Lab Soft News

Using Pathology Informatics Principles to Explore Cancer Biology

My colleague and eminent pathology informatician, Dr. Jules Berman, has just published a book entitled Neoplasms: Principles of Development and Diversity. It is the first book of its kind that applies informatics principles to explore the field of tumor biology and focuses the following two important questions:

• Is cancer a single disease process that is manifested in many different types of tumors, or is cancer many different diseases, all related by excessive cell growth? If all cancer can be characterized by a single disease process, why haven't we isolated the process and cured cancer? If cancer is thousands of different diseases, how can we ever hope to cure all of the different kinds of cancer?

• If cancer is characterized by the progressive accumulation of genetic abnormalities, and if every tumor specimen is genetically unique and distinct from every other tumor specimen, why do tumors fit into precisely named types? Worded another way, why does every unique tumor fall into one of the diagnostic entities (e.g., Warthin tumor, melanoma, lobular carcinoma of breast, and so on) that pathologists are taught to recognize.

Jules has developed a web page that describes the book, including the complete table of contents, and links to the publisher's web site.

Correction of Recent Note on the "Tissue of Origin" of Malignant Lesions

In a recent note on identifying the "tissue of origin" of malignant lesions based on new technology from Pathwork Diagnostics (see: "Tissue of Origin" Genomic Test Receives FDA Approval), I received the following comment from Glenda G. Anderson:

As founder of Pathwork and its technology, it is very encouraging to hear that you have found the raw agreement indices to be "important descriptive statistics with unique common-sense value". We worked particularly hard on this aspect of the product with a hope to accomplish just that. You likely share my belief that result reporting is an oft overlooked, but critically important element of good clinical diagnostic product design. Wanted to offer one correction. The test uses a high density oligonucleotide microarray (in fact, a GeneChip), not a tissue microarray. A test of this sort requires the high level of platform reproducibility available only from the former.

The press release that my original note was based on referred to the underlying technology of the test as a "microarray" and I jumped to the incorrect conclusion that this meant a "tissue microarray" rather than, as Glenda now indicates, a high density oligonucleotide microarray (i.e., a GeneChip).

Here is a definition for a DNA microarray from the Wikipedia:

A DNA microarray is a high-throughput technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target. In standard microarrays, the probes are attached to a solid surface by a covalent bond to a chemical matrix....The solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip or colloquially Affy chip when an Affymetrix chip is used....DNA microarrays can be used to measure changes in expression levels or to detect single nucleotide polymorphisms (SNPs)....

Here are more details about the test from the company (see: Pathwork Diagnostics’ Core Technology):

The Pathwork Tissue of Origin Test is currently available through Pathwork Diagnostics Laboratory and an FDA-cleared kit version of the test will be available to clinical laboratories. For the test, frozen tissue biopsy specimens are processed on the company’s custom Pathchip microarray. Test processing includes RNA extraction, amplification, hybridization and scanning. The data is analyzed using the Pathwork Tissue of Origin Test’s proprietary analytics, and test results reports are provided to both the oncologist and pathologist. Pathwork Diagnostics’ microarray tests run on the proven, commercially available Affymetrix GeneChip  System. A version of the test for formalin-fixed, paraffin-embedded (FFPE) tumor specimens and needle biopsy specimens, including core and fine needle aspiration (FNA), will be available through Pathwork’s CLIA laboratory later this year.

Note the exciting news that the test will soon be available for formalin-fixed, paraffin-embedded (FFPE) tumors as well as FNA specimens.

Operational Issue Relating to Endo-Microscopy: Some Early Speculation

In a previous note (see: Progress in Diagnoses with Endo-Microscopy), I discuss some of the evolving science and technology underlying endo-microscopy. Using this technique, a probe is inserted into suspicious lesions discovered during colonoscopy. This probe functions as a confocal microscope and provides a detailed examination of the lesion at the cellular level. After reading this note, Bev MD posted the following comment:

As a pathologist, I would have a few questions about the sensitivity/specificity of this technique. How would it label cells that we consider "dysplastic' for instance? What would be its false negative rate, risking nonremoval of a cancerous polyp that has invaded by the time of a re-exam? Given the relative ease of resecting most polyps, I can't see much benefit except for saving the pathologist's bill, given that the endoscopist would now charge more using this technique, plus a polyp excision charge for ones deemed "malignant". Or am I missing something?

All good questions but perhaps somewhat premature given that this technology is still in the research stage and therefore has not entered into standard clinical practice. However and as I speculated in my note, the major potential and obvious advantage of endo-microscopy is its  ability to determine real-time whether borderline lesions are benign or malignant. It would then be possible to resect malignant and dysplastic lesions during the procedure or immediately afterward with a surgical procedure.

I can't help drawing an analogy between this new approach and fine-needle aspiration (FNA) of "lumps and bumps" in the breast, thyroid, and lung lesions. The cells aspirated from the lesion are immediately examined by a cytopathologist and the patient is spared a traumatic surgical procedure for purposes of diagnosis. In the case of endoscopy, recent news (see: Flat Colon Lesions Identified And Removed Using Colonoscopy) suggests that the diagnosis of gross lesions in the colon may be more challenging than previously thought. Here is an excerpt from the article:

A study released this week from researchers at the Veterans Affairs Palo Alto Healthcare System in California shows that non-polypoid colorectal neoplasms or flat colon lesions, are more common in Americans than previously thought and may have a greater association with cancer compared to polypoid neoplasms or the more commonly diagnosed colorectal polyp.

So where does all of this leave us, admittedly at an early stage in the development of the endo-microscopy? Here are some of my early ideas:

• Research on endo-microscopy will undoubtedly continue, perhaps resulting in an extension of the diagnostic capabilities of colonoscopy and with the added ability to determine, perhaps with certainty, the diagnosis of colonic lesions real-time.
• It is quite possible, following the example of FNA, that the microscopic diagnosis during endoscopy will be turfed to pathologists who will participate in the procedure. Given that the majority of colonoscopies reveal no suspicious lesions at all, it could make perfect sense for pathologists to provide such support.
• Pathologists are now commonly joining gastroenterology groups to diagnose the tissue samples obtained during various procedures such as endoscopy. If endo-microcopy eventually takes hold, the technology may act as an incentive for more gastroenterology groups to recruit their own pathologists.

Microchip Separation of Circulating Tumor Cells as a New Cytopathology Technique

A recent landmark paper published in Nature (Isolation of rare circulating tumour cells in cancer patients by microchip technology) opens up new horizons in pathobiology, cytopathology, and diagnostic medicine relating to the separation and identification of circulating tumor cells (CTCs). Below is summary statement of the work copied from the abstract:

Here we describe the development of a unique microfluidic platform (the 'CTC-chip') capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions, and without requisite pre-labelling or processing of samples.

More details about this approach to cellular diagnosis will certainly be brought to light over time as the technology continues to mature. In this note, however, I want to focus on some semantic issues pertaining to this technology. To be more specific, I want to discuss what term could and should be used to describe the collection of CTCs by microchips. My first instinct was to refer to the process as a biopsy but this is clearly not the case. Here's a definition of the term biopsy that I have patched together after some web browsing:

A biopsy is the removal and examination of a tissue sample obtained for diagnostic purposes from a patient using a scalpel or needle.

When I discussed this semantic issue with my colleague Ul Balis, one of the authors of the article cited above, we came to the conclusion that the isolation and identification of CTCs by the chip is more closely akin to a cytopathology process than to surgical pathology with its reliance on biopsies or sampling of a resected organ. However and in the usual case with cytopathology, the cells being sampled are obtained by exfoliative sampling (Pap or buccal smears, for example) or needle aspiration as in the case of fine needle aspiration (FNA). This latter procedures, in light of the definition above, actually seems more like a surgical biopsy although the sample consists of aspirated cells or cellular aggregates.

So what term should we now use to describe the process of malignant cell collection using the CTC-chip? Ul Balis has come up with what I consider the perfect neologism to describe the process: endofoliative cytology. The prefix endo- implies, as is certainly the case, that the collected CTCs became detached from, or migrate from, tumors in deep organs, enter the blood stream, and then adhere to the chip microposts.

If the CTCs isolated by the chip are found to be truly representative of the tumor cells remaining in-situ, we may now be entering what may be called a pre-biopsy era when a solid tumor diagnosis can be made totally on the basis of CTCs and without the need for a "cutting" biopsy with its uncertainty and morbidity. The proponents of molecular imaging are pursuing this same goal but without the need for any tissue or cellular sample.

HPV Testing More Accurate Than Pap Smears in Diagnosing Cervical Cancer

When I read a recent article (see: NEJM Report Finds HPV Test More Effective Than Pap in Determining Cervical Cancer Risk), it dawned on my that we may have reached a tipping point in terms of using only morphologic observation as the primary basis for diagnosing malignant lesions. The article focuses on cervical Pap smears and suggests that this technique should not be used as a front-line approach for diagnosing pre-cancerous and cancerous cervical lesions. Here is an excerpt from it (boldface emphasis mine):

The first randomized, controlled study in North America of HPV testing as a stand-alone screen concluded that it is almost 40 percent more accurate than traditional cytology (the Pap "smear") in identifying women with advanced cervical disease, according to a report published ... in The New England Journal of Medicine. The study, which involved more than 10,000 Canadian women age 30-69, found that the HPV test's sensitivity - its ability to accurately identify women with pre-cancerous cervical cells or cancer - was 94.6 percent, compared to 55.4 percent for the Pap. HPV (humanpapilloma virus) is the primary cause of cervical cancer. This seminal study used QAIGEN's Hybrid Capture 2 High-Risk HPV DNA test...."We already knew before conducting this study that the sensitivity of the Pap left a lot to be desired," stated one of the study's authors...."However, 55.4 percent accuracy is only slightly above chance. Flipping a coin gives you 50 percent."...Under the currently recommended guidelines, screening that includes HPV testing may be performed at longer intervals than when the Pap is used alone. However, the authors of the NEJM report concluded that co-testing "only marginally improved sensitivity compared with HPV testing alone". The conclusions of [the study] reinforce a growing number of other studies showing greater sensitivity for HPV testing and suggesting its use as the primary, front-line screen -- with the Pap reserved for follow-up evaluation.

As noted in a previous note (see: Defining Test Sensitivity and Specificity for the General Public), sensitivity is the ability of a test to detect a true positive. The sensitivity of QAIGEN's Hybrid Capture 2 High-Risk HPV DNA test was 94.6% and that of the standard cervical Pap test was little better than a coin flip. The authors of this paper went on to predict that the detection of cervical cancer will henceforth involve consumer education, HPV vaccination, and DNA testing of cervical cells. It appears that we are witnessing the twilight of the cervical Pap smear except for follow-up of patients with diagnosed disease.

For me, the take-home lesson here is that the days are numbered for diagnosing clinically significant lesions only on a morphologic basis. Such observations will need to be correlated with genetic, proteomic, or immunohistochemical analysis of the tissue or cells under review or a search for a viral agent. Surgical pathology is thus becoming more like clinical pathology. It's not clear at this time how many other malignant lesions beyond cervical cancer will be discovered to have a viral or bacterial etiology. Nevertheless, it's clear that major technical and scientific forces are at work that will reshape the practice of surgical and cytopathology.

Irish Pap Smears Imported by Quest

In an effort to allay the anxiety of a large number of Irish lasses, Quest Diagnostics has begun to import pap smears from the auld sod (see: Cervical smears sent to US for testing). Below is an excerpt from the article (boldface emphasis mine):

The cervical smears of up to 30,000 Irish women are being sent to the United States for testing in a bid to avert a crisis in Irish laboratories....[T]he National Hospitals Office ...has signed a contract with Quest Diagnostics Laboratory in Dallas,Texas, [to process these tests]....In Cork hospitals, there is a backlog of between 10,000 and 12,000 tests and HSE South said the maximum waiting time for results was six months. [The] medical director of Well Woman Clinics in Dublin, said that the waiting time for results from the Royal College of Surgeons in Ireland (RCSI) was currently four months, but that a one month turnaround for cervical smear results is best practice. By sending the tests to the US, the NHO claims the entire backlog will be cleared in the first half of this year. Quest Diagnostics processes 55 million smear tests a year.

I have the following comments about this article:

• Don't you just love this statement: "a one month turnaround for cervical smear results is best practice [in Ireland]." How long would a cytology lab last in the U.S. with that kind of TAT?
• My guess is that the Irish health system will rapidly adapt to the convenience of shipping their pap smears to Texas and that this is more apt to be a permanent arrangement than the stopgap approach to clear up the backlog in Ireland, as it is described in the article.
• I have written a previous note (see: Need for Pap Test May Be Fading) about how the HPV vaccine will reduce the incidence of cervical carcinoma among women in the U.S. and hence the demand for pap smears. Perhaps this Irish pap smear initiative will be a "dress rehearsal" for a more generalized phenomenon with pap smears shipped to the U.S. from multiple other countries.
• How about this yearly pap smear volume for Quest -- 55 million!

Need for Pap Test May Be Fading

An interesting topic for discussion is how the HPV vaccine and the genetic HPV test, now a common component of the Pap test, will affect the future practice of cytopathology. The New York Times recently covered this important issue (see: Pap Test, a Mainstay Against Cervical Cancer, May Be Fading). Below is an excerpt from the article (bold face emphasis mine):

The Pap smear, an annual ritual for many women and the mainstay of cervical cancer prevention for more than half a century, may start to fade in importance. It will not disappear for many more years, if ever. But a newer genetic test that detects human papillomavirus, or HPV, which causes cervical cancer, is starting to play a bigger role in screening. The new vaccine [for HPV] could also deal a longer-term blow to Pap testing, which works by detecting abnormal cells from the cervix that could be on their way to becoming cancerous....The vaccine, approved only for girls and women 9 to 26 years old, does not protect against all strains of HPV that cause cancer.

If a precancerous lesion is present, the Pap test will detect it only 50 percent to 80 percent of the time. Pap testing is effective only because it is done often; a lesion can take 10 years to turn into a cancer, so a yearly test will probably find it in time. As more women get the HPV vaccine, however, the number of lesions will decline, making the Pap test more costly per cancer case detected....

Pap testing is going to break down in a world in which vaccination decreases the prevalence of lesions, said [an oncology expert]... Some gynecologists say having women come for annual Pap testing brings them in for other needed examinations....The perception is that the annual Pap smear drives volume, said ... a gynecological oncologist at [Kaiser]. "I don't see anyone raising their hand volunteering to give that up." At Kaiser, where doctors' pay does not depend on customer volume, the genetic HPV test is offered with Pap testing to every woman 30 and older. If both tests are negative, a woman does not come back for further screening for three years. The savings from less frequent screening more than pay for the extra cost of the HPV test.

I know that there have been ongoing discussions within the pathology professional societies about the future role and importance of cytopathology as the volume of cervical specimens begins to decline. The good news is that this change will not take place precipitously due both to the force of habit of clinicians and patients and also due to the natural history of virally-induced cervical dysplasia and cancer. However, it seems unlikely that the volume of urine specimens as well as fine needle and peritoneal/pleural fluid aspirations will ever make up for the slow but certain decline in cervical specimens. It's not clear what the discipline of cytopathology will look like when the dust settles.

For me, the second very interesting point in the article above is that both clinicians and pathologists have cause to worry about what is referred to as the "breakdown" of Pap testing. This is because the annual Pap test for four decades or more has been an important stimulus for women's annual physical examinations. The appropriate response to this complaint is relatively simple and applies to both men and women. Broad cancer surveillance and wellness testing will be accomplished in the future by the use of large biomarker test panels, a topic that has been addressed here in detail in the past. This will be a change for the better, given the relatively low sensitivity and specificity of the Pap smear (see: Defining Test Sensitivity and Specificity for the General Public) alluded to in the article above. Moreover, the Pap test only screened for a single type of neoplasm.