Tools tackle cancer's complexity

Science Article

Flow cytometry, laser capture, DNA array uncover hidden cell differences

Technician Cintia deBarros uses a Hutch flow cytometer to sort cells. The equipment directs the cells into a pair of tubes that then go to the researcher. Photo by Michelle Hruby

Cancer is often described as a collection of diseases, striking many organ systems. But its complexity goes far beyond that definition.

Leukemia - once thought to be a single disease - is known to be a collection of about 20 cancers that differ in their aggressiveness and response to therapies. There is evidence that other cancers likely consist of multiple forms. And even within a single tumor, abnormal cells with distinct features may coexist.

Discovering the underlying genetic differences for this complexity, which may lead to improved diagnostic tests and customized cancer therapy, is one of the most challenging problems facing oncology researchers today.

But state-of-the-art technologies for cell and genetic analysis available at the Hutchinson Center - including flow cytometry, laser capture microdissection and DNA array analysis - are helping scientists uncover the hidden cellular differences that define cancers of the breast and other organs.

Diverse disease

"Breast cancer is such a diverse disease - there are so many different cell populations in a tumor," said Dr. Elizabeth Schubert, a staff scientist in Dr. Peggy Porter's lab. "The key thing is to be able to separate the types of cells from one another. That's critical to our studies."

Schubert and other Hutch scientists who study tumor complexity work with tissue obtained from biopsies and surgeries. Their precious samples are small and must be analyzed with highly sensitive techniques.

One of the biggest workhorses for tumor analysis is the flow cytometer, which is used to determine physical characteristics of cells - including size, shape, DNA content and protein content - as they move one by one past a detector.

Using this technique, scientists hope to identify biological markers-observable changes in DNA or protein-that can be used to detect cancer earlier and to predict and monitor response to therapy.

Successful in Barrett's

The approach has been tremendously successful in Hutch studies of Barrett's esophagus, a pre-cancerous condition that puts patients at risk for esophageal cancer.

Using flow cytometry, Dr. Brian Reid's research group can follow the genetic changes that occur in precancerous cells and use this information to predict a patient's risk of developing cancer. Those high-risk patients can then be monitored rigorously, allowing most cancer cases to be detected so early that patients have a high chance of survival.

Because flow cytometry allows scientists to "see" the invisible changes inside cells, the technique is much more sensitive - and accurate - than standard visual analysis that is used to determine whether a tumor is benign or malignant.

When used to analyze samples from many different women, flow cytometry may reveal molecular markers that distinguish different types of breast cancer.

"We already know from pathologists who look at tumor samples that there are different types of breast cancers," Schubert said.

But the molecular changes responsible for thesedifferences have yet to be determined.

Crucial to studies for characterizing multiple forms of breast cancer is a large patient population that has provided, in addition to tumor tissue, information about family history and behaviors that may reveal clues about cancer risk.

"A lot of what we do feeds back into other work done here at the Hutch," Schubert said.

"We have the power of the epidemiology studies led by Janet Daling's group, and we hope to correlate molecular tumor markers with data from their studies. For instance, was a woman pre- or post-menopausal at the time of diagnosis? We hope a combination of epidemiologic and molecular data will help define risk groups and add important information for clinical management."

Tissue samples

After obtaining tumor samples, Porter's group confirms the patient's initial diagnosis using standard methods in which the tissue is reacted with agents that stain the cells to reveal markers that diagnose breast cancer. Schubert then takes tiny samples for flow cytometry to determine if the cancerous cells have DNA abnormalities.

The Hutch solid-tumor flow cytometry shared resource helps prepare the tumor samples by treating them with chemicals that allow the DNA to fluoresce, allowing the amount of DNA to be quantified, said Jeri Glogovac, a project manager overseeing the development of the solid-tumor flow cytometry facility.

Changes in DNA content are likely to be some of the earliest alterations in cells that predict cancer. For example, in patients with Barrett's esophagus, changes in DNA content can effectively predict progression to cancer.

When tumors are small, they may have changes too subtle to be detected by flow cytometry, Schubert said.

Laser capture microdissection

Laura Cousens, a technician in Porter's lab, uses a method called laser capture microdissection to separate cells of interest from small tumors. The abnormal cells are identified visually, under a microscope. With the use of a laser, cells are embedded in the surface of a resin-coated cap and pulled from the tissue for further testing.

Once cells of interest are sorted, they can be analyzed in more detail for known or suspected mutations. Also, with DNA arrays, they can be used to discover new mutations.

DNA arrays are used to examine thousands of genes in the cell simultaneously. Comparing gene expression patterns between normal and tumor cells reveals genes present at abnormal levels in cancerous tissue.

While an ultimate goal of the research is to develop better diagnostic and prognostic tests for patients, Schubert said the projects are also fascinating from a basic research standpoint.

"We're likely to learn some of the basic biology of tumors, such as, how do tumors metastasize?" she said. "That's a fundamental question in cancer biology. There is still a lot we don't understand, and finding better markers is essential."

---

WHAT'S A FLOW CYTOMETER DO?

A flow cytometer determines the physical characteristics of cells, including:

• Size
• Shape
• DNA content
• Protein content

Center News Table of Contents