Archive for the ‘Imaging-based Cancer Patient Management’ Category

CancerBase.org – The Global HUB for Diagnoses, Genomes, Pathology Images: A Real-time Diagnosis and Therapy Mapping Service for Cancer Patients – Anonymized Medical Records accessible to anyone on Earth

Reporter: Aviva Lev-Ari, PhD, RN


UPDATED on 10/29/2019

Pan-cancer computational histopathology reveals mutations, tumor composition and prognosis

Yu Fu1, Alexander W Jung1, Ramon Viñas Torne1, Santiago Gonzalez1,2, Harald Vöhringer1, Mercedes Jimenez-Linan3, Luiza Moore3,4, and Moritz Gerstung#1,5 # to whom correspondence should be addressed 1) European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, UK. 2) Current affiliation: Institute for Research in Biomedicine (IRB Barcelona), Parc Científic de Barcelona, Barcelona, Spain. 3) Department of Pathology, Addenbrooke’s Hospital, Cambridge, UK. 4) Wellcome Sanger Institute, Hinxton, UK 5) European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.


Dr Moritz Gerstung European Molecular Biology Laboratory European Bioinformatics Institute (EMBL-EBI) Hinxton, CB10 1SA UK. Tel: +44 (0) 1223 494636 E-mail: moritz.gerstung@ebi.ac.uk


Pan-cancer computational histopathology reveals mutations, tumor composition and prognosis

Here we use deep transfer learning to quantify histopathological patterns across 17,396 H&E stained histopathology image slides from 28 cancer types and correlate these with underlying genomic and transcriptomic data. Pan-cancer computational histopathology (PC-CHiP) classifies the tissue origin across organ sites and provides highly accurate, spatially resolved tumor and normal distinction within a given slide. The learned computational histopathological features correlate with a large range of recurrent genetic aberrations, including whole genome duplications (WGDs), arm-level copy number gains and losses, focal amplifications and deletions as well as driver gene mutations within a range of cancer types. WGDs can be predicted in 25/27 cancer types (mean AUC=0.79) including those that were not part of model training. Similarly, we observe associations with 25% of mRNA transcript levels, which enables to learn and localise histopathological patterns of molecularly defined cell types on each slide. Lastly, we find that computational histopathology provides prognostic information augmenting histopathological subtyping and grading in the majority of cancers assessed, which pinpoints prognostically relevant areas such as necrosis or infiltrating lymphocytes on each tumour section. Taken together, these findings highlight the large potential of PC-CHiP to discover new molecular and prognostic associations, which can augment diagnostic workflows and lay out a rationale for integrating molecular and histopathological data.



Key points

● Pan-cancer computational histopathology analysis with deep learning extracts histopathological patterns and accurately discriminates 28 cancer and 14 normal tissue types

● Computational histopathology predicts whole genome duplications, focal amplifications and deletions, as well as driver gene mutations

● Wide-spread correlations with gene expression indicative of immune infiltration and proliferation

● Prognostic information augments conventional grading and histopathology subtyping in the majority of cancers



Here we presented PC-CHiP, a pan-cancer transfer learning approach to extract computational histopathological features across 42 cancer and normal tissue types and their genomic, molecular and prognostic associations. Histopathological features, originally derived to classify different tissues, contained rich histologic and morphological signals predictive of a range of genomic and transcriptomic changes as well as survival. This shows that computer vision not only has the capacity to highly accurately reproduce predefined tissue labels, but also that this quantifies diverse histological patterns, which are predictive of a broad range of genomic and molecular traits, which were not part of the original training task. As the predictions are exclusively based on standard H&E-stained tissue sections, our analysis highlights the high potential of computational histopathology to digitally augment existing histopathological workflows. The strongest genomic associations were found for whole genome duplications, which can in part be explained by nuclear enlargement and increased nuclear intensities, but seemingly also stems from tumour grade and other histomorphological patterns contained in the high-dimensional computational histopathological features. Further, we observed associations with a range of chromosomal gains and losses, focal deletions and amplifications as well as driver gene mutations across a number of cancer types. These data demonstrate that genomic alterations change the morphology of cancer cells, as in the case of WGD, but possibly also that certain aberrations preferentially occur in distinct cell types, reflected by the tumor histology. Whatever is the cause or consequence in this equation, these associations lay out a route towards genomically defined histopathology subtypes, which will enhance and refine conventional assessment. Further, a broad range of transcriptomic correlations was observed reflecting both immune cell infiltration and cell proliferation that leads to higher tumor densities. These examples illustrated the remarkable property that machine learning does not only establish novel molecular associations from pre-computed histopathological feature sets but also allows the localisation of these traits within a larger image. While this exemplifies the power of a large scale data analysis to detect and localise recurrent patterns, it is probably not superior to spatially annotated training data. Yet such data can, by definition, only be generated for associations which are known beforehand. This appears straightforward, albeit laborious, for existing histopathology classifications, but more challenging for molecular readouts. Yet novel spatial transcriptomic44,45 and sequencing technologies46 bring within reach spatially matched molecular and histopathological data, which would serve as a gold standard in combining imaging and molecular patterns. Across cancer types, computational histopathological features showed a good level of prognostic relevance, substantially improving prognostic accuracy over conventional grading and histopathological subtyping in the majority of cancers. It is this very remarkable that such predictive It is made available under a CC-BY-NC 4.0 International license. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. bioRxiv preprint first posted online Oct. 25, 2019; doi: http://dx.doi.org/10.1101/813543. The copyright holder for this preprint signals can be learned in a fully automated fashion. Still, at least at the current resolution, the improvement over a full molecular and clinical workup was relatively small. This might be a consequence of the far-ranging relations between histopathology and molecular phenotypes described here, implying that histopathology is a reflection of the underlying molecular alterations rather than an independent trait. Yet it probably also highlights the challenges of unambiguously quantifying histopathological signals in – and combining signals from – individual areas, which requires very large training datasets for each tumour entity. From a methodological point of view, the prediction of molecular traits can clearly be improved. In this analysis, we adopted – for the reason of simplicity and to avoid overfitting – a transfer learning approach in which an existing deep convolutional neural network, developed for classification of everyday objects, was fine tuned to predict cancer and normal tissue types. The implicit imaging feature representation was then used to predict molecular traits and outcomes. Instead of employing this two-step procedure, which risks missing patterns irrelevant for the initial classification task, one might directly employ either training on the molecular trait of interest, or ideally multi-objective learning. Further improvement may also be related to the choice of the CNN architecture. Everyday images have no defined scale due to a variable z-dimension; therefore, the algorithms need to be able to detect the same object at different sizes. This clearly is not the case for histopathology slides, in which one pixel corresponds to a defined physical size at a given magnification. Therefore, possibly less complex CNN architectures may be sufficient for quantitative histopathology analyses, and also show better generalisation. Here, in our proof-of-concept analysis, we observed a considerable dependence of the feature representation on known and possibly unknown properties of our training data, including the image compression algorithm and its parameters. Some of these issues could be overcome by amending and retraining the network to isolate the effect of confounding factors and additional data augmentation. Still, given the flexibility of deep learning algorithms and the associated risk of overfitting, one should generally be cautious about the generalisation properties and critically assess whether a new image is appropriately represented. Looking forward, our analyses revealed the enormous potential of using computer vision alongside molecular profiling. While the eye of a trained human may still constitute the gold standard for recognising clinically relevant histopathological patterns, computers have the capacity to augment this process by sifting through millions of images to retrieve similar patterns and establish associations with known and novel traits. As our analysis showed this helps to detect histopathology patterns associated with a range of genomic alterations, transcriptional signatures and prognosis – and highlight areas indicative of these traits on each given slide. It is therefore not too difficult to foresee how this may be utilised in a computationally augmented histopathology workflow enabling more precise and faster diagnosis and prognosis. Further, the ability to quantify a rich set of histopathology patterns lays out a path to define integrated histopathology and molecular cancer subtypes, as recently demonstrated for colorectal cancers47 .

Lastly, our analyses provide It is made available under a CC-BY-NC 4.0 International license. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

bioRxiv preprint first posted online Oct. 25, 2019; doi: http://dx.doi.org/10.1101/813543.

The copyright holder for this preprint proof-of-concept for these principles and we expect them to be greatly refined in the future based on larger training corpora and further algorithmic refinements.



July 27, 2016
world map illustration
Illustration by Tricia Seibold and iStock/liuzishan

During his 2016 State of the Union address, President Barack Obama called on Vice President Joe Biden – who had months earlier lost his son Beau to brain cancer – to head a “moonshot” to significantly accelerate research into the disease. The president said he wanted to harness the spirit of American innovation that took us from zero to landing a man on the moon in a decade to similarly find new ways to prevent, diagnose and treat cancer.

One of those intrigued by that call to action was Stanford’s Jan Liphardt, an associate professor of bioengineering who specializes in biophysics, the tumor microenvironment and data analysis. Stanford Engineering talked to Liphardt about how he came to be involved with the moonshot and his approach to using data and the voice of patients to better understand cancer and how it can be treated, and how sharing information can better inform the course of cancer research.

How did you get involved in the National Cancer Moonshot?

In March, after the president’s charge, the vice president challenged scientists, doctors, industry and patients to give their best ideas to the moonshot. The White House also reached out to a few outsiders, myself included. The White House instructions were unusual: “Do something big and different. There is no money and you have 87 days. Go.”

I like a challenge, and this was a chance to serve, even in the face of administrative hurdles. So I looked for advice, teammates and support. Russ Altman, a colleague at Stanford, suggested it was time to give patients a way to volunteer their own health data in order to help find cures. I collaborated with Peter Kuhn, a professor of medicine and engineering at the University of Southern California, who’s known for carefully listening to cancer patients, advocates and their supporters. In short order we had links with advocates like AnneMarie Ciccarella, Sonja Durham, Lori Marx-Rubiner, Jack Whelan and Jack Park. That’s how we got to CancerBase.org.

What’s the idea the team came up with?

We thought for about a week: What would matter to the patients that Stanford and other research institutions serve? What would scale? Well, we’re not going to run a clinical trial, go near protected health information, invent a new drug or write a research proposal. There’s no time for that. Whatever it was, it had to be useful, scalable, legal and different. That pointed to data, the web, patients and decisions.

One thing jumped out: Right now, there’s significant friction in medical data sharing. People all over the world can already effortlessly share other kinds of information – pictures, movies, ideas, stories, tweets. Increasingly, they are using the same tools to share personal medical information. It’s remarkable what cancer patients already share: diagnoses, genomes, pathology images. But that information is not yet widely used to understand where they are with their diseases.

Ideally, everyone, including scientists and doctors, would have as much information as possible at their fingertips. Many patients think when they give data for research, magically scientists all over the world can dig into this information, find patterns and help. The practical reality is that it’s nearly impossible for any one scientist to access the amounts of data they would like.

So that’s the simple idea: A global map and give patients the tools they need to share their data – if they want to. They can donate information for the greater good. In return, we make a simple promise: When you post data, we’ll anonymize them and make them available to anyone on Earth in one second. We plan to display this information like real-time traffic data. HIPAA doesn’t apply to this direct data-sharing. The patients can give us whatever information they want, and they can tell us what they want us to do with it. We’re a conduit. Their data belong to them, not to us.

How does it work?

Today we ask just five basic questions. Over time we will add more. You join, give some information, and we’ll put you on a global map. Right now, some of the things we don’t know about cancer are incredibly simple: Where is everyone on Earth with cancer? How old are they? What is their diagnosis? Did their cancers metastasize? Global, instantaneous data sharing is the story.

In a second phase, we are going to see if we can plot all the information just like Waze does for traffic. Our role is to synthesize the information and plot it in ways that ordinary people can understand. Think of it this way – patients want to be able to chart their treatment path. Who went straight, who went left? People just getting on the highway are curious about what people did who came before them, and what happened to those people. Did they arrive at the destination easily and promptly? We’re a real-time diagnosis and therapy mapping service for cancer.

You say that giving patients a way to share their health data is important to help finding cures. Why?

Let me give you a specific example. At Stanford, I’m part of a team of cancer biologists and clinicians funded by the Stanford Cancer Institute to think about the next generation of screening for breast cancer in the U.S. Every year, the U.S. uses mammography to screen more than 40 million women for breast cancer. In this project, it quickly became clear that there is currently no central, easy-to-access repository of mammograms for research use.

That’s a major lost opportunity – our nation spends billions on screening, but we don’t store, share and analyze this information in a scalable and simple manner. In the traditional approach, our team would spend several hundred thousand dollars, and about three years, to assemble perhaps 1,000 mammograms. We would then use this tiny dataset to try to find something interesting, but since the dataset is so small, we would be blind to rare features of breast cancer and its predictors. It clearly makes a lot more sense to compare and explore 100 million images.

This sounds completely impossible until you realize that Instagram users upload 58 million images every day. Once you start to think about supposedly intractable research problems from a web or social networking perspective, new possibilities open. Imagine, for example, if there were a simple way for every single woman on Earth to upload and share her de-identified mammogram? Or more generally, imagine a world in which patients have the tools to globally share de-identified health data, if they want to. That’s exactly the idea behind CancerBase – let’s just give people those tools and see what happens.

How much data and how many people are needed to make this viable?

We think we are going to need several tens-of-thousands of members. There are approximately 50 million people on Earth with a cancer diagnosed in the last five years, and 200 million more people have an immediate family member with cancer. Almost 2 billion people are active on Twitter and Facebook – a quarter of the world’s population. If just a few percent of those people sign up, we could do something no one on Earth has done before.

Are there hopes to create a “developer community,” people who find ways to use your data that you didn’t even think about or have the time to work on?

Definitely. As much as we think we can predict what these data are useful for, we don’t really know. By making the anonymized data available to everyone within one second, they might start to do things that we never dreamed of. The more eyes look at these data, the better off everyone will be. The dream is to have cancer-relevant medical data flow unimpeded around the world in seconds, so that everyone, wherever they are, can see and use this information.



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3D Imaging of Cancer Cells

Larry H. Bernstein, MD, FCAP, Curator



3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions

Dark Daily Apr 8th 2016        Jon Stone



3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions.

New technology from researchers at the University of Texas Southwestern Medical Center enables the ability to study cancer cells in their native microenvironments.

Imaging research is one step closer to giving clinicians a way to do high-resolution scans of malignant cells in order to diagnose cancer and help identify useful therapies. If this technology were to prove successful in clinical studies, it might change how anatomic pathologists and radiologists diagnose and treat cancer.

Researchers at the University of Texas Southwestern Medical Center developed a way to create near-isotropic, high-resolution scans of cells within their microenvironments. The process involves utilizing a combination of two-photonBessel beams and specialized filtering.

New Imaging Approach Could be Useful to Both Pathologists and Radiologists

In a recent press release, senior author Reto Fiolka, PhD, said “there is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned. Our microscope is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor.”

In a study in Developmental Cell, Erik S. Welf, PhD, et al, described the new microenvironmental selective plane illumination microscopy (meSPIM). When developing the technology, the team outlined three goals:

1. The microscope design must not prohibitively constrain microenvironmental properties.

2. Spatial and temporal resolution must match the cellular features of interest.

3. Spatial resolution must be isotropic to avoid spatial bias in quantitative measurements.

This new technology offers pathologists and medical laboratory scientists a new look at cancer cells and other diseases. The study notes that meSPIM eliminates the influence of stiff barriers, such as glass slide covers, while also allowing a level of control over both mechanical and chemical influences that was previously impossible.

Early meSPIM Research Reveals New Cell Behaviors

Early use of meSPIM in observing melanoma cells is already offering new insights into the relationship between the cell behavior of cellular- and subcellular-scale mechanisms and the microenvironment in which these cells exist. The study notes, “The ability to image fine cellular details in controllable microenvironments revealed morphodynamic features not commonly observed in the narrow range of mechanical environments usually studied in vitro.”

One such difference is the appearance of blebbing. Created by melanoma cells and lines, these small protrusions are thought to aid in cell mobility and survival. Using meSPIM, observers could follow the blebbing process in real-time. Formation of blebs on slides and within an extracellular matrix (ECM) showed significant differences in both formation and manipulation of the surrounding microenvironment.

The team is also using meSPIM to take a look at membrane-associated biosensor and cytosolic biosensor signals in 3D. They hope that investigation of proteins such as phosphatidylinositol 3-kinase (PI3K) and protein kinase C will help to further clarify the roles these signals play in reorientation of fibroblasts.

meSPIM combined with computer vision enables imaging, visualization, and quantification of how cells alter collagen fibers over large distances within an image volume measuring 100 mm on each side. (Photo Copyright: Welf and Driscoll et al.)

The research team believes this opens new possibilities for studying diseases at a subcellular level, saying, “Cell biology is necessarily restricted to studying what we can measure. Accordingly, while the last hundred years have yielded incredible insight into cellular processes, unfortunately most of these studies have involved cells plated onto flat, stiff surfaces that are drastically different from the in vivo microenvironment …

“Here, we introduce an imaging platform that enables detailed subcellular observations without compromising microenvironmental control and thus should open a window for addressing these fundamental questions of cell biology.”

Limitations of meSPIM

One significant issue associated with the use of meSPIM is the need to process the large quantity of data into useful information. Algorithms currently allow for automatic bleb detection. However, manual marking, while time consuming, still provides increased accuracy. Researchers believe the next step in improving the quality of meSPIM scans lie in computer platforms designed to extract and process the scan data.

Until this process is automated, user bias, sample mounting, and data handling will remain risks for introducing errors into the collected data. Yet, even in its early stages, meSPIM offers new options for assessing the state of cancer cells and may eventually provide pathologists and radiologists with additional information when creating treatment plans or assessments.


Seeing cancer cells in 3-D (w/ Video)



Cancer in 3-D


Extracted surfaces of two cancer cells. (Left) A lung cancer cell colored by actin intensity near the cell surface. Actin is a structural molecule that is integral to cell movement. (Right) A melanoma cell colored by PI3-kinase activity near the cell surface. PI3K is a signaling molecule that is key to many cell processes. Credit: Welf and Driscoll et al.

Cancer cells don’t live on glass slides, yet the vast majority of images related to cancer biology come from the cells being photographed on flat, two-dimensional surfaces—images that are sometimes used to make conclusions about the behaviour of cells that normally reside in a more complex environment. But a new high-resolution microscope, presented February 22 in Developmental Cell, now makes it possible to visualize cancer cells in 3D and record how they are signaling to other parts of their environment, revealing previously unappreciated biology of how cancer cells survive and disperse within living things.

“There is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned,” says senior author Reto Fiolka, an optical scientist at the University of Texas Southwestern Medical Center. “Our is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor environments.”

Read more at: http://phys.org/news/2016-02-cancer-cells-d-video.html#jCp

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Laser Therapy Opens Blood-Brain Barrier

Curator: Larry H. Bernstein, MD, FCAP


Laser Surgery Opens Blood-Brain Barrier to Chemotherapy


ST. LOUIS, March 11, 2016 — A laser probe has been used to open the brain’s protective cover, enabling delivery of chemotherapy drugs to patients with glioblastoma — the most common and aggressive form of brain cancer.

In a pilot study conducted by the Washington University School of Medicine in St. Louis, Mo., 14 patients with glioblastoma underwent minimally invasive laser surgery to treat a recurrence of their tumors. Heat from the laser was already known to kill brain tumor cells but, unexpectedly, the researchers found that the technology penetrated the blood-brain barrier.

“The laser treatment kept the blood-brain barrier open for four to six weeks, providing us with a therapeutic window of opportunity to deliver chemotherapy drugs to the patients,” said neurosurgery professor Eric Leuthardt, MD, who also treats patients at Barnes-Jewish Hospital. “This is crucial because most chemotherapy drugs can’t get past the protective barrier, greatly limiting treatment options for patients with brain tumors.

The team is still closely following the patients, though early results indicate they are doing better on average, in terms of survival and clinical outcomes, than what the researchers would expect with other treatment methods.

Glioblastomas are one of the most difficult cancers to treat. Most patients diagnosed with this type of brain tumor survive just 15 months, according to the American Cancer Society.

The research is part of a larger phase II clinical trial that will involve 40 patients. Twenty patients were enrolled in the pilot study, 14 of whom were found to be suitable candidates for the minimally invasive laser surgery, a technology that Leuthardt helped pioneer.

The laser technology was approved by the FDA in 2009 as a surgical tool to treat brain tumors. The Washington team’s research marks the first time the laser has been shown to disrupt the blood-brain barrier, which shields the brain from harmful toxins but inadvertently blocks potentially helpful drugs, such as chemotherapy.

As part of the trial, doxorubicin, a widely used chemotherapy, was delivered intravenously to 13 patients in the weeks following the laser surgery. Preliminary data indicate that 12 patients showed no evidence of tumor progression during the short, 10-week time frame of the study. One patient experienced tumor growth before chemotherapy was delivered; the tumor in another patient progressed after chemotherapy was administered, the researcher reported.

The laser surgery was well-tolerated by the patients in the trial; most went home one to two days afterward, and none experienced severe complications. The surgery was performed while a patient lies in an MRI scanner, providing the neurosurgical team with a real-time look at the tumor. Using an incision of only 3 mm, a neurosurgeon robotically inserted the laser to heat up and kill brain tumor cells at a temperature of about 150 °F.

“The laser kills tumor cells, which we anticipated,” said Leuthardt. “But, surprisingly, while reviewing MRI scans of our patients, we noticed changes near the former tumor site that looked consistent with the breakdown of the blood-brain barrier.”

Leuthardt confirmed and further studied these imaging findings with study co-author Dr. Joshua Shimony, a professor of radiology at Washington University.

The researchers, including co-corresponding author Dr. David Tran, a neuro-oncologist now at the University of Florida, performed follow-up testing, which showed that the degree of permeability through the blood-brain barrier peaked one to two weeks after surgery but that the barrier remained open for up to six weeks.

Other successful attempts to breach the barrier have left it open for only a short time — about 24 hours — not long enough for chemotherapy to be consistently delivered, or have resulted in only modest benefits, the researchers said. The laser technology leaves the barrier open for weeks — long enough for patients to receive multiple treatments with chemotherapy. Further, the laser only opens the barrier near the tumor, leaving the protective cover in place in other areas of the brain. This has the potential to limit the harmful effects of chemotherapy drugs in other areas of the brain, the researchers said.

The findings also suggest that other approaches, such as cancer immunotherapy — which harnesses cells of the immune system to seek out and destroy cancer — could also be useful for patients with glioblastomas.

The researchers are planning another clinical trial that combines the laser technology with chemotherapy and immunotherapy, as well as trials to test targeted cancer drugs that normally can’t breach the blood-brain barrier.

The research was published in Plos One (doi: 10.1371/journal.pone.0148613).


Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.


Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (Ktrans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.


In all 14 evaluable patients, Ktrans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.


The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration  

ClinicalTrials.gov NCT01851733

Citation: Leuthardt EC, Duan C, Kim MJ, Campian JL, Kim AH, Miller-Thomas MM, et al. (2016) Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier. PLoS ONE 11(2): e0148613.  http://dx.doi.org:/10.1371/journal.pone.0148613

Glioblastoma (GBM) is the most common and lethal malignant brain tumor in adults [1]. Despite advanced treatment, median survival is less than 15 months, and fewer than 5% of patients survive past 5 years [2, 3]. Effective treatment options for recurrent GBM remain very limited and much of research and development efforts in recent years have focused on this area of greatly unmet needs. Up to 90% of recurrent tumors develop within the 2–3 cm margin of the primary site and are thought to arise from microscopic glioma cells that infiltrate the peritumoral brain region prior to resection of the primary tumor [4, 5]. Therefore elimination of infiltrative GBM cells in this region likely will improve long-term disease control.

Inadequate CNS delivery of therapeutic drugs due to the blood brain barrier (BBB) has been a major limiting factor in the treatment of brain tumors. The presence of contrast enhancement on standard brain MRI qualitatively reflects a disrupted state of the BBB. For this reason, drug access to the viable contrast enhanced tumor rim is likely significantly higher than to the peritumoral region, which usually does not have contrast enhancement [6, 7]. Evidence supporting this hypothesis came from studies in which drug levels of cytotoxic agents were sampled in tumors and the surrounding brain tissue at the time of surgery or autopsy. Drug concentrations were at the highest in the enhancing portion of tumors, and then rapidly decreased up to 40 fold lower by 2–3 cm distance from the viable tumor edge [810]. Overall, these observations suggest that the BBB and its integrity negatively correlate with delivery and potentially therapeutic effects of BBB impermeant drugs.

To circumvent the BBB problem in local drug delivery, recent approaches have focused on bypassing it. A previously described method is the use of Gliadel, a polymer wafer impregnated with the chemotherapeutic agent carmustine (BCNU) and placed intra-operatively in the resection cavity to bypass the BBB. This approach resulted in a statistically significant but modest survival advantage in both newly diagnosed and recurrent GBM [1113]. The modest benefit of Gliadel could be due to the short duration of drug delivery as nearly 80% of BCNU is released from the wafer over a period of only 5 days [14]. This observation further supports the notion that the BBB is critical to chemotherapy effect. However, Gliadel is not widely utilized as it requires an open craniotomy and can impair wound healing. Another approach of bypassing the BBB is the convection-enhanced delivery system in which a catheter is surgically inserted into the tumor to deliver chemotherapy [15]. This procedure requires prolonged hospitalization to maintain the external catheter to prevent serious complications and as a result has not been used extensively.

The role of hyperthermia in inducing BBB disruption has been previously described in animal models of CNS hyperthermia. In a rodent model of glioma, the global heating of the mouse’s head to 42°C for 30 minutes in a warm water bath significantly increased the brain concentration of a thermosensitive liposome encapsulated with adriamycin chemotherapy [16]. To effect more locoregional hyperthermia, retrograde infusion of a saline solution at 43°C into the left external carotid artery in the Wistar rat reversibly increased BBB permeability to Evans-blue albumin in the left cerebral hemisphere [17]. In another approach, neodymium-doped yttrium aluminum garnet (Nd:YAG) laser-induced thermotherapy to the left forebrain of Fischer rats resulted in loco-regional BBB disruption as evidenced by passage of Evans blue dye, serum proteins (e.g. fibrinogen & IgM), and the chemotherapeutic drug paclitaxel for up to several days after thermotherapy [18]. The effect of hyperthermia on the BBB of human brain has not been examined.

Here we describe an approach to induce sustained, local disruption of the peritumoral BBB using MRI-guided laser interstitial thermal therapy, or LITT. The biologic effects and correlation with MRI findings of LITT have been studied in both animal and human models since the development of LITT over twenty years ago. A well-described zonal distribution of histopathological changes with corresponding characteristic MR imaging findings centered on the light-guide track replace the lesion targeted for thermal therapy. The central treatment zone shows development of coagulative necrosis with complete loss of normal neurons or supporting structures immediately following therapy, corresponding to hyperintense T1-weighted signal intensity relative to normal brain [1922]. The peripheral zone of the post-treatment lesion is characterized by avid enhancement with intravenous gadolinium contrast agents, which peaks several days following thermal therapy and persists for many weeks after the procedure. Gadolinium contrast enhancement in the brain following LITT is due to leakage of gadolinium contrast into the extravascular space across a disrupted BBB [2023]. The perilesional zone of hyperintense signal intensity of FLAIR-weighted images develops within 1–3 days of thermal treatment and persists for 15–45 days [22].

We demonstrate that in addition to cytoreductive ablation of the main recurrent tumor, hyperthermic exposure of the peritumoral region resulted in localized, lasting disruption of the BBB as quantified by dynamic contrast-enhanced MRI (DCE-MRI) and serum levels of brain-specific enolase (BSE), thus providing a therapeutic window of opportunity for enhanced delivery of therapeutic agents.

Table 1. Patient Baseline Demographics and Characteristics.
TMZ/RT: Stupp protocol of 60 Gy radiotherapy plus concurrent 75mg/m2 daily temozolomide. Doxorubicin treatment: Timing of 20mg/m2 IV weekly doxobubicin treatment after LITT. Early = Starting within 1 week after LITT; Late = Starting at 6 weeks after LITT.  http://dx.doi.org:/10.1371/journal.pone.0148613.t001
Quantitative measurement of LITT-induced peritumoral BBB disruption by DCE-MRI

Brain MRI obtained within 48 hours following LITT showed the targeted tumor replaced by a post-treatment lesion corresponding to the volume of treated tissue on intraoperative thermometry maps. The post-treatment lesion lost the original rim of tumor-associated contrast enhancement and instead demonstrated central hyperintense T1-weighted signal compared to the pre-treated tumor and normal brain and a faint, newly developed discontinuous rim of peripheral contrast enhancement extending beyond the original tumor-associated enhancing rim (Fig 2A). These findings are consistent with a loss of viable tumor tissue caused by LITT, thus achieving an effective cytoreduction similar to open surgical resection. Of note, the rim of new peripheral contrast enhancement persisted for at least the next 28 days (Fig 2B–2E). Perilesional edema qualitatively evaluated on FLAIR-weighted images increased from pretreatment imaging at week 2 and persisted at week 4 following LITT (Fig 2F–2I). Perilesional edema decreased on subsequent MRI examinations. These findings qualitatively indicate that peritumoral BBB is disrupted by LITT and that the disruption peaks within approximately 2 weeks after the procedure.


Fig 3 demonstrates the Ktrans time curves for our cohort of patients. In all subjects the Ktrans in the ROIs within the enhancing ring around the ablated tumor is highly elevated in the first few days after the procedure and then progressively decreases at approximately the 4-week time point. The bottom right subplot in Fig 3 is an average of the Ktrans time courses from all the subjects with adjacent curves indicating the plus and minus one standard error of the mean curves. This figure demonstrates the peak Ktrans value immediately after the LITT procedure with persistent elevation out to about 4 weeks. Radiographically, persistent contrast enhancement and FLAIR hyperintensity were observed well past 6 weeks and in many cases more than 10 weeks later. Several patients had recurrent tumor by radiographic criteria (increasing size of the edema and enhancing area around the tumor site) and these patients also demonstrated a corresponding increase in the Ktrans value. These recurrences occurred after the 10-week mark and thus were not included in Fig 3. Importantly no difference in the pattern of Ktrans tracing was consistently observed between the 10 patients receiving late doxorubicin treatment and the 4 patients receiving early doxorubicin treatment. In summary, these results indicate that the peritumoral BBB disruption as measured by Ktrans peaked immediately after LITT and persisted above baseline for an additional 4 weeks.


To optimize the ELISA assay for BSE, we collected sera from 3 patients with a newly diagnosed low-grade (WHO grade 2) glioma before and after their planned craniotomy and surgical resection, and determined serum concentrations of BSE. WHO grade 2 gliomas were chosen for the optimization because as they are generally non-contrast enhanced tumors on brain MRI, tumor-associated BBB is relatively intact and consequently, serum concentrations of brain-specific factors are predicted to be low pre-operatively and to then rise post-operatively due to the BBB compromise from the surgery. Serum BSE concentrations were low prior to surgery and then, as predicted, consistently increased after open craniotomy and tumor resection, thus indicating that this method had adequate sensitivity in detecting changes in serum levels of BSE due to disruption of the BBB (Fig 4).

Fig 4. Optimization of the BSE ELISA assay for measuring BBB disruption.

Serum concentrations of BSE before and after open craniotomy for surgical debulking in 3 subjects (A, B, and C) with a low-grade glioma, WHO grade II. *p<0.05.  http://dx.doi.org:/10.1371/journal.pone.0148613.g004


Fig 5. BBB disruption induced by LITT as measured by serum biomarkers
Serum concentrations of BSE for each of the 14 evaluable subjects in the study (A-N) and as the mean + SEM (O) as a function of time in days from the LITT procedure. In 7/14 subjects, serum BSE levels slightly decreased immediately after LITT, then in 13/14 subjects, serum BSE levels rose shortly after LITT, peaked between 1–3 weeks after LITT, and then decreased by the 6-week time point. In Patient #12, serum BSE concentration increased at week 10 coincident with an increased Ktrans at the same time point, consistent with a recurrent tumor as demonstrated on diagnostic MR imaging. Patient #15’s serum BSE concentration began to rise by week 4, consistent with early multifocal recurrent disease as demonstrated on diagnostic MR imaging.  http://dx.doi.org:/10.1371/journal.pone.0148613.g005

LITT is a minimally invasive neurosurgical technique that achieves effective tumor cytoreduction of brain tumors using a laser to deliver hyperthermic ablation. Here we have demonstrated that an unexpected, potentially useful effect of LITT is its ability to also disrupt the BBB in the peritumoral region that extends outwards 1–2 cm from the viable tumor rim. Importantly, the disruption persists in all 14 evaluable, treated patients for up to 4 weeks after LITT as measured quantitatively by DCE-MRI and up to 6 weeks as measured by serum levels of the brain-specific factor BSE. These observations indicate that after LITT there is a window during which enhanced local delivery of therapeutic agents into the desired location (i.e. peritumoral region) can potentially be achieved.

In all of the patients in this series, the peaks of serum concentrations of BSE showed wider variations and were delayed from several days to 1–2 weeks following the peak of BBB disruption as measured by Ktrans. The wider variations and delay of BSE concentrations lead to relatively low correlation coefficients between the two parameters and could be explained by: 1) the higher data point resolution for the serum values versus DCE-MRI values (weekly versus biweekly, respectively); 2) interval physiologic breakdown of thermally ablated tissue coupled with subsequent diffusion and equilibration between the intracranial and peripheral compartments; and 3) high inter-tumor heterogeneity among patients resulting in a wide variation in the rates at which ablated tissues of different compositions are broken down and released into the circulation. Whether these differences may be in part due to tumor-related factors such as IDH1/2 mutations and MGMT promoter methylation is unclear due to the small number of subjects. More importantly, both methods showed that the peritumoral BBB disruption induced by LITT was temporary, decreasing soon after peaking and being resolved by 4–6 weeks in most patients. In addition, although no significant difference in all the BBB measurement parameters was observed between the early and late doxorubicin treatment arms, the number of evaluable subjects was too small to allow generalization at this time.

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President Carter’s Status

Author: Larry H. Bernstein, MD, FCAP



Most Experts Not Surprised by Carter’s Status 

But early response does not mean ‘cure’




by Charles Bankhead
Staff Writer, MedPage Today


Former President Jimmy Carter’s announcement that he is free of metastatic melanoma surprised many people but, not most melanoma specialists contacted by MedPage Today.

With the evolution of modern radiation therapy techniques and targeted drugs, more patients with metastatic melanoma achieve complete and partial remissions, including remission of small brain metastases like the ones identified during the evaluation and initial treatment of Carter. However, the experts — none of whom have direct knowledge of Carter’s treatment or medical records — cautioned that early remission offers no assurance that the former president is out of the woods.

“If I had a patient of my own with four small brain mets undergoing [stereotactic radiation therapy], I would tell them that I fully expected the radiation to take care of those four lesions,” said Vernon K. Sondak, MD, of Moffitt Cancer Center in Tampa. “The fact that President Carter reports that it has done just that is not a surprise to me at all.

“I would also tell my patient that the focused radiation only treats the known cancer in the brain, and that if other small areas of cancer are present, they will likely eventually grow large enough to need radiation or other treatment as well, and that periodic brain scans will be required to monitor for this possibility.”

Carter also is being treated with the immune checkpoint inhibitor pembrolizumab (Keytruda), which is known to stimulate immune cells that then migrate to tumor sites to eradicate the lesions, noted Anna Pavlick, DO, of NYU Langone Medical Center in New York City.

“Melanoma is no longer a death sentence, and we are really changing what happens to patients,” said Pavlick. “It really is amazing.”

Carter’s melanoma story began to emerge in early August when he had surgery to remove what was described as “a small mass” from his liver. Following the surgery, Carter announced that his doctors had discovered four small melanoma lesions in his brain, confirming a suspicion the specialists had shared with him at the time of the surgery.

Carter subsequently underwent focused radiation therapy to eradicate the brain lesions and initiated a 12-week course of treatment with pembrolizumab. The radiation therapy-targeted therapy combination was a logical option for Carter, given observations that the PD-L1 inhibitor has synergy with radiation, noted Stergios Moschos, MD, of the University of North Carolina Lineberger Comprehensive Cancer Center at Chapel Hill.

“I have seen this in other patients with metastatic melanoma,” said Gary K. Schwartz, MD, of Columbia University Medical Center in New York City. “It is remarkable but absolutely possible within the realm of immunotherapy today.”

Although Carter’s announcement is undeniably good news, the optimism should be tempered by a long-term perspective, suggested Nagla Abdel Karim, MD, PhD, of the University of Cincinnati Medical Center.

“We do have similar stories; however, we would be careful to call it a ‘complete remission’ and ‘disease control’ and not a ‘cure,’ so far,” said Karim. “We would resume therapy and follow-up any autoimmune side effects. Most important is the quality of life, which he seems to enjoy, and we are very happy with that.”

Darrell S. Rigel, MD, also of NYU Langone Medical Center, represented the lone dissenter among specialists who responded to MedPage Today‘s request for comments.

“I’m happy for him, but it’s very unusual, especially in older men, who usually have a worse prognosis,” said Rigel. “He is on a new drug that may have a little more promise, but there is no definitive cure at this point.”



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Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator



GEN News Highlights   Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer




The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]


Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The investigators discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.


Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma


SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma


Cortical GABAergic excitation contributes to epileptic activities around human glioma


Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.


Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.


Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies


Magnetic Resonance Metabolic Imaging of Glioma


Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (68). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 1012).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (1419). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 1719). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.
A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.
The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (7678). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (8183). These so-called IL-15 DCs can be derived from CD14+monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4+ helper T cells licenses DCs and enables them to prime CD8+ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.
Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.


Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …


The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42).  …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….


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Immunopathogenesis Advances in Diabetes and Lymphomas

Larry H Bernstein, MD, FCAP, Curator




 Science team says they’ve taken another step toward a potential cure for diabetes

Wednesday, January 27, 2016 | By John Carroll
Building on years of work on developing new insulin-producing cells that could one day control glucose levels and cure diabetes, a group of investigators led by scientists at MIT and Boston Children’s Hospital say they’ve developed a promising new gel capsule that protected the cells from an immune system assault.

Dr. Jose Oberholzer, a professor of bioengineering at the University of Illinois at Chicago, tested a variety of chemically modified alginate hydrogel spheres to see which ones would be best at protecting the islet cells created from human stem cells.

The team concluded that 1.5-millimeter spheres of triazole-thiomorphine dioxide (TMTD) alginate were best at protecting the cells and allowing insulin to seep out without spurring an errant immune system attack or the development of scar tissue–two key threats to making this work in humans.

They maintained healthy glucose levels in the rodents for 174 days, the equivalent to decades for humans.

“While this is a very promising step towards an eventual cure for diabetes, a lot more testing is needed to ensure that the islet cells don’t de-differentiate back toward their stem-cell states or become cancerous,” said Oberholzer.

Millions of diabetics have effectively controlled the chronic disease with existing therapies, but there’s still a huge unmet medical need to consider. While diabetes companies like Novo ($NVO) like to cite the fact that a third of diabetics have the disease under control, a third are on meds but don’t control it well and a third haven’t been diagnosed. An actual cure for the disease, which has been growing by leaps and bounds all over the world, would be revolutionary.

Their study was published in Nature Medicine.

– here’s the release
– get the journal abstract


Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

Arturo J Vegas, Omid Veiseh, Mads Gürtler,…, Robert Langer & Daniel G Anderson

Nature Medicine (2016)   http://dx.doi.org:/10.1038/nm.4030

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy4. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier56. However, clinical implementation has been challenging because of host immune responses to the implant materials7. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Subject terms: Regenerative medicine  Type 1 diabetes

Figure 1: SC-β cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.close

(a) Top, schematic representation of the last three stages of differentiation of human embryonic stem cells to SC-β cells. Stage 4 cells (pancreatic progenitors 2) co-express pancreatic and duodenal homeobox 1 (PDX-1) and NK6 homeobox 1…


Potential Cure for Diabetes Discovered  
http://www.rdmag.com/news/2016/01/potential-cure-diabetes-discovered   01/27/2016

Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.  See —

Bubble Technique Could Create Type 1 Diabetes Therapy


Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.

Previous treatments for this disease have involved injecting beta cells from dead donors into patients to help their pancreas generate healthy-insulin cells, writes STAT. However, this method has resulted in the immune system targeting these new cells as “foreign” so transplant recipients have had to take immune-suppressing medications for the rest of their lives.

The first paper published in the journal Nature Biotechnology explained how scientists analyzed a seaweed extract called alginate to gauge its effectiveness in supporting the flow of sugar and insulin between cells and the body. An estimated 774 variations were tested in mice and monkeys in which results indicated only a handful could reduce the body’s response to foreign invaders, explains STAT.

The other paper in the journal Nature Medicine detailed a process where scientists developed small capsules infused with alginate and embryonic stem cells. A six-month observation period revealed this “protective bubble” technique “began to produce insulin in response to blood glucose levels” after transplantation in mice subjects with a condition similar to type 1 diabetes, reports Gizmodo.

Essentially, this cured the mice of their diabetes, and the beta cells worked as well as the body’s own cells, according to the researchers. Human trials could still be a few years away, but this experiment could yield a safer alternative to insulin injections.


Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

Arturo J Vegas, Omid Veiseh, Joshua C Doloff, et al.

Nature Biotechnology (2016)    http://dx.doi.org:/10.1038/nbt.3462

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1, 2, 3, 4, 5, 6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.


Video 1: Intravital imaging of 300 μm SLG20 microcapsules.

Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules.

Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.


Clinical Focus on Follicular Lymphoma: CAR T-Cells Active in Relapsed Blood Cancers

MedPage Today

CAR T-Cells Active in Relapsed Blood Cancers

Complete responses in half of patients

by Charles Bankhead

Patients with relapsed and refractory B-cell malignancies have responded to treatment with modified T-cells added to conventional chemotherapy, data from an ongoing Swedish study showed.

Six of the first 11 evaluable patients achieved complete responses with increasing doses of chimeric antigen receptor (CAR)-modified T-cells that target the CD19 antigen, although two subsequently relapsed.

Five of the six responding patients received preconditioning chemotherapy the day before CAR T-cell infusion, in addition to chemotherapy administered up to 90 days before T-cell infusion to reduce tumor-cell burden. The remaining five patients received only the earlier chemotherapy, according to a presentation at the inaugural International Cancer Immunotherapy Conference in New York City.

“The complete responses in lymphoma patients despite the fact that they received only low doses of preconditioning compared with other published data surprised us,” Angelica Loskog, PhD, of Uppsala University in Sweden, said in a statement. “The strategy of both providing tumor-reductive chemotherapy for weeks prior to CAR T-cell infusion combined with preconditioning just before CAR T-cell infusion seems to offer promise.

CAR T-cells have demonstrated activity in a variety of studies involving patients with B-cell malignancies. Much of the work has focused on patients with leukemia, including trials in the U.S. B-cell lymphomas have proven more difficult to treat with CAR T-cells because the diseases are associated with higher concentration of immunosuppressive cells that can inhibit CAR T-cell activity, said Loskog. Moreover, blood-vessel abnormalities and accumulation of fibrotic tissue can hinder tumor penetration by therapeutic T-cells.

Each laboratory has its own process for modifying T-cells. Loskog and colleagues in Sweden and at Baylor College of Medicine in Houston have developed third-generation CAR T-cells that contain signaling domains for CD28 and 4-1BB, which act as co-stimulatory molecules. In preclinical models, third-generation CAR T-cells have demonstrated increased activation and proliferation in response to antigen challenge. Additionally, they have chosen to experiment with tumor burden-reducing chemotherapy, a preconditioning chemotherapy to counter the higher immunosuppressive cell count in lymphoma patients.

Loskog reported details of an ongoing phase I/IIa clinical trial involving patients with relapsed or refractory CD19-positive B-cell malignancies. Altogether, investigators have treated 12 patients with increasing doses (2 x 107 to 2 x 108 cells/m2) of CAR T-cells. One patient (with mixed follicular/Burkitt lymphoma) has yet to be evaluated for response. The remaining 11 included three patients with diffuse large B-cell lymphoma (DLBCL), one with follicular lymphoma transformed to DLBCL, two with chronic lymphocytic leukemia, two with mantle cell lymphoma, and three with acute lymphoblastic leukemia.

All of the patients with lymphoma received standard tumor cell-reducing chemotherapy, beginning 3 to 90 days before administration of CAR T-cells. Beginning with the sixth patient in the cohort, patients also received preconditioning chemotherapy (cyclophosphamide/fludarabine) 1 to 2 days before T-cell infusion to reduce the number and activity of immunosuppressive cells.

Cytokine release syndrome is a common effect of CAR T-cell therapy and occurred in several patients treated. In general, the syndrome has been manageable and has not interfered with treatment or response to the modified T-cells.

On the basis of the data produced thus far, the investigators have proceeded with patient evaluation and enrollment. They have already begun cell production for the next patient that will be treated with autologous CAR T-cells.

Although laboratories have their own cell production techniques, the treatment strategy has broad applicability to the treatment of B-cell malignancies, said Loskog.

“The results using different CARs and different techniques for manufacturing them is very similar in the clinic, in terms of initial complete response,” she told MedPage Today. “By using 4-1BB as a co-stimulator in the CAR intracellular region, it seems possible to achieve long-term complete responses in some patients. However, preconditioning of the patients with chemotherapy to reduce the regulatory immune cells seems crucial for effect.”

In an effort to manage the effect of patients’ immunosuppressive cells, the investigators have begun studying each the immune profile before and after treatment. Preliminary results suggest that the population of immunosuppressive cells increases over time, which has the potential to interfere with CAR T-cell responses.

“Especially for lymphoma, it may be crucial to deplete such cells prior to CAR infusion,” said Loskog. “It may even be necessary with supportive treatment for some time after CAR T-cell infusion. A supportive treatment needs to specifically regulate the suppressive cells while sparing the effect of CARs.”

The immunotherapy conference is jointly sponsored by the American Association for Cancer Research, the Cancer Research Institute, the Association for Cancer Immunotherapy, and the European Academy of Tumor Immunology.


PET-CT Best for FL Response Assessment

PET-CT associated with better progression-free and overall survival rates in follicular lymphoma.

Kay Jackson

PET-CT (PET) rather than contrast-enhanced CT scanning should be considered the new gold standard for response assessment after first-line rituximab therapy for high-tumor burden follicular lymphoma (FL), a pooled analysis of a central review in three multicenter studies indicated.

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Cancer Companion Diagnostics

Curator: Larry H. Bernstein, MD, FCAP


Companion Diagnostics for Cancer: Will NGS Play a Role?

Patricia Fitzpatrick Dimond, Ph.D.


Companion diagnostics (CDx), in vitro diagnostic devices or imaging tools that provide information essential to the safe and effective use of a corresponding therapeutic product, have become indispensable tools for oncologists.  As a result, analysts expect the global CDx market to reach $8.73 billion by 2019, up from from $3.14 billion in 2014.

Use of CDx during a clinical trial to guide therapy can improve treatment responses and patient outcomes by identifying and predicting patient subpopulations most likely to respond to a given treatment.

These tests not only indicate the presence of a molecular target, but can also reveal the off-target effects of a therapeutic, predicting toxicities and adverse effects associated with a drug.

For pharma manufacturers, using CDx during drug development improves the success rate of drugs being tested in clinical trials. In a study estimating the risk of clinical trial failure during non-small cell lung cancer drug development in the period between 1998 and 2012 investigators analyzed trial data from 676 clinical trials with 199 unique drug compounds.

The data showed that Phase III trial failure proved the biggest obstacle to drug approval, with an overall success rate of only 28%. But in biomarker-guided trials, the success rate reached 62%. The investigators concluded from their data analysis that the use of a CDx assay during Phase III drug development substantially improves a drug’s chances of clinical success.

The Regulatory Perspective

According to Patricia Keegen, M.D., supervisory medical officer in the FDA’s Division of Oncology Products II, the agency requires a companion diagnostic test if a new drug works on a specific genetic or biological target that is present in some, but not all, patients with a certain cancer or disease. The test identifies individuals who would benefit from the treatment, and may identify patients who would not benefit but could also be harmed by use of a certain drug for treatment of their disease. The agency classifies companion diagnosis as Class III devices, a class of devices requiring the most stringent approval for medical devices by the FDA, a Premarket Approval Application (PMA).

On August 6, 2014, the FDA finalized its long-awaited “Guidance for Industry and FDA Staff: In Vitro Companion Diagnostic Devices,” originally issued in July 2011. The final guidance stipulates that FDA generally will not approve any therapeutic product that requires an IVD companion diagnostic device for its safe and effective use before the IVD companion diagnostic device is approved or cleared for that indication.

Close collaboration between drug developers and diagnostics companies has been a key driver in recent simultaneous pharmaceutical-CDx FDA approvals, and partnerships between in vitro diagnostics (IVD) companies have proliferated as a result.  Major test developers include Roche Diagnostics, Abbott Laboratories, Agilent Technologies, QIAGEN), Thermo Fisher Scientific, and Myriad Genetics.

But an NGS-based test has yet to make it to market as a CDx for cancer.  All approved tests include PCR–based tests, immunohistochemistry, and in situ hybridization technology.  And despite the very recent decision by the FDA to grant marketing authorization for Illumina’s MiSeqDx instrument platform for screening and diagnosis of cystic fibrosis, “There still seems to be a number of challenges that must be overcome before we see NGS for targeted cancer drugs,” commented Jan Trøst Jørgensen, a consultant to DAKO, commenting on presentations at the European Symposium of Biopathology in June 2013.

Illumina received premarket clearance from the FDA for its MiSeqDx system, two cystic fibrosis assays, and a library prep kit that enables laboratories to develop their own diagnostic test. The designation marked the first time a next-generation sequencing system received FDA premarket clearance. The FDA reviewed the Illumina MiSeqDx instrument platform through its de novo classification process, a regulatory pathway for some novel low-to-moderate risk medical devices that are not substantially equivalent to an already legally marketed device.

Dr. Jørgensen further noted that “We are slowly moving away from the ‘one biomarker: one drug’ scenario, which has characterized the first decades of targeted cancer drug development, toward a more integrated approach with multiple biomarkers and drugs. This ‘new paradigm’ will likely pave the way for the introduction of multiplexing strategies in the clinic using gene expression arrays and next-generation sequencing.”

The future of CDxs therefore may be heading in the same direction as cancer therapy, aimed at staying ahead of the tumor drug resistance curve, and acknowledging the reality of the shifting genomic landscape of individual tumors. In some cases, NGS will be applied to diseases for which a non-sequencing CDx has already been approved.

Illumina believes that NGS presents an ideal solution to transforming the tumor profiling paradigm from a series of single gene tests to a multi-analyte approach to delivering precision oncology. Mya Thomae, Illumina’s vice president, regulatory affairs, said in a statement that Illumina has formed partnerships with several drug companies to develop a universal next-generation sequencing-based oncology test system. The collaborations with AstraZeneca, Janssen, Sanofi, and Merck-Serono, announced in 2014 and 2015 respectively, seek to  “redefine companion diagnostics for oncology  focused on developing a system for use in targeted therapy clinical trials with a goal of developing and commercializing a multigene panel for therapeutic selection.”

On January 16, 2014 Illumina and Amgen announced that they would collaborate on the development of a next-generation sequencing-based companion diagnostic for colorectal cancer antibody Vectibix (panitumumab). Illumina will develop the companion test on its MiSeqDx instrument.

In 2012, the agency approved Qiagen’s Therascreen KRAS RGQ PCR Kit to identify best responders to Erbitux (cetuximab), another antibody drug in the same class as Vectibix. The label for Vectibix, an EGFR-inhibiting monoclonal antibody, restricts the use of the drug for those metastatic colorectal cancer patients who harbor KRAS mutations or whose KRAS status is unknown.

The U.S. FDA, Illumina said, hasn’t yet approved a companion diagnostic that gauges KRAS mutation status specifically in those considering treatment with Vectibix.  Illumina plans to gain regulatory approval in the U.S. and in Europe for an NGS-based companion test that can identify patients’ RAS mutation status. Illumina and Amgen will validate the test platform and Illumina will commercialize the test.

Treatment Options

Foundation Medicine says its approach to cancer genomic characterization will help physicians reveal the alterations driving the growth of a patient’s cancer and identify targeted treatment options that may not have been otherwise considered.

FoundationOne, the first clinical product from Foundation Medicine, interrogates the entire coding sequence of 315 cancer-related genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers.  Based on current scientific and clinical literature, these genes are known to be somatically altered in solid cancers.

These genes, the company says, are sequenced at great depth to identify the relevant, actionable somatic alterations, including single base pair change, insertions, deletions, copy number alterations, and selected fusions. The resultant fully informative genomic profile complements traditional cancer treatment decision tools and often expands treatment options by matching each patient with targeted therapies and clinical trials relevant to the molecular changes in their tumors.

As Foundation Medicine’ s NGS analyses are increasingly applied, recent clinical reports describe instances in which comprehensive genomic profiling with the FoundationOne NGS-based assay result in diagnostic reclassification that can lead to targeted drug therapy with a resulting dramatic clinical response. In several reported instances, NGS found, among the spectrum of aberrations that occur in tumors, changes unlikely to have been discovered by other means, and clearly outside the range of a conventional CDx that matches one drug to a specific genetic change.

TRK Fusion Cancer

In July 2015, the University of Colorado Cancer Center and Loxo Oncology published a research brief in the online edition of Cancer Discovery describing the first patient with a tropomyosin receptor kinase (TRK) fusion cancer enrolled in a LOXO-101 Phase I trial. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors.

While the authors say TRK fusions occur rarely, they occur in a diverse spectrum of tumor histologies. The research brief described a patient with advanced soft tissue sarcoma widely metastatic to the lungs. The patient’s physician submitted a tumor specimen to Foundation Medicine for comprehensive genomic profiling with FoundationOne Heme, where her cancer was demonstrated to harbor a TRK gene fusion.

Following multiple unsuccessful courses of treatment, the patient was enrolled in the Phase I trial of LOXO-101 in March 2015. After four months of treatment, CT scans demonstrated almost complete tumor disappearance of the largest tumors.

The FDA’s Elizabeth Mansfield, Ph.D., director, personalized medicine staff, Office of In Vitro Diagnostics and Radiological Health, said in a recent article,  “FDA Perspective on Companion Diagnostics: An Evolving Paradigm” that “even as it seems that many questions about co-development have been resolved, the rapid accumulation of new knowledge about tumor biology and the rapid evolution of diagnostic technology are challenging FDA to continually redefine its thinking on companion diagnostics.” It seems almost inevitable that a consolidation of diagnostic testing should take place, to enable a single test or a few tests to garner all the necessary information for therapeutic decision making.”

Whether this means CDx testing will begin to incorporate NGS sequencing remains to be seen.

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Low Energy Photon Intra-Operative Radiotherapy System

Larry H. Bernstein, MD, FCAP, Curator



The Dosimetric Characteristics and Potential Limitation in Clinical Application of a Low Energy Photon Intra-Operative Radiotherapy System

Spring Zhou Editor at Scientific Research Publishing        https://www.linkedin.com/grp/post/143951-6069684489951391748


Purpose: To investigate the dosimetric characteristics of a low energy photon intra-operative radiotherapy (IORT) system and explore its potential limitation in clinical application.

Methods: A special water phantom, a parallel-plate ionization chamber and an electrometer were used to measure the depth dose rate, isotropy of dose distribution in X/Y plane, dosimetry reproducibility of bare probe and spherical applicators of different size which were used in comparison with the system data.

Results: The difference in depth dose rate between the measurement and system data for bare probe is -2.16% ± 1.36%, the range of the relative deviation for isotropy in the X/Y plane is between -1.9% and 2.1%. The difference in depth dose rate, transfer coefficient, isotropy in X/Y plane between the measurement and system data for the whole set of spherical applicators is -10.0% – 2.3%, -8.9% – 4.2% and -1.6% – 2.6%, respectively. Higher surface dose rate and steeper gradient depth dose are observed in smaller spherical applicators. The depth dose rate and isotropy for bare probe and spherical applicators have been shown good reproducibility. The uncertainty of measurement is associated with the positioning accuracy, energy response, noise current and correction function f’(R).

Conclusions: Thorough commissioning of the low energy photon IORT system helps us better understand the dosimetry characteristics, verify the system data, obtain adequate data for clinical application and routine quality assurance. The steep gradient depth dose and limited treatment range may restrain its potential in clinical application.



The Dosimetric Characteristics and Potential Limitation in Clinical Application of a Low Energy Photon Intra-Operative Radiotherapy System

IJMPCERO> Vol.4 No.2, May 2015    DOI: 10.4236/ijmpcero.2015.42023

Zhenhua Xiao, Ouyang Bin, Zhenyu Wang, Botian Huang, Bixiu Wen*


Cite this paper

Xiao, Z. , Bin, O. , Wang, Z. , Huang, B. and Wen, B. (2015) The Dosimetric Characteristics and Potential Limitation in Clinical Application of a Low Energy Photon Intra-Operative Radiotherapy System. International Journal of Medical Physics, Clinical Engineering and Radiation Oncology, 4, 184-195. doi:10.4236/ijmpcero.2015.42023.
Intra-operative radiotherapy (IORT) delivers single high dose radiation directly to the tumor bed within a relative short period of treatment time during the surgical operation, which requires a higher surface dose to protect the deep normal tissues. It often uses high energy electron beam or low energy photon beam. Modern IORT is usually delivered in the operating room, which requires that the device is light weighted and easy to move with high quality of radiation protection. Dedicated IORT device commercially available includes Mobetron® MeV electron beam system [1] (Intra Op Medical Corporation, California, USA), NOVAC™ 7 system (New Radiant Technology SpA, Italy) and the INTRABEAM® 50 kV X-ray device (Carl Zeiss Medical Company, Germany).
Since the results from the randomized TARGIT A trial were published, IORT has been applied for locally advanced or recurrent rectal cancer [2], superficial cutaneous malignancies [3] and as full dose partial breast irradiation (PBI) or as a boost after whole-breast radiatherapy (WBI) in early stage breast cancer [4] [5]. Vaidya J.S. et al. have analysis overall survival of using as single-dose targeted intraoperative radiotherpay (TARGIT) versus fractionated external beam radiotherapy (EBRT) for breast cancer. For patients enrolled at 33 centers in 11 countries, 1721 patients were randomised to TARGIT and 1730 to EBRT. The 5-year risk for local recurrence in the conserved breast was 3.3% (95% CI 2.1 – 5.1) for TARGIT versus 1.3% (0.7 – 2.5) for EBRT (p = 0.042). Wound-related complications were much the same between groups but grade 3 or 4 skin complications were significantly reduced with TARGIT (four of 1720 vs 13 of 1731, p = 0.029) [6]. TARGIT-B (for boost, ISRCTN43138042) is an ongoing multicenter randomised controlled trial that began in 2013, which is testing the replacement of EBRT boost to the tumor bed by a TARGIT boost given during surgery. An ongoing open registry study, TARGIT-R (for registry, ISRCTN91179875), began in 2013, aiming to monitor the long-term effectiveness and safety of the patients treated with TARGIT following breast conserving surgery for early breast cancer [7].
Eaton D.J. et al. described the dosimetry of the INTRABEAM® system with the spherical applicators [8]. Maxime Goubert et al. have reported dosimetric behavior of the system equipped with flat and surface applicators [3]. There still remains controversary for the dosimetric characteristics and clinical application of IORT. In this paper we introduce the commissioning test of the INTRABEAM® 50 kV x-ray system by investigating the dosimetric characteristics of the low energy photon IORT system and explore its potential limitation in clinical application.
The device for measurement includes a dedicated water tank (see in Figure 1) with radiation protection design (Carl Zeiss Surgical GmbH), a parallel plate ionization chamber (volume: 0.0053 cm3, type 34013, PTW, Freiburg, Germany) and a dosimeter (UNIDOS E, PTW). Two waterproof measuring chambers in the water tank were designed to measure depth dose rate and isotropy respectively. The ionization chamber(IC) is inserted with the ionization holder into the measuring chamber, which is closed with cover during measurement. “dIC” is designated as “distance between the entrance foil and chamber top” in PTW calibration certificate. The thickness of measuring chamber was printed in the user manual as “dH”. The air gap “dA” between the upper surface of ionization chamber housing and the inside of measuring chamber wall is constantly 0.5 mm. Those distances are considered in measuring the depth dose rate at particular position. The XRS is fixed into the platform which can be adjusted vertically with range of 10 cm and precision of 0.001 mm. The lowest surface of the probe was defined as the isocenter of 0 mm in depth. Due to the certain thickness of the holder wall of the ionization chamber, the measurement in Z direction ranges from 2.0 mm to 44.0 mm with 0.5 mm increment when the distance is less than 40.0 mm, 1.0 mm for 40.0 mm or higher with a period of 1 minute for each point of measurement. The platform of the water tank is also designed with a turntable structure in the X/Y plane for measuring the isotropic dose distribution. The XRS can be rotated with the platform every 45˚ to the total of 8 angles (initial position was defined as 0˚ during measurements). The X, Y directions can be located by fine tune to align the central axis of the bare source probe with the center of the ionization chamber. The position of X, Y directions were kept unchanged for isotropic measurement.
3.1. Depth Dose Rate of Bare Probe The depth dose rate for the bare probe was measured three times. Figure 2 depicts the dose rate in Gy/min or error of measurement in percentage as a function of distance between the surface of XRS probe and the ionization chamber monitor-node. The red line represents the average measurement for three times, the data in blue line are obtained from the operation system and the repeatability of the measurements for 3 times are shown in percentage of error in black line. As shown in Figure 2(a) & Figure 2(b), the maximum deviation of three times measurement for bare probe ranges from 0.14% to 1.3%. The value of dose rate measured is higher than that obtained from the operation system when the distance between the surface of XRS probe to the ionization chamber monitor-node is <5.0 mm; the value measured is lower than that from the operation system when the distance is 10.0 mm or more. The average error between the actual measurement and the system is −2.16% ± 1.36% ranging from −3.65% to 2.83%.
3.2. Isotropy of Bare Probe Figure 3(a) depicts isotropic dose in X/Y plane for bare probe, the value of each angle was normalized to the average of 8 measurements at the specific distance. The consistency for the value of each measurement angle is relatively good. As shown in Figure 3(b), the relative deviation of measurements for four times ranged from −1.9% to 2.1%. The deviation of measurement at 90˚ (−1.9%) and 225˚ (2.1%) were relatively larger. The tendency of the measuring error is consistent with good repeatability.
Figure 1. Schematic diagram of commissioning setup for a spherical applicator of 4.5 cm in diameter (water tank).
Figure 2. The depth dose rate (a) and the error in percentage; (b) measured in comparison with data from operation system for bare probe. The red line represents the average of measurement for three times, blue line depicts the data from the operation system, the black line for the deviation of measurement with bare probe in percentage error.
Figure 3. Isotrophy in X/Y plane for bare probe. (a) Normalized value and relative deviation in 8 measurement angles. The hollow circle represents the normalized value, and solid dots the relative percentage deviation of each measurement point magnified 20 times; (b) Relative percentage deviation for 4 times measurement.
3.3. Depth Dose Rate of Spherical Applicators Figure 4(a) depicts the curve of dose rate measured as a function of the distance from the surface of spherical applicators to ionization chamber monitor-node for 8 types of spherical applicators. The dose rate is the highest at the surface of the applicators which falls as the distance between the surfaces of spherical applicators to ionization chamber monitor-node increases. The dose rate varies significantly with the size of the applicator. For the applicator with smaller diameter the dose rate is higher with relative shorter treatment time if the same radiation dose is delivered. As the diameter of the applicator increases, the falling gradient in dose rate slows down. From Figure 4(a), we also observe that the curves of the dose rate overlaps for applicators in diameters 2.5 cm and 3.5 cm, 3.0 cm and 4.0 cm, respectively.
Figure 4(b) shows the curve of depth dose rate as a function of distance between the isocenter of spherical applicator and ionization chamber monitor-node. Obvious separation of dose rate was observed. At the same distance from the isocenter, the dose rate is smaller in the applicators of diameter ≤3.0 cm than those of diameter >3.0 cm. The difference in depth dose rate, transfer coefficient and isotropy between measurements and system data is presented in Table 1. The average deviation in depth dose rate between measurements and system data for the applicators of diameter ≤3.0 cm is in the range from −5.1% to −4.8%; it reduces to −2% – −0.1% with the increase in diameter of the applicators.
Figure 4. The curve of depth dose rate measured for each spherical applicator. (a) The curve of depth dose rate of different distance from the surface of spherical applicators to ionization chamber monitor-node; (b) The curve of depth dose rate of different distance from the isocenter of spherical applicator to ionization chamber monitor-node.
Table 1. The difference in depth dose rate, transfer coefficient and isotropy for spherical applicators.
Diameters (cm)   ……
Deviation of depth dose rate (range) (%) ……
Deviation of transfer coefficiency (range) (%) …….
Isotropy (range) (%) …….
3.4. Transfer Coefficient of Spherical Applicators The transfer coefficient of spherical applicator is defined as ratio between the depth dose rates with or without applicator at the same distance to the source isocenter. The depth dose rate can be obtained by multiplication of the bare source dose rate and the conversion coefficient. Figure 5 is the curve for deviation in transfer coefficient between measured values and the system data for spherical applicators. The average deviation of transfer coefficient at the same distance between the isocenter of spherical applicator and ionization chamber monitor-node for applicators ≤3.0 cm ranges from −2.6% to −2.2%; whereas the average deviation for tors >3.0 cm is between 0.8% and 2.4%. The more detailed data for difference in transfer coefficient between measurements and system data are presented in Table 1 for each individualized applicator.
3.5. Isotropy of Spherical Applicator As shown in Table 1, the average deviation in isotropy between measurements and system data of different spherical applicator’s X/Y plane ranges from −1.4% to 2.6% which shows no obvious change as the diameter of applicator increases. Figure 6(a) & Figure 6(b) depicts the representative isotropy for dose distribution in X/Y plane for a spherical applicator of diameter 4.5 cm which shows the consistency of the measurement values.
Figure 5. The curve for deviation in transfer coefficient between measured values and the system data for spherical applicators.
Figure 6. The representative isotropy in X/Y plane for a ϕ 4.5 cm spherical applicator. (a) Point view of normalized value and the relative deviation; (b) The percentage relative deviation of 3 times measurements.
3.6. Repeatability of Spherical Applicator The Dose rate was measured in 3 times for 4.5 cm spherical applicator, the deviation in repeatability ranges from 0.2% to 0.7%, which shows good repeatability and it gradually gets better as the depth increases. As shown in Figure 7(a) & Figure 7(b), poorer repeatability and increased deviation of error are observed when the distance >15 mm from the surface.
Figure 7. The curve for measurement value of depth dose rate and comparison chart with system data for ϕ 4.5 cm spherical applicator. (a) The depth dose rate for ϕ 4.5 cm spherical applicator. The red line intends for the average 3 measurement values, the blue line for the system value; (b)The error in percentage for the measurements in 3 times.
3.7. Potential Limitation of Clinical Application Figure 8 depicts the graphic of dose distribution for the spherical applicator in diameter 4.5 cm. The doses measured at the distance of 0.2, 0.5, 1.0, and 2.0 cm are 15.3, 10.7, 6.4 and 2.7 Gy, respectively when surface dose of 20 Gy is prescribed. Table 2 lists the depth dose value for spherical applicators when the surface dose of 20 Gy is prescribed, which shows fast dose falling with gradual increasing depth. The doses range from 35.0% to 56.0% at 5 mm from the surface of the spherical applicators and fall to 16.5% – 34.0% at 10 mm and 5.5% – 15.5% at 20 mm.
Figure 8. Dose distribution for a ϕ 4.5 cm spherical applicator when the surface dose of 20 Gy is prescribed.
Table 2. Depth dose rate (Gy) at serial typical depths for spherical applicators when surface dose of 20 Gy is prescribed.
Diameter (cm)
Distance from surface (mm) 0 2 5 10 15 20
Dose measurement tools used for INTRABEAM system include water tank/ionization chamber [10] [11], film/ solid water phantom [11], Thermo-luminescence [12], etc. Water tank/ionization chamber has the highest accuracyamong those instruments [13]. Schneider et al. have compared homogeneity of each dose distribution and depth-dose measurements for flat and surface applicators using film dosimetry in a solid water phantomand a soft X-ray ionization chamber in a water tank [10]. One of the important factors influencing the accuracy of the measurement is the relative position error of X-ray source to ionization chamber due to sharp attenuation of low energy X-ray in the water. The minimal position change will lead to relatively large measurement deviation. As shown in depth dose rate curve fitting function and differential coefficient data, the dose gradient at 3 and 10 mm from the source isocenteris 60%/mm and 24%/mm, respectively. The position error of ±0.1 mm will lead to deviation of dose rate at ±6% and ±2.4%, respectively.
Eaton et al. have reported that the positioning of X-ray source is the most important factor that affects the measurement result and the measurement accuracy may be affected by the ionization chamber’s volume effects in the area of steep dose falling, which may account for the poorer repeatability of measurement near the isocenter [13]. Other factors influence the accurate measurement including the energy response of ionization chamber [13], low voltage of X-ray [14], etc. The spectrum of X-ray approaching to the surface of the probe is complex ranging from 0 to 50 kV with a large number of low energy kV X-ray [14] (<20 kV) which showed rapid attenuation in water [15] resulting in significant difference at different depth and the measurement deviation could reach to ±2.2% due to the energy response [9].
Another important reason for poor repeatability is that the measurement can be affected by noise current. The sensitive volume of ionization chamber is only 0.0053 cm3, and the ionization chamber is supposed to collect only approximately 25 pC charge per minute at the depth of 35 mm during the measurement of X-ray source, which has led to low signal-to-noise ratio influenced by noise current. The uncertainty of measurement for ionization chamber could reach to ±3.4% [9]. The accuracy and repeatability of measurement will gradually decrease with further increase in the distance from the X-ray source since ionization chamber collects less charge per minute (about 16.5 pC).
The type of ionization chamber, design of measuring chamber for water tank, method for calculation and condition of acquisition system for absorbed dose are different between users and company system. The calculation of absorbed doses, transfer coefficient and isotropy in X/Y plane for the measurement data requires the manufacturer to provide the correction function f'(R) for bare probes and spherical applicators of different size. The f'(R) value is different at different depth which may introduce correction error, the error for uncertainty of f'(R) is relatively larger when the depth is shallower. The average of uncertainty for f'(R) is ±7.8%.
Armoogum et al. analyzed the factors that affect the measurement result including temperature and atmospheric pressure correction factor, ionization chamber position deviation, ionization chamber current, the chamber/dosimeter calibration factor, output drift and calibration of absorbed dose [9]. Among these factors positional deviation is the most significant one. The estimation of total uncertainty for all these factors can reach to ±10.8%. Our data have shown that deviation between the measurement data and the system ranges from −10.0% to −5.0% for spherical applicator ≤3 cm in diameter and within ±5.0% for the applicators>3 cm in diameter, which are comparable with the data reported in literatures [9].
Our data have also shown a relative larger deviation of isotropy in X/Y plane measured at 90˚ (1.9%) and 225˚ (2.1%). The possible reasons include: 1) the probe is a hollow needle with 100 mm long and 3.2 mm outside diameter which may be bended during the operation with need to be calibrated before each use since system requirements of probe bending value is less than 0.1 mm; 2) there may exist certain errors during the rotation of the water tank platform, which may lead to different distance from the tip of the probe to the ionization chamber monitor units in different angles; 3) the noise current may influence the correct measurements (the dosimeter charge is about 16.5 pC/min without applicators).
The clinical application of IORT with INTRABEAM system is determined by the size and category of applicators. The biggest diameter for spherical and tablet applicators is 5.0 cm and 6.0 cm, respectively. The surrounding wall is embedded with thin metal sheath interiorly for spherical applicators of diameter ≤3 cm. Low energy X-ray can be attenuated rapidly through the metal sheath. The gradient of dose rate value for spherical applicators in diameter ≤3 cm were larger than that for applicators with diameter >3cm when measured at the same distance from the isocenter.
Our results have shown that the steep dose gradient exists from 0 cm, the surface of the applicator to 1.0 cm; the higher surface dose rate and the greater dose gradient have been observed for the smaller applicators. When the region of treatment is too large, it will be very difficult to calculate accurate dose distribution due to lack of beam bridging technology with over- or under-dose. The limited region and depth of treatment may restrain from its potential in clinical application. Vaidya JS et al. have reported in TARGIT A, a phase III clinical trial that for patients with early breast cancer <2 cm the tumor local control rate of IORT is not inferior to external beam radiotherapy after breast conserving surgery; external beam radiotherapy should be considered for patients with tumor of 2 – 3 cm or with poorer prognostic factors [6]. Sperk E et al. have reported that patient selection for IORT should be restrictive when provided as accelerated partial breast irradiation (APBI) [4].
The system only uses water for dose calculation whereas homogeneity of human structure cannot be revised. The calculated dose value and the deviation will be very big in different tissue due to the spectrum chacteristics of the low energy photon (≤50 kV); the dose distribution will be influenced by the air gap between tissues and the applicator [14]. Monte Carlo modelling could be used in comparison with result from ionization chambers, radiochromic film and other dosimeters such as TLDs on the subsequent periodic QA tests for the INTRABEAM system [8]. There exist great difficulties for external beam radiotherapy when needed to supplement the postoperative radiotherapy especially for the accurate dose calculation and precise delineation of target and surrounding normal tissue irradiated especially for the nerves and blood vessels. All these factors mentioned above may seriously restrict INTRABEAM system to be widely used in clinical practice.
5. Conclusion In summary, thorough commissioning of INTRABEAM system helps us better understand the dosimetry characteristics, verify the system data and a quire adequate data for clinical application and routine quality assurance. It is necessary to establish the benchmark for long term quality assurance based on the measurement data. The characteristics of high dose at the surface of applicator, great dose gradient and limited treatment range may restrain from its potential in wide clinical application


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[2] Klink, C.D., Binnebosel, M., Holy, R., Neumann, U.P. and Junge, K. (2014) Influence of Intraoperative Radiotherapy (IORT) on Perioperative Outcome after Surgical Resection of Rectal Cancer. World Journal of Surgery, 38, 992-996.    http://dx.doi.org/10.1007/s00268-013-2313-1
[3] Goubert, M. and Parent, L. (2015) Dosimetric Characterization of INTRABEAM((R)) Miniature Accelerator Flat and Surface Applicators for Dermatologic Applications. Physica Medica, 31, 224-232. http://dx.doi.org/10.1016/j.ejmp.2015.01.009
[4] Sperk, E., Astor, D., Keller, A., Welzel, G., Gerhardt, A., Tuschy, B., et al. (2014) A Cohort Analysis to Identify Eligible Patients for Intraoperative Radiotherapy (IORT) of Early Breast Cancer. Radiation Oncology, 9, 154.
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[6] Vaidya, J.S., Wenz, F., Bulsara, M., Tobias, J.S., Joseph, D.J., Keshtgar, M., et al. (2014) Risk-Adapted Targeted Intraoperative Radiotherapy versus Whole-Breast Radiotherapy for Breast Cancer: 5-Year Results for Local Control and Overall Survival from the TARGIT—A Randomised Trial. Lancet, 383, 603-613.    http://dx.doi.org/10.1016/S0140-6736(13)61950-9
[7] Williams, N.R., Pigott, K.H., Brew-Graves, C. and Keshtgar, M.R. (2014) Intraoperative Radiotherapy for Breast Cancer. Gland Surgery, 3, 109-119.
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[9] Armoogum, K.S., Parry, J.M., Souliman, S.K., Sutton, D.G. and Mackay, C.D. (2007) Functional Intercomparison of Intraoperative Radiotherapy Equipment—Photon Radiosurgery System. Radiation Oncology, 2, 11.
[10] Schneider, F., Clausen, S., Tholking, J., Wenz, F. and Abo-Madyan, Y. (2014) A Novel Approach for Superficial Intraoperative Radiotherapy (IORT) Using a 50 kV X-Ray Source: A Technical and Case Report. Journal of Applied Clinical Medical Physics, 15, 4502.
[11] Ebert, M.A., Asad, A.H. and Siddiqui, S.A. (2009) Suitability of Radiochromic Films for Dosimetry of Very-Low Energy X-Rays. Journal of Applied Clinical Medical Physics, 10, 2957.    http://dx.doi.org/10.1120/jacmp.v10i4.2957
[12] Soares, C., Drupieski, C., Wingert, B., Pritchett, G., Pagonis, V., O’brien, M., et al. (2006) Absorbed Dose Measurements of a Handheld 50 kVP X-Ray Source in Water with Thermoluminescence Dosemeters. Radiation Protection Dosimetry, 120, 78-82.    http://dx.doi.org/10.1093/rpd/nci622
[13] Eaton, D.J. (2012) Quality Assurance and Independent Dosimetry for an Intraoperative X-Ray Device. Medical Physics, 39, 6908-6920.   http://dx.doi.org/10.1118/1.4761865
[14] Ebert, M.A. and Carruthers, B. (2003) Dosimetric Characteristics of a Low-kV Intra-Operative X-Ray Source: Implications for Use in a Clinical Trial for Treatment of Low-Risk Breast Cancer. Medical Physics, 30, 2424-2431.
[15] Ebert, M.A., Carruthers, B., Lanzon, P.J., Haworth, A., Clarke, J., Caswell, N.M., et al. (2002) Dosimetry of a Low-kV Intra-Operative X-Ray Source Using Basic Analytical Beam Models. Australasian Physical Engineering Sciences in Medicine, 25, 119-123.   http://dx.doi.org/10.1007/BF03178772


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Dense Breast Mammogram

Larry H. Bernstein, MD, FCAP, Curator




The Problem With Mammograms



Hallie Leighton had dense breasts — a fact she discovered only in her late 30s, via a mammogram. She grew up in an Ashkenazi family in New York, pursued a career in writing and worked with organizations promoting peace between Israelis and Arabs. By 2013 she was making a documentary on her father Jan Leighton, an actor who set the record as an actor for appearing in the most roles (2,407 according to the 1985 Guinness Book of World Records). She was never able to complete it. She died that year, at the age of 42.

Every woman in Leighton’s family had breast cancer, so she began getting annual mammograms at 35 — five years earlier than the recommended age. In 2009 the results of Leighton’s mammogram came in as “negative” or “normal”; by 2013 she was bedridden, undergoing her final days of chemotherapy.

When Leighton was first diagnosed in 2010, her doctor told her, “You have breast cancer, and it was there in 2009.” The tumor in Leighton’s breast went undiscovered until it was palpable — and at that point, the cancer was already in stage 4.

Happygram,” a documentary which exposes some of the shortcomings in mammography, chronicles Leighton’s struggle with cancer and the implications of having dense breasts.

“Most women simply aren’t informed that they have dense breast tissue,” said Leighton’s best friend Julie Marron. She wrote and directed the documentary, which is currently screening at film festivals around the country.

Breast density is defined by the relative amount of fat in relation to the amount of connective and epithelial tissue (tissue that lines blood vessels and cavities). When more than 50% of breast tissue is connective and epithelial tissue, instead of fatty tissue, the breasts are considered dense. Mammography is the only way to determine breast density.

“If you have dense breasts, what looks dense on a mammogram looks the same as a cancer would look. It tends to confuse or confound the physician, and reduces the sensitivity of the mammogram,” said Gerald Kolb, founder and president of The Breast Group, which counsels clients on different technologies in breast care. “Hallie Leighton’s breasts looked like snowballs; there was no chance they were going to find anything with the mammogram.”

Forty percent of women who are screened for breast cancer have dense breast tissue. These women also account for more than 70% of all invasive cancers. “Mammograms are not very effective screening tools for these women, as they miss between 50% and 75% of all invasive cancers in dense breast tissue,” Marron said. “This is obviously a very critical issue when you are dealing with a population that is more likely to develop cancer.”

Ashkenazi women are even more at risk. They are 1.6 times more likely than the general population to have dense breast tissue, according to Kolb. Moreover, one in 40 Ashkenazi women will test positive for one or both of BRCA gene mutations responsible for breast cancer. For the general population, that number is between one in 350 and one in 800.The BRCA 1 or 2 genes don’t cause cancer, they fight cancer, Kolb says. But if the gene is mutated, the body is not as well equipped to fight the cancer.

“A woman with a BRCA mutation has a lifetime risk of around 33% to 87%, depending on the gene and mutation,” Marron said. “Compare this to a lifetime risk of 12% for developing breast cancer for the overall population.” BRCA gene mutations can be inherited from either or both parents, and therefore they can be present in men as well as in women.

Breast density and BRCA gene mutations are not directly related, but both independently present an increased susceptibility to breast cancer.

“The biggest risk is that a doctor is not going to find the cancer when it’s really small,” Kolb said. When a tumor is detected at a centimeter or smaller, there’s a 95% cure rate. But if the cancer is the size of a golf ball by the time it’s detected, Kolb says, the woman has a 60% chance of living for five years, and then her mortality increases dramatically.

The good news is that mammography isn’t the only method of detecting breast cancer; the bad news is that very few people know this. “What we’re trying to do in the density movement is give women enough information so they can ask appropriate questions of a doctor,” Kolb said.

Kolb advises high-risk women to get a genetic risk analysis, which can be performed by a genetic counselor or a radiologist. He advises getting the risk analysis as early as age 25, but doing so is a personal decision. Not every woman is emotionally prepared to know the results.

“Mammography is a starting point,” said Dr. Dennis McDonald, a California-based women’s imager. Additionally, doctors recommend that women with dense breasts get an MRI, which McDonald says is reserved for high-risk women. It’s an expensive, invasive and time-consuming procedure that requires the injection of fluid in order to read the MRI. As of yet, doctors do not know the side effects of getting an annual MRI.

“A doctor should have started [Leighton] on an MRI right away. She was high risk and they chose to just monitor with a mammogram,” Kolb said. “That’s insufficient.”

Breast ultrasound is another alternative for women with dense breast tissue. “Most of the time, breast density doesn’t present a problem [with ultrasounds],” McDonald said. Though the ultrasound is effective in detecting cancer, he says the downside is that radiologists are often not that comfortable with the technology, simply because they have little experience with it. There are also a lot of false positives, he adds, which result in unnecessary exams or biopsies.

As “Happygram” documents, informing women of their breast density and of alternatives to mammography is a highly charged political issue.

“The whole breast cancer industry has grown up around mammograms,” Marron said. “Physicians weren’t educated on [breast density], deliberately so to a certain extent, and refused to inform patients on this issue, which is really outrageous if you think about it.” Marron says that doctors are required by law and ethical guidelines to inform patients of “material” medical information. “There is no legitimate reason that women have not been informed of this information,” she noted.

After Leighton’s diagnosis, she wanted to ensure that other women didn’t suffer the same misfortune of all-too-late tumor discovery on account of dense breast tissue. She gave media interviews, lobbied in Albany and starred in “Happygram,” all the while undergoing chemotherapy. She died four months after the Breast Density Information Bill passed in New York.

The law requires that every mammography report given to a patient with dense breasts inform the patient in plain language that she has dense breast tissue and that she should talk to her physician about the possible benefits of additional screenings. In New York, the first state in the nation to pass this kind of law, at least 2,500 women with dense breasts and invasive breast cancer received “normal” or “negative” results on their mammograms.

Similar legislation has been passed in more than 20 states throughout the country, but not without objection. Many well-intentioned radiologists, poorly informed about alternative screening options, feared that telling women the limitations of mammography would cause them to lose faith in it altogether and not get tested. Others argued that the information would make women anxious, and that it wouldn’t be fair for those who couldn’t afford additional testing. And still further arguments against informing women were possibly impacted by financial considerations, Marron added.

“Women aren’t getting the benefit of full notification across the board yet,” Marron said. “I think that has to change through education. That’s the primary reason we made this movie. There’s been so much resistance within the medical community to telling women. Change isn’t going to come from the medical community, it has to come from the patients.”

Ashkenazi women shouldn’t panic, Kolb says, but they need to carefully examine their breast density and alternative screening options: “Anytime you have a preventative tragedy like that, you have to do everything in your power to stop it from happening.”

Madison Margolin is a freelance writer based in New York. She writes frequently for the Village Voice.

Read more: http://forward.com/culture/324003/the-problem-with-mammograms/#ixzz3qufQOSmn

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developments in medical spectroscopy

Larry H. Bernstein, MD, FCAP, Curator




Using QCLs for MIR-Based Spectral Imaging — Applications in Tissue Pathology
A quantum cascade laser (QCL) microscope allows for fast data acquisition, real-time chemical imaging and the ability to collect only spectral frequencies of interest. Due to their high-quality, highly tunable illumination characteristics and excellent signal-to-noise performance, QCLs are paving the way for the next generation of mid-infrared (MIR) imaging methodologies.

Using QCLs for MIR-Based Spectral Imaging — Applications in Tissue Pathology




Efficient Spectroscopic Imaging Demonstrated In Vivo
Although optical spectroscopy is routinely used study molecules in cell samples, it is currently not practical to perform in vivo. Now, a converted Raman spectroscopy system has been used to reveal the chemical composition of living tissues in seconds.

Efficient Spectroscopic Imaging Demonstrated In Vivo




Broadband Laser Aimed at Cancer Detection
Covering a wide swath of the mid-infrared region, a new laser system offers greater spectral sensitivity.

Broadband Laser Aimed at Cancer Detection




Using QCLs for MIR-Based Spectral Imaging — Applications in Tissue Pathology

A quantum cascade laser (QCL) microscope allows for fast data acquisition, real-time chemical imaging and the ability to collect only spectral frequencies of interest. Due to their high-quality, highly tunable illumination characteristics and excellent signal-to-noise performance, QCLs are paving the way for the next generation of mid-infrared (MIR) imaging methodologies.


H. Sreedhar*1, V. Varma*2, A. Graham3, Z. Richards1, F. Gambacorata4, A. Bhatt1,
P. Nguyen1, K. Meinke1, L. Nonn1, G. Guzman1, E. Fotheringham5, M. Weida5,
D. Arnone5, B. Mohar5, J. Rowlette5
1 Department of Bioengineering, University of Illinois at Chicago
2 Department of Pathology, University of Illinois at Chicago
3 Department of Bioengineering, University of Illinois at Urbana-Champaign
4 Department of Chemical Engineering, University of Illinois at Chicago
5 Daylight Solutions, San Diego
*Contributed Equally

Real-time, MIR chemical imaging microscopes could soon become powerful frontline screening tools for practicing pathologists. The ability to see differences in the biochemical makeup across a tissue sample greatly enhances a practioner’s ability to detect early stages of disease or disease variants. Today, this is accomplished much as it was 100 years ago — through the use of specially formulated stains and dyes in combination with white light microscopy. A new MIR, QCL-based microscope from Daylight Solutions enables real-time, nondestructive biochemical imaging of tissues without the need to perturb the sample with chemical or heat treatments, thus preserving the sample for follow-on fluorescence tagging, histochemical staining or other “omics” testing within the workflow.
MIR chemical imaging is a well-established absorbance spectroscopy technique; it senses the relative amount of light that molecules absorb due to their unique vibrational resonances falling within the MIR portion of the electromagnetic spectrum (i.e., wavelengths from approximately 2 to 15 µm). This absorption can be detected with a variety of MIR detector types and can provide detailed information about the sample’s chemical composition.

The most common instrument for this type of measurement is known as a Fourier transform infrared (FTIR) spectrometer. FTIR systems use a broadband MIR light source, known as a globar, to illuminate a sample; the absorption spectrum is generated by the use of interferometry. Throughout the past decade, FTIR systems have incorporated linear arrays and 2D focal plane arrays (FPAs) in a microscope configuration to enable a technique known as chemical imaging.

With this approach, the illumination beam is expanded across a sample area, and the data produced is transformed into a hyperspectral data cube — a 2D image of the sample with an absorption profile associated with every pixel. This is a very versatile technique that allows the detailed spatial distribution of chemical content to be analyzed across a sample. Recently, this technique has proved to be very useful within the biomedical imaging sector for label-free, biochemical analyses of cells, tissue and biofluids.

While FTIR microscopy now is established as a powerful technique for a wide variety of applications, the instruments used for this methodology are fundamentally limited by the brightness of the globar source. Users looking to maximize the signal-to-noise ratios, and the associated resolutions of the images produced are forced to use synchrotron facilities, which replace the globar light source with a MIR beam generated by a particle accelerator. This approach can yield excellent results but clearly is not practical for benchtop applications; it is particularly unfit for biomedical imaging applications within clinical settings.

The recent advent of QCLs has provided an ideal light source for next-generation MIR microscopy. They are compact, semiconductor-based lasers that produce high-brightness light in the MIR region. The devices can be manufactured in an external cavity configuration to provide broadly tunable output with a narrow spectral bandwidth at each frequency. In this configuration, a QCL can be tuned across the MIR spectrum to sequentially capture an absorption profile for chemical identification.

Daylight Solutions’ IR microscope incorporates a broadly tunable and high-brightness QCL light source (it is an order of magnitude brighter than a synchrotron), a set of high numerical aperture (NA) diffraction-limited objectives, and an uncooled microbolometer FPA into a compact, benchtop instrument, as shown in Figure 1. The instrument provides rapid, high-resolution chemical images across very large fields of view and also provides a real-time chemical imaging mode. By overcoming the physical size, camera cooling and data collection time requirements of FTIR-based instruments, the microscope is positioned to bring MIR microscopy beyond research settings and into clinical use.

Schematic of a quantum cascade laser (QCL) microscope.

Figure 1. Schematic of a quantum cascade laser (QCL) microscope. Courtesy of Daylight Solutions.

Dr. Michael Walsh of the University of Illinois at Chicago (UIC) conducts research within the pathology department’s Spectral Pathology Lab, which has been using the IR microscope for the past several months. Walsh has been focused on developing chemical imaging techniques, with the ultimate goal of improving diagnoses within the field of tissue pathology.

Currently, the state-of-the-art method-ology used for the diagnosis of most solid-organ diseases is to extract a tissue sample via a biopsy. Tissue inherently has very little contrast and needs to be stained with dyes or probes to visualize and identify cell types and tissue structures. The field of pathology is based on examining the stained tissues, typically using white light, to determine if the tissue morphology deviates from a normal pattern. If the tissue looks abnormal, the disease state may be further subclassified by grade or by predicted outcome. However, the field of pathology is limited by the information that can be derived from the stained tissues and the subjective interpretation of the tissue by a highly trained pathologist.

Spero microscope.

Spero microscope. Courtesy of Daylight Solutions.

UIC’s Spectral Pathology Lab is focused on identifying areas in pathology where current techniques fail, or where there is a need for additional diagnostic or prognostic information that can help improve patient care. Potentially, MIR imaging is a very valuable adjunct to the current practice of pathology. Rather than using only stains, MIR imaging can interrogate the entire biochemistry of the tissue and render a diagnosis in an objective fashion. Traditionally, MIR imaging with an FTIR system has been limited by slow data acquisition speeds and the need to collect the entire spectral data cube. QCL imaging with the Spero microscope has the potential to speed up the data acquisition of images obtained from a tissue sample and to collect only the spectral frequencies of interest. The device also provides real-time imaging of samples at 30 fps, which could allow pathologists to very rapidly identify areas of interest on a tissue biopsy in a manner that is similar to their current clinical workflows. Some examples of the comparison of FTIR-derived and QCL-derived images from multiple organ tissues of interest are presented.

H&E-stained image of a mouse brain section on IR reflective slide, with selected regions labeled: hypothalamus, thalamus, and dentate gyrus.
Figure 2.
(a) H&E-stained image of a mouse brain section on IR reflective slide, with selected regions labeled: hypothalamus, thalamus, and dentate gyrus. (b) Transflectance QCL IR image of same region, prior to staining, at 1652 cm−1, in which the thalamus is clearly distinguished from surrounding regions. (c) Same region at 1548 cm−1. (d) Same region at 1500 cm−1. Courtesy of University of Illinois at Chicago (UIC)/Spectral Pathology Lab.

A tissue section from a mouse brain was scanned using the Spero microscope’s high-magnification objective (12.5×; 0.7 NA; 1.4 × 1.4-µm pixels) at various MIR frequencies in transflection mode, as shown in Figure 2. The tissue then was stained using hematoxylin and eosin (H&E), the most common stain in histopathology, and is displayed in Figure 2a. Using the H&E stain, regions were identified in the brain (thalamus, dentate gyrus and hypothalamus) that correlated with structures in the IR image. By illuminating the tissue at various wavelengths, discrete tissue features exhibit contrast due to the difference in absorption, as highlighted in the IR images taken at 1652, 1548 and 1500 cm−1 in Figure 2b-d, respectively. The microscope also makes it possible to visualize tissue at these individual wavelengths in real time. The identification of cell types and their biochemical changes is of particular interest in neuropathology.

Transmission FTIR image of a 4-µm thick section from a human liver tissue microarray on barium fluoride at 1650 cm-1.
Figure 3.
(a) Transmission FTIR image of a 4-µm thick section from a human liver tissue microarray on barium fluoride at 1650 cm-1. The image was taken with 64 coadditions of successive scans. (b) Transmission image from the Spero microscope of the same tissue at 1652 cm-1, both baseline corrected between 1796 cm-1 and 904 cm-1. In both images, the bright white stripe dividing the tissue core roughly in half is a region of fibrosis (red arrow), while the rest of the tissue on either side is composed primarily of hepatocytes (blue arrow). Courtesy of UIC/Spectral Pathology Lab.

A single biopsy core obtained from human liver tissue was scanned in transmission mode on a barium fluoride substrate by an Agilent Cary 600 Series FTIR microscope (Figure 3a). The FTIR image was acquired using a 36× Cassegrain collecting objective and a 15× Cassegrain condenser for a pixel size of 2.2 × 2.2 µm. Figure 3b shows the same liver core acquired using the Spero microscope with the high-magnification collecting objective (12.5×, 0.7 NA) and condenser objective for a pixel size of 1.4 × 1.4 µm. High-definition IR imaging enables clear contrast and identification of the band of fibrosis in the center of the core and the surrounding regions of liver cells, known as hepatocytes, and is indicated within Figure 3a-b. Acquisition of IR imaging data at the diffraction limit enables chemical information to be recorded from tissue structures at the single-cell level, allowing accurate characterization of individual tissue components, different cell types, varied disease states or other aspects of a tissue section.

Averaged spectra for regions of interest corresponding to the hepatocytes and the fibrotic area on the FTIR image in Figure 3a.
Figure 4.
(a) Averaged spectra for regions of interest corresponding to the hepatocytes and the fibrotic area on the FTIR image in Figure 3a. Spectra have been truncated from 1800 to 900 cm-1, normalized to 1650 cm-1, and baseline corrected between 1796 and 904 cm-1. (b) Averaged spectra for regions of interest corresponding to the hepatocytes and the fibrotic area on the Spero microscope image in Figure 3b. Spectra have been normalized to 1652 cm-1 and baseline corrected between 1796 and 904 cm-1.

Figure 4 displays average spectra calculated from homogenous tissue regions that describe hepatocytes and fibrosis within the liver tissue core shown in Figure 3. The spectra acquired from both FTIR and QCL systems are very similar. Walsh is focused on developing spectral classifiers that can aid pathologists in making very difficult diagnoses in the precancerous stages of liver cancer.

H&E-stained section of human colon tissue, and FTIR (with 16 coadditions) and Spero microscope transmission images of a 4-µm thick serial section of the same sample on barium fluoride. FTIR image shown at 1650 cm-1, Spero microscope image shown at 1652 cm-1.
Figure 5.
H&E-stained section of human colon tissue, and FTIR (with 16 coadditions) and Spero microscope transmission images of a 4-µm thick serial section of the same sample on barium fluoride. FTIR image shown at 1650 cm-1, Spero microscope image shown at 1652 cm-1. The red circle indicates mucin, the green circle indicates malignant colon carcinoma epithelium, and the blue circle indicates fibroblastic stroma. The raw spectra (taken from single pixels in approximately the same location for each of the three tissue features) are shown below their respective IR images. The FTIR spectra were truncated to match the Spero microscope’s spectral range of 1800 to 900 cm-1. Courtesy of UIC/Spectral Pathology Lab.

Point spectra from individual pixels were obtained and compared from a human colon sample on barium fluoride scanned in transmission on the same FTIR and QCL systems, which is shown in Figure 5. A serial section was obtained and stained with H&E to identify the different tissue structures. Using the H&E image as a reference, spectra from mucin (red), malignant colon carcinoma epithelium (green) and fibroblastic stroma (blue) were collected from a single pixel at approximately the same location. The unprocessed QCL and FTIR spectra are shown directly beneath their respective images. The FTIR system has an FPA size of 128 × 128 detector elements, while the Spero system has a microbolometer of 480 × 480 detector elements. Therefore, the FTIR image was collected as a mosaic and then stitched together.

FTIR and Spero microscope spectra from a single pixel of mucin, from the tissue shown in Figure 5.
Figure 6.
(a) FTIR and Spero microscope spectra from a single pixel of mucin, from the tissue shown in Figure 5. (b) FTIR and Spero microscope spectra from a single pixel of malignant colon carcinoma epithelium, from the same tissue. (c) FTIR and Spero microscope spectra from a single pixel of fibroblastic stroma. All spectra have been normalized (FTIR to 1650 cm-1, Spero to 1652 cm-1) and baseline corrected between 1796 and 904 cm-1, with the FTIR spectra truncated to match the Spero microscope’s spectral range of 1800 to 900 cm-1. Note that pixels for each tissue feature were located in approximately the same region, and that the two images have different pixel sizes (2.2 × 2.2 µm for FTIR, 1.4 × 1.4 µm for Spero microscope). Courtesy of UIC/Spectral Pathology Lab.

The spectra obtained from the regions of interest depicted in Figure 5 were preprocessed, as shown in Figure 6. The data was peak height normalized to the Amide I band. The FTIR data and QCL data were processed using a simple, two-point linear baseline correction between 1796 and 904 cm−1. Figure 6a-c shows the processed data from single pixels looking at the biochemistry of mucin, malignant colon carcinoma epithelium and fibroblastic stroma, respectively. The spectra from the QCL and FTIR systems are very similar on an individual-pixel level.

Finally, Figure 7 shows the scan of a frozen prostate tissue section captured with the microscope. Once thawed, the system can quickly image these sections at a single frequency of interest. The real-time capabilities of the system combined with the capacity for scanning frozen samples could someday allow for the analysis of samples in a time-critical intraoperative setting.

Transflectance scan of a 5-µm frozen human prostate tissue section on Kevley low-emissivity substrate captured with the Spero microscope.

Figure 7. Transflectance scan of a 5-µm frozen human prostate tissue section on Kevley low-emissivity substrate captured with the Spero microscope. Visualized with a false color map at 1640 cm-1. Data was baseline corrected between 1796 and 904 cm-1. Courtesy of UIC/Spectral Pathology Lab. University of Illinois at Chicago — Spectral Pathology Lab members, from left to right: David Martinez, Francesca Gambacorta, Vishal Varma, Andrew Graham and Michael Walsh. Courtesy of Daylight Solutions.

While there has been significant interest in MIR imaging for pathology applications for a number of years1-5, the technology has lacked the maturity to be ready for clinical implementation due to slow scanning speeds, low spatial resolutions and by a lack of computational power to fully handle large multispectral datasets. The Spero microscope, coupled with modern computing power, overcomes these limitations. The information detailed above demonstrates that the quality of the images and spectra obtained from the instrument are similar to those offered by FTIR imaging methods but with the additional benefits associated with the use of a QCL-based system. Recent advances in large multielement FPAs6-8) and high-resolution imaging approaches9-11 for tissue pathology have made this a much more attractive approach for fast and detailed image acquisition. QCLs represent the next step toward clinical implementation — they have demonstrated fast data acquisition, live-imaging capabilities and the ability to collect only spectral frequencies of diagnostic value.

Meet the authors

Michael Walsh holds a PhD in biological sciences and is an assistant professor at the University of Illinois at Chicago in Chicago; email: walshm@uic.edu. Matthew Barre is the business development manager at Daylight Solutions in San Diego; email: mbarre@daylightsolutions.com. Benjamin Bird is an applications scientist at Daylight Solutions in San Diego; email: bbird@daylightsolutions.com.


1. D.C. Fernandez et al. (2005). Infrared spectroscopic imaging for histopathologic recognition. Nat Biotechnol, Vol. 23, Issue 4, pp. 469-474.

2. C. Matthaus et al. (2008). Chapter 10: Infrared and Raman microscopy in cell biology. Methods Cell Biol, Vol. 89, pp. 275-308.

3. C. Kendall et al. (2009). Vibrational spectroscopy: a clinical tool for cancer diagnostics. Analyst, Vol. 134, Issue 6, pp. 1029-1045.

4. C. Krafft et al. (2009). Disease recognition by infrared and Raman spectroscopy. J Biophotonics, Vol. 2, Issue 1-2, pp. 13-28.

5. F.L. Martin et al. (2010). Distinguishing cell types or populations based on the computational analysis of their infrared spectra. Nat Protoc, Vol. 5, Issue 11, pp. 1748-1760.



Broadband Laser Aimed at Cancer Detection

Covering a wide swath of the mid-infrared, a new system offers greater spectral sensitivity


MUNICH, Sept. 25, 2015 — Mid-infrared (MIR) light is rich with molecular “fingerprint” information that can be used to detect substances from atmospheric pollutants to cancer cells.

While some lasers already operate in this region, enabling a variety of spectroscopy applications, their linewidth is relatively narrow, which limits the types of substances they can detect at any given moment.

Now a team of researchers from Germany and Spain has developed a laser system with phase-coherent emission from 6.8 to 16.4 μm and output power of 0.1 W. That is broad and powerful enough, they said, to detect subtle signs of cancer early in its development.

Molecules absorb portions of the MIR spectrum in ways that are unique to their atomic structures, and their absorption patterns provide a means of identifying the molecules with great specificity, even in low concentrations.


Emission spectrum

The emission spectrum of the laser and corresponding molecular fingerprint regions. Courtesy of the Institute of Photonic Sciences (ICFO).

“Cancer causes subtle modification in protein structure and content within a cell,” said professor Dr. Jens Biegert, a group leader at the Institute of Photonic Sciences (ICFO) in Barcelona. “Looking at only a few nanometer range, the probability of detection is extremely low. But comparing many of such intervals, one can have an extremely high confidence level.”

The new laser system generates MIR pulses via difference-frequency generation driven by the nonlinearly compressed pulses of a Kerr-lens mode-locked Yb:YAG thin-disc oscillator. It features a repetition rate of 100 MHz and pulse durations of 66 fs — so short that the electric field oscillates only twice per pulse.


Staff scientist Dr. Ioachim Pupeza (left) and postdoctoral researcher Oleg Pronin helped develop a laser system that emits ultrashort pulses of mid-infrared light. These pulses can be used to detect trace molecules in gaseous and liquid media. Courtesy of Thorsten Naeser/Ludwig Maximilian University.

“Since we now possess a compact source of high-intensity and coherent infrared light, we have a tool that can serve as an extremely sensitive sensor for the detection of molecules, and is suitable for serial production,” said project leader Dr. Ioachim Pupeza, a staff scientist at Ludwig Maximilian University of Munich (LMU).

The LMU and ICFO researchers aim to use their MIR laser to identify and quantify disease markers in exhaled air. Many diseases, including some types of cancer, are thought to produce specific molecules that end up in the air expelled from the lungs.

“We assume that exhaled breath contains well over 1000 different molecular species,” said Dr. Alexander Apolonskiy, an LMU group leader.

However, the amount of molecular biomarkers present in exhaled breath is extraordinarily low, meaning a diagnostic tool would need to be capable of detecting concentrations of at least one part per billion. The next step will be to couple the new laser system with a novel amplifier that would increase its brightness and boost sensitivity one part per trillion.

Detecting MIR signatures

The laser’s output spans more than one octave. Until now, the researchers said, such broadband emission has only been available from large-scale synchrotron sources.

Other more compact MIR sources, such as quantum cascade lasers (QCLs), have narrower linewidths. Tuning them to different sensing bands is time consuming, and combining multiple QCLs emitting in different parts of the MIR would be cost-prohibitive, Biegert said.

Meanwhile, the laser system’s 100-MHz pulse train is hundreds to thousands of times more powerful than state-of-the-art frequency combs that emit in the same range, the researchers said.

Detecting broadband MIR signals presents its own problems, however. Detectors for this region have poor signal-to-noise ratios unless cooled with liquid nitrogen, the researchers said.

In this case, electro-optical sampling proved to be a better option. Well-established for the terahertz range, the technique is less common in the fingerprint region.

“In the MIR range, there are not many groups who have implemented this already, because you need a broadband, phase-stable MIR pulse and an ultrashort sample pulse at the same time, which is quite challenging,” Pupeza said.

Having solved that problem with their broadband laser, the team now could use electro-optical sampling to extract the data they wanted.

In a nutshell, the process works like this: The electric field of an MIR pulse alters the birefringence of a crystal. This change can be measured by observing how the polarization of slightly shorter near-infrared (NIR) pulse is changed while propagating through the same crystal at the same time. In the end, only the NIR pulse is measured directly.

“Therefore, one big advantage is low-noise detection in the NIR, even though one obtains information on spectral components in the MIR,” said Ioachim Pupeza. “You only need to perform a Fourier transform numerically to get the spectrum of the pulse once you have its electric field.”

The research was published in Nature Photonics (doi: 10.1038/nphoton.2015.179).



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