Molecular Mysteries in the Blood

Molecular diagnostics are increasingly impacting the clinical lab—and few disciplines are changing as rapidly or as readily as hematopathology. It’s a natural combination; high rates of genetic mutations¹ and disease heterogeneity² coupled with minimally invasive access to samples makes molecular analysis an efficient and effective approach to diagnosis, prognosis, monitoring, and treatment selection for diseases of the blood. But just how much value does it bring to the lab—and what molecular tests should every lab have on its menu?

Moving into molecular diagnostics

“The landscape of molecular diagnostics in hematopathology has undergone significant transformation due to advancements in next-generation sequencing (NGS) technologies, the development of targeted therapies, and the increasing understanding of the genetic basis of hematologic malignancies,” says Kamran Mirza, professor of hematopathology and Godfrey D. Stobbe Professor of Pathology Education at the University of Michigan. “High-throughput sequencing has become more accessible and affordable, enabling comprehensive genomic profiling of hematologic cancers. Additionally, the integration of molecular data with clinical and pathological findings has enhanced the accuracy of diagnoses and the ability to monitor minimal residual disease (MRD).”

Sanam Loghavi, assistant professor of hematopathology at the University of Texas MD Anderson Cancer Center, agrees. “The scaling, decreased cost, and rapid adoption of novel molecular diagnostic assays in CLIA-certified laboratories has enabled more accurate classification and risk stratification and identification of therapeutic targets in solid tumors and hematologic neoplasms.”

And molecular testing is still on the rise. Loghavi points to the adoption of optical genome mapping^3–5 and RNA sequencing in clinical laboratories as an example; both technologies can be instrumental in the diagnostic workup of patients with suspected hematologic neoplasms. New, high-precision techniques like droplet digital PCR for detecting MRD are increasingly common. And, more broadly, the routine use of comprehensive NGS for lymphoid and myeloid malignancies is revealing a wide range of mutations and structural changes that might previously have gone unnoticed—and might one day offer new therapeutic targets.

Choosing the right tests

Key molecular tests labs should have in their hematopathology arsenals include:

• NGS (for comprehensive genomic profiling; Loghavi recommends both DNA sequencing and RNA fusion panels)
• cytogenetics (for identifying chromosomal abnormalities via karyotyping)
• fluorescence in situ hybridization (for detecting specific chromosomal abnormalities)
• optical genome mapping (for detecting structural variations at high resolution)
• PCR and real-time PCR (for detecting specific gene mutations, such as BCR::ABL1 fusions in chronic myeloid leukemia)
• flow cytometry (for immunophenotyping and MRD assessment)
• gene expression profiling (for specific conditions such as diffuse large B-cell lymphoma)

Labs that have the basics in place and want to move to the next level of molecular diagnostics for hematopathology should consider offering comprehensive panels for hematologic malignancies, including targeted treatment markers and MRD detection, or investing in high-throughput sequencing instruments and related bioinformatics infrastructure for advanced NGS.

But equipment alone is not enough. Ensuring that staff are trained in molecular techniques and bioinformatics analysis can keep labs at the forefront of the discipline, as can collaborating with other institutions to stay abreast of the latest advancements. Rigorous quality control measures and participation in external proficiency testing programs can help labs ensure that their results are accurate and reliable.

When molecules matter

For patients with hematologic malignancies, the importance of molecular hematopathology doesn’t stop at diagnosis and treatment selection. “Our classification and risk-stratification systems are largely based on genetics, as are our methods for disease monitoring,” explains Loghavi. MRD tracking and genetic markers can reflect treatment response and signal disease recurrence before symptoms appear. Treatment plans can also be modified in light of a patient’s molecular response—for instance, by adjusting tyrosine kinase inhibitors in chronic myeloid leukemia patients based on BCR::ABL1 levels. Changes in molecular markers can even redefine a patient’s prognosis during and after treatment.

But which patients need disease monitoring? “The indications for longitudinal testing are not standardized and can be subject to misinterpretation,” Loghavi warns. “We need better standardized guidelines to help guide these decisions.”

There are some situations, however, in which molecular testing is typically unwarranted. Mirza says, “In cases where the clinical and morphological findings clearly indicate a benign condition, molecular testing might not be necessary. Additionally, when the cost of testing outweighs the potential clinical benefit, especially in resource-limited settings, or when molecular results are unlikely to influence clinical management or outcomes, it may offer no benefit.”

Common mistakes in molecular testing

The first challenge that can impact molecular testing happens before the sample even reaches the laboratory. Poor sample quality can lead to inaccurate results—or no results at all—making proper sample collection and handling crucial. Common preanalytical errors include insufficient sample collection volumes, inadequate mixing, clotting, hemolysis, and diluted or contaminated samples.⁶

In the laboratory, Mirza points out several errors that can lead to inaccuracies. “Relying solely on one type of test without integrating clinical, morphological, and additional molecular data can lead to incomplete or incorrect diagnoses,” he cautions. “Additionally, misunderstanding the clinical significance of certain mutations or variants, particularly variants of unknown significance, can lead to inappropriate clinical decisions. Finally, failing to implement stringent quality control measures can result in false positives or negatives.”

How do you know when to call in an expert hematopathologist? Ideally, always. “In my opinion, every patient with a hematologic neoplasm should have their biopsy interpreted by a hematopathologist or a pathologist with expertise in hematopathology,” says Loghavi. However, not every laboratory has a subspecialist on staff. For those without ready access, Mirza suggests four key considerations for determining when expert consultations are needed:

1. Case complexity: “Complex cases with ambiguous findings should involve a hematopathology subspecialist.”
2. Experience and expertise: “Assess the lab’s experience and expertise in performing and interpreting the specific test in question.”
3. Resource availability: “Determine whether the lab has the necessary resources, including equipment and trained personnel, to perform the test accurately.”
4. Quality assurance: “Ensure that the lab can meet quality standards and interpret results in the context of the patient’s clinical presentation.”

Our molecular future

“The treatment landscape of hematologic neoplasms is rapidly evolving, with many new targeted therapies on the horizon for our patients,” Loghavi says. “However, our treatment strategies are only as good as our diagnostic capabilities. We need to be aware of these novel therapies and stay at the forefront of developing clinical assays to facilitate personalized treatment strategies for our patients.”

Mirza adds, “It’s essential to emphasize the importance of continuous education and staying up to date with the latest advancements in molecular hematopathology. Ongoing professional development is crucial for providing the best patient care—and collaboration between patient-facing physicians, pathologists, and researchers can drive innovation and improve diagnostic accuracy and treatment outcomes.”

References:

1. Feusier JE et al. Large-scale identification of clonal hematopoiesis and mutations recurrent in blood cancers. Blood Cancer Discov. 2021;2(3):226–237. doi:10.1158/2643-3230.BCD-20-0094.
2. Chu MP et al. Addressing heterogeneity of individual blood cancers: the need for single cell analysis. Cell Biol Toxicol. 2017;33(2):83–97. doi:10.1007/s10565-016-9367-4.
3. Soler G et al. Optical genome mapping in routine cytogenetic diagnosis of acute leukemia. Cancers (Basel). 2023;15(7):2131. doi:10.3390/cancers15072131.
4. Valkama A et al. Optical genome mapping as an alternative to FISH-based cytogenetic assessment in chronic lymphocytic leukemia. Cancers (Basel). 2023;15(4):1294. doi:10.3390/cancers15041294.
5. Puiggros A et al. Optical genome mapping: a promising new tool to assess genomic complexity in chronic lymphocytic leukemia (CLL). Cancers (Basel). 2022;14(14):3376. doi:10.3390/cancers14143376.
6. Iqbal MS et al. Preanalytical errors in a hematology laboratory: an experience from a tertiary care center. Diagnostics (Basel). 2023;13(4):591. doi:10.3390/diagnostics13040591.