Biochemical Tumor Markers: Are New Testing Guidelines Fit for Purpose?

The recent publication of the Clinical and Laboratory Standards Institute (CLSI) guideline C65, Biochemical Tumor Marker Testing, provides a necessary foundation for the global standardization of protein-based oncology diagnostics.

In a landscape often distracted by the rapid emergence of molecular genomics, CLSI C65 refocuses the laboratory on the "total testing process" for the most frequently utilized soluble markers. However, while the document achieves technical excellence in the markers it covers, its strategic omissions – specifically regarding plasma cell dyscrasias and the nuances of radiation therapy monitoring – highlight a critical need for expanded scope in future iterations.

What is included?

CLSI C65 is explicitly designed to provide guidance for ten commonly measured soluble biochemical tumor markers that circulate in blood and are typically measured in serum or plasma. These markers include:

• Prostate-specific antigen (PSA), both total and free

• Carcinoembryonic antigen (CEA)

• Cancer antigen (CA) 19-9

• CA125 and human epididymis protein 4 (HE4)

• α-fetoprotein (AFP)

• Human chorionic gonadotropin (hCG)

• CA15-3 and CA27.29 (the latter noted as essentially equivalent for breast cancer management)

• Thyroglobulin

• Calcitonin

The guideline's strength lies in its rigorous approach to the testing lifecycle, particularly its focus on pre-examination factors. It estimates that up to 70 percent of laboratory errors occur in this phase, often due to poor specimen quality.

To mitigate this, CLSI C65 provides detailed stability data (eg, AFP is stable at 4°C for 7 days, while calcitonin requires immediate freezing). The guidance also highlights the importance of specimen timing, noting, for instance, that CA125 should not be sampled during menstruation or within two weeks of pelvic surgery.

What about nucleic acid-based markers?

A key characteristic of CLSI C65 is its narrow focus on biochemical analytes. The document explicitly states that molecular techniques are not discussed. This exclusion of DNA and nucleic acid-based markers (such as circulating tumor DNA) is a deliberate structural choice, as molecular guidelines are maintained in separate CLSI portfolios.

While this approach ensures technical depth for protein assays, it maintains a divide in the clinical laboratory that may become increasingly artificial as multi-modal "liquid biopsy" approaches – combining proteins, DNA, and RNA-based investigations – become the standard of care.

A critical omission: serum free light chains

Despite its comprehensive treatment of ten markers, CLSI C65 notably omits serum free light chains (FLCs). Although they are biochemical proteins measured via automated immunoassays, FLCs are not included in the guideline's primary list. This is a significant gap given FLCs' role in managing plasma cell dyscrasias, such as multiple myeloma.

FLCs have a short half-life of 3 to 5 hours, compared to intact immunoglobulins, making them highly sensitive markers of early treatment response or relapse. In excluding these markers, the laboratory community lacks a CLSI-standardized framework for the unique reporting requirements of FLCs. Examples include the kappa:lambda ratio and the specific reference intervals required for patients with renal impairment – a common complication in myeloma.

The radiotherapy blind spot

The document defines monitoring as the "assessment of the effectiveness of treatment." However, the guideline does not provide specific kinetics or clinical decision limits tailored to radiation therapy (RT). Standard biochemical responses, such as the "PSA bounce" after prostate radiotherapy or the use of the "Phoenix Criteria" (nadir + 2.0 ng/mL) to define biochemical failure, are absent from the document's reporting and interpretation sections.

Furthermore, Table 3 in CLSI C65, which lists nonmalignant conditions associated with marker elevations, includes factors such as age, smoking, and pregnancy, but omits the physiological "noise" or systemic inflammatory responses often induced by localized radiation. Without standardized guidance on post-RT kinetics, there is a risk of misinterpreting transient spikes as disease progression, potentially leading to unnecessary interventions.

Considerations for the next edition

CLSI C65 Edition 1 is an important first-of-its-kind document that brings much-needed standardization to the most high-volume tumor markers in clinical use. Its focus on analytical interferences, method standardization, and workflow path provides a safeguard against common laboratory pitfalls. However, to truly reflect the evolving needs of oncology, the future second edition should bridge the "biochemical-molecular" divide and address the missing pillars of protein diagnostics.

Future editions provide an opportunity to integrate guidance documents into a multi-modal framework of standardized oncology laboratory medicine, incorporating cross-disciplinary knowledge. Inclusion of FLCs would provide a complete framework for hematologic oncology, while expanding the kinetics of tumor markers to include treatment-specific responses, such as RT, would elevate the guideline from an analytical standard to a truly comprehensive clinical tool.

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