Operating Within Molecular Margins

Today, we still rely on a century-old technique – microscopic review of H&E stained frozen sections – to analyze tissue in an intraoperative setting. And while the value and diagnostic expertise provided by the pathologists who use such traditional techniques are unquestionable, knowledge is advancing around us. Despite progress in other fields, we lack advanced tools in pathology that allow us to quickly assess the molecular make-up of biopsy specimens during a tumor resection. Consequently, molecular information remains hidden in the tissue until later diagnostic evaluation with immunohistochemistry, genetic analyses or other molecular techniques. The ability to conduct molecular analysis during surgery would offer big advantages to pathologists from a cost and workflow perspective, but more importantly, it could have a life-saving impact on the patient.

Stepping away from tradition

In the operating theatre, time is of the essence; there is a real need for creative new approaches that allow pathologists and surgeons to make diagnostic decisions based on detailed molecular information. Using mass spectrometry (MS), we have been able to rapidly detect molecules and distinguish tumor from normal tissue during surgical procedures in real-time (1).

DESI MS in action

Desorption electrospray ionization (DESI) targets the tissue surface with a stream of charged solvent droplets, which extract molecules from the sample and introduce them into the mass spectrometer. An MS profile is quickly acquired in a line scan or a more detailed two-dimensional molecular image, a fact that extends its use to tissue sectioned on a slide. The spatially resolved data can then be overlaid on top of a histology image for validation of the methodology outside of the operating room, which allows correlation of histology with signatures (multiple peaks) or specific single peaks that target one molecule. Intraoperatively, single point analyses are performed in seconds, providing molecular information on the tissue at stake. We believe this approach has several advantages over traditional molecular evaluation:

• It requires minimal to no sample preparation, and can be performed in ambient air conditions
  
• It can reliably measure small molecules, such as lipids and metabolites, with masses below 1,000 Daltons
  
• It can acquire useful molecular information in a matter of seconds.

It’s important to note that this two-dimensional molecular imaging approach allows us to validate MS data against the gold standard of histopathology, which also offers real value in research.

By using lipid profiles acquired by DESI MS, we have been able to discriminate different types of brain tumors (for example, meningioma from glioma), different gliomas subtypes (for example, astrocytoma from oligodendroglioma) and different grades of tumor (for example, WHO grade II glioma from WHO grade IV glioma).

In our latest study, we used DESI MS to detect a single metabolite that is generated by gliomas with mutations in isocitrate dehydrogenase (IDH) 1 and 2 genes (1), found in the high majority of low grade gliomas. These mutated enzymes generate an oncometabolite – 2-hydroxyglutarate (2-HG) – which accumulates to high levels in these gliomas and can be used to trace tumor cells.

DESI MS approach, step-by-step

The full clinical protocol is described in our recent paper (1); however, we can summarize the DESI MS process in three key steps: 1) Smear or touch prep from tissue on  glass slide; 2) Place glass slide on the instrument; 3) Acquire data in single points analysis intraoperatively; (2D imaging in the lab).

We have been able to reproducibly detect 2-HG in regions containing tumor, but not in normal tissue or regions of hemorrhage, which supports the use of DESI MS in defining the margins of a tumor and, therefore, guiding surgery. The high sensitivity and specificity was exciting to see, as was the ability to detect tumor cell concentration down to under 5 percent. We have validated our findings using a complete DESI MS analysis system installed in the Advanced Multimodality Image Guided Operating (AMIGO) suite at the Brigham and Women’s Hospital (BWH) in Boston, MA, USA (see Figure).

Mutations in IDH1 and IDH2 are not only found in gliomas but also in intrahepatic cholangiocarcinomas, acute myelogenous leukemias (AML) and chondrosarcomas, so we believe the detection of 2-HG or other metabolites with DESI MS could be useful in other clinical applications. Moreover, DESI MS could have applications in the diagnosis of a broad range of tumor types and could also provide a good alternative to intraoperative MRIs – without the associated high cost and disruption to standard operative workflows.

Tools of the future?

We hope that our work will pave the way for further development and clinical trial testing of metabolite guided surgical approaches; we have proof of principal studies underway both in the area of brain tumors as well as for resection of breast cancer (our manuscript on this study will soon by published in PNAS). These are the first steps in what could be a revolution in the way we conduct surgery. Admittedly, implementation of these technologies will require a long period of rigorous testing and validation. As the expertise using these approaches increases, validation studies will be required to determine the elements of pathology practice that might be redundant and those where complementary information is added to existing modalities. Indeed, it seems likely that the new intraoperative approaches being developed by us and other groups around the world will provide truly complementary tools – based on mass spectrometry and other analytical tools – for the pathologists and surgeons of tomorrow.

Corporate Backing

Waters Corporation recently announced an exclusive agreement with US-based instrument manufacturer Prosolia for DESI technology for clinical mass spectrometry applications (June 2014). One month later, it announced its acquisition of rapid evaporative ionization mass spectrometry (REIMS) technology from MediMass. Clearly, the company sees real potential in the technology. Here, Jeff Mazzeo, Senior Director, Health Science Business, Waters Division tells us why.

What promise have you seen so far with use of ambient ionization mass spec technology in surgical applications?

During an operation to remove cancerous tissue, surgeons can be unsure of exactly where the diseased tissue ends and healthy tissue begins. The result is that healthy issue is sometimes excised, or worse, parts of a tumor are missed and a follow up operation must be scheduled to remove the remaining malignant tissue. I believe the work conducted by Santagata, Agar and team, as well as work by Zoltan Takats (2), have shown that ambient ionization MS has the potential to one day transform surgical resection procedures.

Do you foresee any immediate challenges to more routine use of  MS in this setting?

Like many, we are encouraged by the early research with DESI and REIMS techniques. While still in their conceptual stages of development, the technologies must continue to demonstrate application benefits to a range of diseased tissues in much larger patient populations. There is also ongoing development work to be completed to make the instruments more feasible for routine surgical use in terms of installation, maintenance and use. Lastly, regulatory strategies must be discussed and agreed in order to develop solutions that can meet regulatory requirements so that clinical trials can be performed.

The advantages for the surgeon and patient are obvious, but what do you think this would mean for the histopathologist?

Just as we believe that MS has the potential to transform surgery, we also believe that imaging MS has the potential to transform histopathology. While more work is needed to correlate the results of MS investigations with traditional histopathology techniques, objective chemical information will hopefully add to the understanding of the morphology of tissue sections.