A bizarre case of tapeworm-derived cancer causing cancer-like tumors in a Colombian man – Behind the scene story of the lab investigation

A bizarre case of tapeworm-derived cancer causing cancer-like tumors in a 41-year-old Colombian man has got lot of media attention recently and emphasized again the role of laboratory analysis in complex case investigations. “The key for the diagnosis of this unusual case was combined use of various laboratory techniques – conventional pathology and advanced molecular biology, and synergy among clinicians, pathologists and lab scientists to solve this mystery,” said Dr. Julu Bhatnagar, Ph.D., one of the primary authors of the report published in the November 5 issue of the New England Journal of Medicine and Team Lead of the Molecular Pathology Section in CDC’s Infectious Diseases Pathology Branch (IDPB). http://www.nejm.org/doi/full/10.1056/NEJMoa1505892.

In 2013, doctors in Colombia asked CDC to help diagnose biopsies with strange pattern from lung tumors and lymph nodes of a HIV positive man who was non-adherent to therapy. The man presented with fatigue, fever, cough, and weight loss of several months' duration, and lung, liver and adrenal nodules and enlarged lymph nodes. “Initial histopathological analysis was very puzzling, as the growth pattern was cancer like, but the cells were about 10 times smaller than a normal human cancer cells and they were fusing together, which is rare for human cells. The question was if we were dealing with an unfamiliar, possibly unicellular, eukaryotic organism or rare type of cancer? So, it was decided to proceed for the molecular analysis of the tissue specimens.” Dr. Bhatnagar said.

“Every year, IDPB receives thousands of tissue specimens to provide assistance in investigations of infectious disease of unknown etiologies, unexplained illnesses/deaths and outbreaks; consequently, the Molecular Pathology Lab has uniquely developed more than 150 polymerase-chain-reaction (PCR)-based assays and other cutting edge techniques such as pyrosequencing, in-situ hybridization (ISH) and laser microdissection for the identification of wide-array of pathogens, including bacteria, viruses, parasites and fungi from tissue specimens. Since in this case, initial suspicion was on plasmodial slime mold because of syncytia formation, I started with some broad-range PCR tests targeting slime mold (Myxogastria) and other eukaryotes using DNA extracted from the tissue specimens. By gel electrophoresis of initial PCRs, I saw some faint bands of DNA (PCR products), I decided to make PCRs less stringent and repeated. This time, I got the bands of DNA that I was able to sequence. The result of sequence analysis was completely surprising, as the DNA sequences matched with Hymenolepis nana, a dwarf tapeworm. I could not believe my own results. I tried another PCR, this time specifically targeting cestodes. The resulting DNA sequencing showed 99% identities with Hymenolepis nana again. I searched the literature and interestingly found a case report from Dr. Olson’s group (Natural History Museum, London) related to H. nana infection that showed an image of similar tiny cells in a HIV-positive man.  Feeling more confident now, I sent the results to Dr. Atis Muehlenbachs, a pathologist handling the case, other pathologists of the group and Dr. Sherif Zaki, Chief of the IDPB. They were as surprised as I was. One of them actually predicted in advance and said, “This is really exotic -- case of the decade!” It was concluded after further review that the tiny cells in the lymph nodes of our patient was certainly very similar to the ones showed in Dr. Olson’s case report. Although there were some unanswered questions, Dr. Muehlenbachs quickly contacted doctors in Colombia and informed about the diagnosis. However, unfortunately, patient was in palliative care at that time and died 72 hours after the diagnosis. No specific treatment was attempted.”

“Even though the patient died, our investigation continued, as there was a question if we could actually localize H. nana DNA in those tiny cells to confirm that the cells originated from a tapeworm,” said Dr. Bhatnagar. “Dr. Zaki suggested attempting in-situ hybridization. I designed and prepared DNA probes using H. nana DNA that we amplified from the patient’s samples. Simultaneously, probes targeting human Alu DNA sequences were prepared. In-situ hybridization was attempted and this time showed what we were expecting - H. nana DNA probe signals were detected in the proliferative tiny cell clusters, while human Alu probe signals detected in the surrounding human tissue. So, this issue was resolved.”

“The next goal was to confirm the species and to obtain more information about the phylogeny,” Dr. Bhatnagar said. “For this, I tried hymenolepidid-species specific PCR targeting mitochondrial gene CO1 and sequenced the DNA obtained from the PCR products. This case had never seized to amaze us – the sequence was beautiful, but I found some gene mutations. I repeated the sequencing twice and the results remained the same. I shared the sequences with Dr. Muehlenbachs, and he almost jumped out of his chair. He thought the mutations were very similar to the ones that we see in human cancer. I could not agree more. All the papers that I read during my early training years while getting my Ph.D. related to the gastrointestinal tumor markers were coming back to mind.”

“Now, the big question was- if the tapeworm had the cancer or muteted geneS for cancer? We thought we could only get the answer by deep-sequencing of the whole genome of the H. nana. But, at that time, we did not have next-generation sequencing facility in our lab. Next day, Dr. Muehlenbachs and I was sitting in the office of Dr. Michael Frace, Ph.D., CDC’s Biotechnology Core facility to discuss about the project. He mentioned that comparative deep sequencing could be attempted, but could we get him DNA from normal H. nana control? Dr. Muehlenbachs contacted Dr. Olson and others to get us control DNA/tissue so that we can prepare specially treated DNA for deep sequencing. Deep sequencing of the specimens from the patient revealed 6 insertional mutations (5 associated with protein coding genes) and comparative analysis identified H. nana structural genomic variants that are compatible with mutations described in cancer.”

“To a certain extent, mystery solved for this case.” said Dr. Bhatnagar. “Still, there are several unanswered questions and I am optimistic that if experts of several different areas work together collaboratively, some day we will find all the answers and unravel many new facts.” However, Dr. Bhatnagar, who was previously involved in several interesting case investigations including Clostridium sordellii septic shock syndrome associated with medical abortions, has cautioned against reading too much from a single case investigation.