The Daily Sign-Out

Field cancerization though precancerous stromal changes

An important question in cancer research is the causes and mechanisms for the development of multifocal carcinomas, especially for non-small cell lung carcinomas (as I recently blogged on).  While a “field cancerization” effect has been postulated as a model for multifocal carcinomas for a long time, it remains enigmatic why cancers develop at one site and not another given that the entire “field” is presumably exposed to the same carcinogenic agent or stimulus.  More specifically, it is also obscure what the role is of the peritumoral stromal changes observed around multifocal carcinomas.  Are these changes a cause or consequence (or, more likely, both) of these tumors?  Under normal circumstances, the epithelial tissue stromal architecture imposes growth restraints upon epithelial structures and exerts a tumor-suppressive effect on “transformed” neoplastic cells.  A similar effect is observed in the metastatic process in which the vast majority of metastatic tumor cells fail to form tumors at distant metastatic sites.  The conventional view has been that the tumor stromal microenvironment is reactive in nature; that is, the tumor stroma develops as a result of molecular cues from the tumor itself.  A recent article in Cell (8 June 2012) by Bing Ho and colleagues presents data suggesting that the loss of mesenchymal Notch/CSL signaling causes stromal changes that are potent stimuli for the development of carcinomas (abstract).

Mice carrying a mesenchymal-specific deletion of CSL-RBP-Jk, a key effector in the Notch signaling pathway, develop spontaneous multifocal squamous cell carcinomas after dermal atrophy and chronic inflammation.  The authors found that CSL-deficient dermal fibroblasts promote increased tumor cell proliferation through upregulation of c-Jun and c-Fos expression.  This, in turn, was associated with higher levels of diffusible growth factors, inflammatory cytokines, and matrix-remodeling enzymes.

The authors then tested these findings with human skin biopsy specimens of multifocal actinic keratosis, a premalignant skin lesion for cutaneous squamous cell carcinoma strongly associated with sun exposure.  Stromal fields adjacent to multifocal actinic keratosis showed decreased Notch/CSL signaling, albeit only with exposure to UVA and not UVB exposure, through DNA methylation and epigenetic silencing of Notch signaling in dermal fibroblasts.  These findings imply that the phenomenon of field cancerization can be mediated through stromal alterations preceding the development of neoplastic epithelial clones—in other words, the stroma is precancerous due to mutagen exposure.

This model would be a fascinating one to pursue for future lung cancer research with both non-small cell and small cell lung cancers since these cancers are all also strongly associated with a known mutagenic exposure, are posited to develop from a field cancerization effect from this exposure, and are frequently associated with multifocal synchronous tumors.  Unfortunately, there isn’t exactly an analogous premalignant “actinic keratosis” lesion for NSCLC, at least one that can be easily recognize and sample, although I think such atypical adenomatous hyperplasia foci may be easier to study in mouse models.  Anyone interested in pursuing this, let me know! ; )

 

Dr. R2D2--Computerized pathologist?

Meet C-Path, the iJobPlodd version of the 21st century pathologist.

The December 3rd-9th, 2011issue of The Economist has an article in this "Technology Quarterly" section entitled, "Indolent or Aggressive: A computerised pathologist that can outperform its human counterparts could transform the field of cancer diagnosis."  Actually, this a is review/interview on an article (abstract) published on 9 November 2011 in Science Translational Medicine by Daphne Koller and colleagues from Stanford.

Unfortunately, it appears that the author of The Economist article failed to consult a practising pathologist to get some context for this study.  Here's an example:

LOOKING for needles in haystacks is boring. But computers do not get bored. Contracting out to machines the tedious business of assessing the dangerousness of cancer cells in histological microscope slides ought thus to be an obvious thing to do. Cervical-cancer smear tests aside, however, such electronic intrusions into the pathology laboratory are limited. Grading cancer cells into “indolent” and “aggressive”, and hazarding an opinion about whether they spell a treatable condition or an untreatable one, has remained the realm of the human expert.

Perhaps I'm being a bit sensitive, but I don't consider looking at a patient's slides "tedious business" and resent this characterization of my practice of medical care!  According to this logic, perhaps patients would be better served having computers undertake the "tedious business' of taking a history or pelvic exams or listening to the heart.  In brief, this is a cutting-edge research article--not a roadmap for radically changing pathology practice in general (or even for the narrow application for breast cancer) .  As an example, it looks only at breast cancer tissue microarrays--not real clinical specimens.

I absolutely do not wish to take away from this article, however.  It is brilliant!  As I re-read it, apart from being green with envy (imagining how great it would be to do this with my own pet, non-small cell lung cancer!), I am picking up more and more fine points of trying to do this kind of work.  Here are my two main points which make this worth recommending:

1. The image features to be measured were not predetermined by a pathologist: the system made a very large set of measurements of the epithelial and stromal components--and their relationships--of the tumor and then the program determined which of these were related to survival outcomes.
2. The confirmation of the crucial role of the stroma in tumor outcome.

The bottom line, after reading this article (which I highly encourage anyone interested in digital pathology or anyone planning on practicing anatomic pathology in the next 20+ years to do), is that pathologists can be reassured of having a "job."  But--the article points to how our job might be significantly impacted by digital imaging and robust image analysis (ahem!--without regarding to FDA, CAP, payment, etc., i.e. the real drivers for adoption and implementation of these technologies).

WCLC 2011 Oral Presentations: Tumor microenvironment

So I'm finally finished sorting through the abstracts from the 14th World Conference on Lung Cancer that I recently attended and this begins a series of posts of what I found of interest from the oral abstract presentations.

There were two presentations that focused on the immune cell response in the tumor microenvironment that were of particular interest to me in that I have a current project looking at CD3+ lymphocyte responses in resected NSCLC through my ongoing work with the Thoracic Oncology Research Group at Rush University Medical Center.

MO 22.01 from session VIII examined tissue microarrays constructed from 479 patients with stage 1 adenocarcinoma and a median follow-up of 4.3 years.  They looked at a wide variety of different immune markers by immunohistochemistry and assessed localization (tumor vs. stromal) of immune cell infiltration, the grade of that immune cell infiltrate (low vs. high), and expression (sum of intensity of staining plus semi-quantitative assessment of distribution).

Results: The 5-yr relapse free survival (RFS) was 82% for the entire cohort.  There was no independent prognostic significance for any subpopulation of immune cells.  However, the ratio between stromal FOXP3+ (Treg cells) and CD3+ (total T cells) and tumor expression of IL7R and IL12Rbeta2 were found to be significant factors in predicting recurrence by univariate analysis.  Tumor expression of IL12Rbeta2 and stromal FOXP3:CD3 were prognostic even in tumors less than 2 cm.  By multivariate analysis, gender, stage (1A/1B), lymphatic invasion, tumor IL12Rbeta2 and stromal FOXP3:CD3 were independent predictors associated with recurrence.

This study highlights the importance of localization of immune cell response but also underscores the challenge for future studies to use more refined quantitative assessments of immune cell subpopulations and relative expression of IHC staining intesity (such as utilizing digital imaging and image analysis). 

MO 22.04, also from session VIII, presented interesting complementary data to the above presentation. This study examined 196 resected stage 1-3A NSCLC using IHC for infiltrating CD8+ and FOXP3+ cells.  They counted cells expressing these markers in tumor and stromal regions in five "randomly selected" high-power-fields and reported their data as the number of cells per mm² for each region.  They used median cell count as the cutoff to define patient subgroups.

Results:  Increased tumor islet CD8+ and stromal FOXP3+ infiltration were associated with better prognosis, whilst increased stromal CD8+ and tumor islet FOXP3+ infiltration were associated with worse prognosis.  Further, high CD8+ tumor islet/stromal infiltration was associated with better prognosis, whilst high FOXP3+ tumor islet/stromal infiltration was associated with worse prognosis.

Again, this study reinforces the crucial importance of identifying the microlocalization of immune cell subpopulations.  But I note several critical issues with methodology in this study that are worth considering for future studies.  First, the investigators did not clarify their methodology as to how high-power-fields were "randomly selected."  Further, their use of the term "tumor islet" suggests a non-random selection of such foci (tumor budding?) for assessment.  As a pathologist, I can safely say that this is quite unlikely "random"--which would undoubtedly lead to bias towards areas of the tumor that show higher concentrations of positively staining cells.  In fact, our Rush group recently began re-evaluating EMT markers assessed by IHC in a group of NSCLC patients who underwent surgical resection to specifically assess and grade distribution and extent of staining at the tumor-stromal interface rather than the global expression of such staining.  The data are being analyzed currently.  Moreover, I have been assessing the lymphocytic response just by H&E and note an association between foci of tumor budding and intensity of lymphocytic response at the tumor invasive front.  Second, manual counting of "events" just doesn't make it for this type of study and (again!) I must reiterate the need to use image analysis for this type of study.

P53 in tumor-associated fibroblasts in invasive ductal carcinoma

Hasebe et al. have authored an interesting article in a recent Modern Pathology (2010;23:662-672) which builds on their earlier work that showed P53 expression in tumor-stromal fibroblasts (TSF) not forming fibrotic foci to be an important predictor of outcome in patients with invasive ductal carcinoma.

The fibrotic focus has been shown to be a prognostic factor in breast cancer.  Moreover, criteria for fibrotic foci proposed by Van den Eynden et al. in 2007 (Dr. Hasebe was a co-author) appear to both clinically significant and reproducible in daily practice.  I am surprised that the use of this feature has not been adopted for routine use since presenting breast cancer cases with prominent fibrotic foci never fails to elicit curiosity and questions in Tumor Board.  Further, a fibrotic focus has been proposed as a marker for tumor hypoxia and angiogenesis.

The purpose of the current study was to determine whether the combined assessment of P53 expression in tumor-stromal fibroblasts both forming and not forming fibrotic foci serves as a significant predictor of outcome in patients with invasive ductal carcinoma using multivariate analysis with well-established prognostic factors.

The authors studied 1,039 consecutive patients at a single Japanese institution who received surgery including axillary dissection; 873 also received conventional chemotherapy, endocrine therapy, or both.  Using FFPE serial sections of each tumor, the authors evaluated 8 different histological factors, including measuring the fibrotic focus using 8 mm as the cutoff per the Van den Eynden criteria.  Immunohistochemical staining for P53 was evaluated using the Dako mouse monoclonal antibody clone DO7 and staining of tumor-stromal fibroblasts was scored according to modified Allred scoring.  Furthermore, the authors stratified P53 staining in tumor-stromal fibroblasts into 3 groups each for those tumors with and without fibrotic foci.

373 out of 1,039 cases had fibrotic foci.  Notably, in 97 of these 373 (26%) P53 staining could not be evaluated because the tumor tissue sections examined by P53 IHC did not contain a fibrotic focus identified initially at routine examination.

The authors found significantly higher P53 Allred scores in TSFs not forming compared with TSFs forming fibrotic foci.  But P53 Allred scores >4 in TSFs forming fibrotic foci were also associated with fibrotic focus diameter > 8 mm: the size previously established as a cut-off for predicting adverse outcome independent of stage.

Based on Allred scores, the authors segregated P53 expression for TSFs forming and not forming fibrotic foci into risk classes.  Patients in pTNM stages I-III with intermediate to high risk classes of P53 expression showed a significantly higher tumor recurrence rate than patients in the low risk classes.  In addition, stage II and III patients with intermediate to high risk classes of P53 expression showed a significantly higher disease-related mortality rate.

By multivariate analysis, for stage I patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci and histological grade 3 were significantly associated with tumor recurrence (but not disease-specific death).  For stage II patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci were associated with increased tumor recurrence and tumor-related death.  Lymphovascular invasion and blood vessel invasion were significantly associated with increased tumor recurrence, whilst pT1 and fibrotic focus > 8 mm were significantly associated with increased tumor-related death.

(Next to) Bottom Line: P53-expression TSFs located both in the inner fibrotic foci an outer invasive front increase the malignant potential of invasive ductal carcinomas across stages I to III.

SO--should we start doing P53 IHC on all invasive ductal carcinomas?  Perhaps not ready yet for prime-time, but this study provides evidence that may, at least in part, explain the prognostic value of fibrotic focus diameter.  Measuring the fibrotic focus, however, may just be ready for prime-time and you may want to consider reviewing the original paper by Van den Eynden et al published in Histopathology 2007;51:440-451.

BUT I do have a few points to consider.  First, this is a single institution paper, so the methodology needs to be independently validated and a larger, multi-insitutional patient cohort needs to be studied to confirm the association between patient outcomes and P53 staining.  Second, other P53 antibodies should be evaluated to optimize the assay.  Third, the number of invasive ductal carcinomas with fibrotic foci that were excluded from the study is disquieting and may be a major source of observational bias.  Finally, the role of image analysis in providing quantitative P53 cut-offs may provide better discrimination between prognostically different groups.

Advanced DNA Robots

Scientists Announce Advanced DNA Robots - WSJ.com

I'm not sure what this directly has to do with my blog.  But I thought it was a really cool story.  I thought about it in connection to recent and near-future blogs about the cancer microenvironment.  Perhaps the stroma surrounding the tumor can provide a road map for cancer cells to invade and migrate.  Just a thought.

1-Cancer microenvironment: the role of stroma in neoplasia (more USCAP 2010)

This post is more from USCAP 2010.  I attended the "Advanced Molecular BIology" Special Course at USCAP on March 23.  One of the highlights was Dr. Rob West's presentation, "The Role of Stroma in Neoplasia."  I also had the pleasure of sharing a van ride with him to Dulles where we had a chance (because of miserable traffic) to talk for a long while about the cancer microenvironment, cancer stem cells, and, of course, our children.  What follows is my summary of his talk.

The role of the stroma in cancer can be summarized by two broad concepts:

• Wound response: "normal wound-healing gone awry"
• Cancer as an organ: altered communication between the cancer cells and the surrounding stroma

What are the components of the cancer microenvironment?

1. Myofibroblasts/fibroblasts
2. Endothelial cells (and bone marrow derived mesenchymal stem cells?)
3. Leukocytes, especially macrophages
4. Extracellular matrix

Myofibroblasts are key players in the cancer microenvironment because they modulate the extracellular matrix "landscape," regulate angiogenesis, and secrete growth factors and cytokines that influence a wide variety of cellular functions of cancer cells.

If the tumor is going to grow, the nascent tumor and surrounding stroma must recruit endothelial cells by expressing angiogenic factors (e.g. VEGF) which tips the balance of pro-angiogenic factors over anti-angiogenic factors.  Hypoxia, p53 mutation, decreased thrombospondin-1, increased HIF are other factors that promote angiogenesis and the angiogenic switch."  Furthermore, there appears to be an "endothelial-to-mesenchymal transition" (EnMT) that involves the development of vascular networks around the tumor composed of "tip cells"--vessel sprouts without vascular lumens.  This process is reminiscent of EnMT that occurs in tissue patterning during embryogenesis and is important for stabilizing the fragile neovasculature at the interface of the tumor and microenvironment.

The extracellular matrix is not a passive scaffold but is recognized now as an active, evolving compartment.  Invasion into the ECM by cancer cells is a multi-step process that involves dissociation and detachment from surrounding cancer cells, attachment to surrounding ECM, degradation of ECM, and migration through the remodeled ECM.  The other cellular components critically influence this entire process.

Macrophages are now recognized as pivotal in promoting tumor cell migration and invasion, angiogenesis, remodeling of the ECM, and the "metastatic niche."  In particular, macrophage subsets are being defined as "tumor-associated macrophages."

To be continued. . .

Cancer microenvironment--An interview with Dr. Robert Weinberg

The Wall Street Journal (subscription required) published an interesting interview with Dr. Robert Weinstein of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts in which he discusses the role of the cancer microenvironment and its potential as a target for therapy.

Although directed toward a lay audience, this presents a well-structured overview and the accompanying podcast gives a little more depth.  It's well-worth a 7-minute time commitment.  I'm working on a near-future blog which will summarize a recent USCAP session I attended on cancer microenvironment and my discussion with the presenter.