Testerman plans to sequence the 16S gene of this bacterium and her postdoc is currently isolating DNA from it. Concerning the animal studies Dr. Testerman noted that most of the mice have been euthanized. While some had tumors she felt that the immune system of the mice had helped control tumor growth to some extent. Dr. Testerman feels that to grow tumors consistently she will need to employ mice that are even more immuno-compromised. Dr. Testerman also reported that her in vitro work is going well. She has been working with conditionally immortalized cells. When their temperature is raised to 37 degrees they die out. However, when bacteria are added cells continue to grow, but growth takes some time. The bacteria that work are Streptococcus and Amycolatopsis.  She feels that she is getting some type of transformation. Dr. Testerman has seen similar results with PMP cells where is takes some time for them to grow. Dr. Testerman is very encouraged by her in vitro results. Dr. Testerman also reported that she is starting work on fractionation of the bacterial lysates and checking whether her cells are tumorigenic.

Dr. Merrell’s postdoc, Faith, has just about completed work on sample selection for microbiome sequencing. She has developed a graphic table that breaks down the specimens by variables such as sex, age, etc. The table will be finished shortly and sent to the group for comments. A discussion about which variables to consider in the break down took place. Dr. Merrell noted that including too many variables can make things difficult.

Dr. McAvoy discussed the new research website that he has put up on the University of Maryland site. The ACPMP Foundation (Appendiceal Cancer and PMP Pals) has been contacted and they have put a link to the site on their web site. These links will help publicize our research with these 2 support groups.

April 28. Prior to the 4/28 conference call Dr.s Merrell, Testerman, Metcalf, and McAvoy held a conference call on March 24 to discuss a path forward to fund our PMP research. Subsequently, on April 11 Dr.s Merrell and Testerman talked with Phil Daschner of NIH about funding. The result of the call was that the best approach might be to split up our RO1 proposal into one or more smaller R21 proposals. R21 grants provide funding for 2 years. If an R21 is submitted to NCI it has to address at least 1 of the 12 provocative questions that NCI posed. Question 10 (How do microbiota affect the response to cancer therapies?) may be the only one that is appropriate for our research. As an alternative an R21 could be submitted to NAIAD and in this case the provocative questions would not have to be addressed. NAIAD has funded cancer research in the past. It was decided that Dr. Testerman would submit an R21 proposal based on aim 2 of our RO1 proposal. Aim 2 involved Dr. Testerman’s  in vitro research. The R21 would focus on 3 of the isolates that she has cultured from PMP tumor specimens. Dr. Testerman plans to submit the proposal for the June application date. Dr. Merrell tentatively plans to submit an R21 on aim 1 of our RO1, which involves his microbiome research. However, due to other proposal writing he is not able to submit his R21 by June. He will submit later in 2017. The group will also consider submitting proposals to the new NORD rfp that is funded by the ACPMP Foundation. This year the Foundation expanded its funding to 3 grants. The deadline for submission is June 23.

June 9. During the 6/9 conference call the group again discussed the path forward to fund our PMP research. Dr. Testerman is writing a proposal to respond to one of the 12 provocative questions posed by NCI (Question 10: How do microbiota affect the response to cancer therapies?) under RFA 17-017.  Dr. Testerman will submit an R21 proposal based on aim 2 of our RO1 proposal. Aim 2 involved Dr. Testerman’s in vitro research. The R21 would focus on isolates that she has cultured from PMP tumor specimens. Dr. Testerman will inject highly tumorigenic cells into mice and add bacteria. Then, she will treat some of the mice with chemotherapy. Dr. Testerman also plans to implant some non-tumorigenic cells and see if her bacteria cause them to become tumorigenic. Her current proposal draft is too long and it needs to be shortened. Only 6 pages are allowed for an R21 proposal. Dr. Testerman plans to submit the proposal for the June 28 application date. Later in 2017, Dr. Merrell plans to submit an R21 to NCI on aim 1 of our RO1 proposal, which involves his microbiome research. He also will submit a letter of intent to the new NORD rfp (request for proposal) that is funded by the ACPMP Foundation. He will propose studying the microbiota of current tumor samples and compare antibiotics treated patients to non-antibiotics treated patients. He will propose studying both DPAM and PMCA. Dr. Testerman is considering a resubmission of her earlier, successful letter of intent to NORD. This year the ACPMP Foundation expanded the funding to 3 grants. The deadline for NORD submission is June 23. Dr. Metcalf is looking into funding from the Colorado Clinical and Transactional Science Institute. She would investigate whether bacteria go along with the metastasizing cells when cancer metastasizes. The research would have to be carried out in Colorado.

Ms. Sittig reported that there were two new abx patients enrolled since the last conference call. Including today there have been two surgeries from which control tissue was harvested since the last conference call. Of the 20 patients that were registered for the abx protocol in 2017, only 13 have undergone surgery. Ms. Sittig also introduced her new colleague, Mary Caitlin King who previously worked with Dr. Diaz-Montes and is familiar with HIPEC.

Period From 7/2017 Through 12/2017

The group held conference calls on 7/21 9/8, 10/27 and 12/15.

July 21. As discussed in the 7/21 call the following actions to secure funding for the PMP research were taken. Dr. Testerman submitted her R21 proposal to NIH. She also submitted a letter of intent to NORD for funding. Dr. Merrell as well submitted a letter of intent to NORD. Dr. Metcalf was not able to make the conference call, but she emailed the group that she is planning to submit a proposal to a pilot program this fall.  The proposal would be through the CU Denver Medical campus, and it will study microbiomes of metastasized colon cancer. Although not PMP, Dr. Metcalf thought that this study would help us further establish microbiomes in tumors of the peritoneal cavity.

Dr. Testerman reported that she recently had success in culturing another new bacterium from PMP tumor specimens. This bacterium has not yet been characterized but it grows better in a diluted solution. Dr. Testerman also euthanized 2 mice. One did not have a tumor. The second showed that ascites had developed. She also saw a bloody vascularized tumor, plus a number of smaller nodules. Slides from this animal have been sent out and they will be evaluated by a pathologist. Dr. Testerman recently received additional tumor samples from Mercy that can be injected into her mice.

Sept. 9. During the 9/8 conference call it was noted that both Dr.s Merrell and Testerman have been asked to submit a full proposal to NORD for funding through the support from the ACPMP Foundation. Dr. Merrell proposed studying the microbiome of PMP tumors and Dr. Testerman will study animal models of PMP. The proposals are due by October 4.

Ms. Sittig reported that 21 patients have now been enrolled in the antibiotics protocol at Mercy Medical Center. This is the same number as were enrolled in our original antibiotics study in 2007, and enrollments continue to take place. In addition 13 patients have been enrolled in the fecal microbiome study and they are split roughly equally between antibiotics patients and controls. The target is to get 24 patients total.

Oct. 27. During the October 27 conference call Dr. Testerman mentioned that her experiments were going better. She has seen that tumors developed in 3 mice, and one appeared to have ascites. She also has several other mice that she is looking at to determine if they also are developing tumors. Dr. Testerman was also able to re-culture tumor cells and she believes these could be implanted in mice. Right now Dr. Testerman has only 1 mouse left and she hopes that funding either from her R21 proposal or the proposal to the ACPMP Foundation will come through so she can obtain additional mice. She has been working with 2 different mouse lines both of which are hairless. Dr. Testerman does not know yet whether one of the two lines is better than the other, but both are better than earlier mice that she studied. In the future she would like to use in vivo imagining to study tumor growth prior to a mouse being euthanized. This would allow her to follow tumor development over time in the living animals. Dr. Testerman also reported that she has cultured a few more bacterial strains from PMP specimens, but as yet no sequencing identification of these bacteria has been done. A new freshman undergraduate student has become a lab member and this student seems to be eager to get involved in research. Dr. Testerman has also implanted tumor from a May 2017 PMP patient who had a second surgery and HIPEC. Dr. Testerman mentioned that her postdoc tried to do PCR on the one ovarian cancer specimen that she has, but the PCR result was negative and no DNA was found. She used a Wizard DNA kit from Promega Corporation. A discussion about DNA preparation for PCR analysis followed and it led into Dr. Merrell’s report.

Dr. Merrell reported that his postdoc, Faith, had started optimization of the DNA preparation for the approximately 100 PMP specimens for which he wants to study their bacterial microbiome. For her microbiome work, his postdoc Faith is using a Mo Bio Soil DNA extraction kit, which can handle 96 specimens at a time. She set up a trial run using 6 tumor/mucin specimens, and varied several experimental parameters, resulting in a total of 23 extractions. When she carried out PCR (DNA amplification), only 5 of the 23 extractions produced 16S DNA PCR products. Dr. Merrell commented that this result was not good. He noted that the cases with higher tumor mass were more likely to produce results. A discussion took place about what might be inhibiting the DNA amplification. Dr. Metcalf asked about what DNA kit was used for our earlier, published microbiome study. Dr. Merrell said it was a Qiagen kit and that only 1 specimen at a time was studied compared to the 96 that he would like to do all at once. A discussion took place about what might be the best approach to carry out the DNA preparation. Dr. Metcalf mentioned that a colleague had successfully used a FastDNA kit (she shared a link for the kit with Dr. Merrell), and perhaps this kit might work for our PMP studies. This kit costs about $300 for 100 DNA preparations.  Dr. Metcalf mentioned that a paper by Geller et al. (Potential role of intra-tumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine) had just been published. This paper discusses DNA extraction methods, and Dr. Metcalf emailed it to the group.

Dr. Testerman mentioned that her R21 proposal to NIH would be reviewed during the first week in November. Both Dr. Testerman and Dr. Merrell’s ACPMP Foundation proposals will be reviewed within the next several weeks.

 Dr. McAvoy mentioned that 3 new ovarian tumor specimens had been sent to USUHS. He also talked about a survey paper that he found on PUBMed that discussed the role of infectious agents in ovarian cancer. He said he would email the paper to the group. One point that Dr. McAvoy made is that if we can show that the ovarian tumors contain bacteria, then our ability to generate funding from agencies like NIH should be greatly enhanced since we would no longer be dealing exclusively with a rare disease. Dr. Metcalf asked whether it might be good for the group to submit a prospective paper on the microbiome of cancer tumors/understanding the microbial ecology of tumors. The group felt that submitting such a paper was a very good idea that was timely. Dr. Merrell suggested that his postdoc, Faith, might be interested in coordinating the writing of the paper and he would ask her about it.

Dec. 15. During the 12/15 conference call Dr. Testerman mentioned that she did not have much new to report on her culturing experiments. However, her animal experiments are going much better. One mouse has developed ascites. Dr. Testerman is also doing in vivo imaging using an infrared probe. She injects the mice, waits 24 hours for a reagent to be cleaved and then images tumors in vivo. In addition to the mouse with ascites, 4 other mice are ‘lighting up,’ which indicates tumor growth. The imaging can also be done ex vivo. Dr. Testerman plans to order additional mice and she would like to harvest tumors from mice and plant them into new mice to keep the tumor line growing. Dr. Testerman mentioned that she is very encouraged by the mouse results that she is achieving. She mentioned that she is considering doing micro RNA analysis on the mouse tumors. Dr. Merrell raised the question as to whether special RNN preparation kits were needed for such analyses. Dr. Testerman said that the micro RNA methodology had improved and she is looking at kits from Qiagen for this analysis. These kits would cost about $350 for 50 preps. She also requested that when they are ready to do these studies that Dr. Merrell send her specimens on dry ice for cases where he receives significant tumor from Mercy Medical. Dr. Merrell suggested that he and Dr. Testerman revisit this issue in 2018.

For the microbiome research Dr. Merrell reported that he did not have much new to convey about the microbiome work. He has collected information on several DNA prep kits and he is in the process of evaluating them.

At the beginning of the conference call both Dr. Testerman and Dr. Merrell reported that their NORD PMP proposals had both been funded. Congratulations! Dr. Merrell’s will involve the PMP tumor microbiome and Dr. Testerman’s PMP animal experiments. Dr. Testerman also reported that her NIH R21 proposal had been scored but that she thought it would not get funded in this round. Getting scored is an important first step in getting NIH funding. Once Dr. Testerman sees the referees’ comments she will be able to respond to them in a new submission next June. Dr. Metcalf also reported that about ½ of the fecal microbiome specimens had been collected at Mercy. This process takes time since some specimens are taken several months after surgery.

In between the 10/27 and 12/15 conference calls a discussion about whether we should publish a short paper or a letter to the editor on the survival of our antibiotic treated PMP patients took place. Dr. Sardi expressed concerns about the manuscript due to the small size of the patient groups studied. Although initial results were encouraging, in order to conclude that pre and post-operative antibiotics are effective in treating PMP results on a larger number of patients are needed. Thus, we do not plan to publish the short paper. We may publish a letter to the editor and whether we do so will be decided in the near future. As a result of this discussion all previous information on the short paper has been removed from this site.