1. Inter-laboratory comparison of beta-2 microglobulin methods: impact of assay variation on multiple myeloma staging
Pasquale Fedele, Kay-Weng Choy, James Doery, Jake Shortt, George Grigoriadis, Zhong Lu.

2. Multiple Myeloma Prognosis
1960s-1970s:
Initial clinical/lab predictors of survival identified.
1975:
Durie/Salmon (DS) system introduced.
1980s:
Serum beta2-microglobulin (β2M) emerged as the single most powerful prognostic factor.
1980s – 2000:
Additional prognostic factors identified.
Combinations used to stratify prognosis groups.
2005:
International Staging System for Myeloma published.
The outcome for patients with multiple myeloma is highly variable, with an overall survival ranging from less than 6 months to greater than 10 years
This variability derives from heterogeneity in both myeloma cell biology and multiple host factors.
Studies conducted in the 1960s and early 1970s identified a number of clinical and laboratory parameters that are independent predictors of survival duration including haemoglobin level, serum calcium, serum creatinine, and severity of bone lesions.
In 1975, Durie and Salmon introduced a staging system, the Durie/Salmon (DS) system, using commonly available clinical parameters that predicted myeloma cell tumour burden
The level and type of monoclonal protein
Haemoglobin
Calcium level
Number of bone lesions (lytic lesions on skeletal survey – observer dependent).
*** Creatinine level further defined lower versus higher risk patients in each of the three tumour mass stages.
In the 1980s, serum beta2-microglobulin emerged as the single most powerful prognostic factor and was considered a simple, reliable predictor of survival duration.
Subsequently, other prognostic factors were introduced, including serum levels of C-reactive protein, albumin, and plasma cell proliferative index.
Combining these factors with serum B2M provided improved prognostic stratification compared with B2M alone – however non consensus on which ones or on cut off values for B2M or other variables.

3. Data collected from 1981 through 2002.
10,750 previously untreated symptomatic myeloma patients from 17 institutions, including sites in North America, Europe and Asia.
Data collected from 1981 through 2002.
Introduction of the ISS
The international staging system (ISS), has largely defined multiple myeloma prognostication since its publication in the Journal of Clinical Oncology in 2005
In this study, clinical and laboratory data were gathered on 10,750 previously untreated symptomatic myeloma patients from 17 institutions, including sites in North America, Europe and Asia.
Data were collected from 1981 through 2002.
Treatment
7,942 patients received standard therapy
2,808 patients received high-dose therapy as initial treatment.

4. Serum β2M, albumin, platelet count, creatinine and age emerged as powerful predictors of survival
Combination of serum β2M and albumin provided the simplest, most powerful and reproducible three stage classification
Serum β2M, albumin, platelet count, creatinine and age emerged as powerful predictors of survival
Combination of serum B2M and albumin provided the simplest, most powerful and reproducible three stage classification
This clearly divided into 3 prognostic groups:
stage I: β2M < 3.5 mg/L plus serum albumin ≥ 3.5 g/dL (median survival, 62 months);
stage II: neither stage I nor III (median survival, 44 months); and
stage III: β2M ≥ 5.5 mg/L (median survival, 29 months).
The ISS has largely defined multiple myeloma prognostication since its publication.
Greipp et al J Clin Oncol (15):

5. Subsequent findings Discordant albumin results previously reported
Subsequent studies have reported significant discordance of albumin results between serum protein electrophoresis (SPEP) compared with both bromcresol green (BCG)2 and bromcresol purple (BCP)3 methods; with overestimation of albumin by SPEP in the setting of a large M-protein. A review of 496 patients found that while ISS staging was discordant between BCG and SPEP in 69 patients, there was no statistically significant difference in survival compared with “true” stage I and II patients4 (albumin does not impact stage III determination).
Snozek et al Clin Chem. 2007; 53(6):
Quick et al Clin Chem. 2009; 55(3):
Review of 496 patients: ISS staging discordant between BCG vs SPEP in 69 patients.
No statistically significant difference in survival compared with “true” stage I and II patients.
Rajkumar et al J Clin Oncol 2008

6. β2M Analysis Several methods of β2M detection:
Nephelometry
Turbidimetry
Chemiluminescence immunoassay
Enzyme immunoassay
Correlation previously thought to be satisfactory
Cf: Radioimmunoassay – poor correlation with other methods.
Tichy et al Neoplasma (6):

7. RCPA Chemical Pathology QAP
β2M (Tumour Marker Program Cycle 39)
There are several methods for testing β2M, including nephelometric, turbidimetric, chemiluminescent immunoassay (CLIA) and enzyme immunoassay (EIA). Examination of local external quality assurance data showed substantial variations in results obtained using different methods. This prompted us to investigate the inter-method variability of β2M results on human samples and determine the implications this has on multiple myeloma ISS staging.

8. Aim
Investigate the inter-method/inter-laboratory variation of β2M on human samples.
Determine the implications this has on ISS prognostic scores.

9. Methods: stability of β2M analyte
10 samples spun down, tested and simultaneously aliquoted for storage at:
Room temp
Refrigerated at 4°C
Frozen at -20°C and later thawed at 37°C
Frozen at -20°C and later thawed at room temp
Results stable after freeze/thawing
We first determined the stability of the β2M analyte to ensure this would not confound our findings. 10 patient samples were spun down, tested and simultaneously aliquoted for storage at different temperature conditions; 1) Room temperature, 2) Refrigerated at 4C, 3) Frozen at -20CC and later thawed at room temperature, 4) Frozen at -20C and later thawed at 37C. While some instability was seen with storage at room temperature, the results were consistent with repeated testing following storage at 4C and -20C (data not shown). In contrast to the results of a previous report on urine β2M, no significant difference was seen between thawing at room temperature and 37C 5.

10. Methods: interlaboratory variability
21 patient serum samples sent frozen to 4 labs
Lab1: Nephelometric (Immage 800)
Lab3: Turbidimetric (Roche Cobas c502a)
Twenty-one patient serum samples were then sent frozen to four laboratories using different methods/instruments for β2M:
Lab1, nephelometric (Beckman Coulter, Immage);
Lab2, chemiluminescent (Immulite 2000);
Lab3, turbidimetric (Roche Cobas c502) and
Lab 4 turbidimetric (Abbott Architect c16200).
All laboratories use the same method principle for serum albumin determination (bromcresol purple).
(actually Cobas c602)
Lab4: Tubidimetric (Abbott Architect c16200)
(Architect c16200)
Lab2: Chemiluminescence Immunoassay (Immunlite 2000)
(Immulite 2000 XPi)

11. Results Lab1: Nephelometric (Immage)
Lab4: Turbidimetric (Architect) (-9.3%) ( p<0.001)
Lab3: Turbidimetric (Cobas)
1.09 (-20.1%) ( p<0.001)
Lab2: CLIA (Immulite)
1.55 (-28.5%) ( p<0.001)
Lab1 produced β2M results consistently higher than other laboratories): a mean …higher than Internal QC was within acceptable limits for each lab.

12. Results – compare with QAP
Furthermore, differences found very closely reflected the method specific biases seen in recent national quality assurance data (RCPA QAP Tumour Marker Program, using … samples) that had prompted our investigation.
Lab3 &
Lab4
Lab2
Lab1

13. Results – ISS scores 1 2 3
Patient
Lab 1
Lab 2
Lab 3
Lab 4
Patient 3 1
Patient 5
Patient 1 2
Patient 2
Patient 4
Patient 6
Patient 8
Patient 9
Patient 10
Patient 12
Patient 13
Patient 14
Patient 15
Patient 19
Patient 21
Patient 7 3
Patient 11
Patient 16
Patient 17
Patient 18
Patient 20
ISS scores concordant by all 4 labs in only 12/21 (57%) patients
Initial ISS survival data (Greipp 2005)
Subsequent ISS prognostic scores derived from these results were concordant in only 12/21 (57%) of cases by all four laboratories (figure 2).
Compared to Lab1, ISS scores were classified one stage lower in 7, 5 and 3 patients by Lab2, Lab3 and Lab4, respectively.
One patient could have been classified either stage I,II or III depending on which laboratory performed the tests.
Albumin methods aligned well and had no impact on the variation in ISS scores, although four additional patients would have had discordant stages had albumin results not placed them in category 2 by all four laboratories. Excluding albumin results, only 8/21 (38%) of ISS β2M scores were concordant across all sites.

14. Discussion – Why is this important
Significant implications on the validity of the ISS for individual patients.
Illustrates difficulties in applying any test or staging system in the “real world”.
Implications for clinical trials:
Patient stratification (ensure same method for β2M)
Comparison between trials.
These findings have significant implications on the validity of the myeloma international staging system, as well as the results of subsequent clinical studies that have utilized the ISS to stratify risk groups. Perhaps more importantly, it illustrates the risk of assumption when applying any test or staging system to a broader population in the real world and real laboratories. As clinicians, we must understand the limitations of investigations we order.

15. Discussion Harmonisation of β2M methods is needed.
Currently no recognised international reference material available.
β2M method in the initial ISS study not reported.
involved 17 sites across 3 continents – unlikely methodology was concordant across all institutions.
These findings have significant implications on the validity of the myeloma international staging system, as well as the results of subsequent clinical studies that have utilized the ISS to stratify risk groups. Perhaps more importantly, it illustrates the risk of assumption when applying any test or staging system to a broader population in the real world and real laboratories. As clinicians, we must understand the limitations of investigations we order.

16. Conclusion Re-validation of the ISS system required:
Difficulties in standardisation of β2M methods.
Myeloma treatment and supportive care measures have advanced significantly since the original ISS study.
It seems appropriate that we re-evaluate the prognostic information we give to our patients.
Harmonisation of methods for assessment of β2M is required. Unfortunately there is no recognized international standard for beta-2 microglobulin determination for which other methods can be calibrated to. Re-validation of the ISS staging system in a large scale prospective study with clearly defined methods of β2M and albumin testing may be required. Given the addition of the immunomodulatory drugs and proteasome inhibitors to our armamentarium against myeloma; as well as improvements in supportive care, including the use of bisphosphonates and around autologous and reduced-intensity conditioning allogeneic stem cell transplant in recent years; it seems appropriate that we re-evaluate the prognostic information we give to our patients.

17. Acknowledgements Monash Medical Centre Healthscope Alfred Hospital
Kay-Weng Choy
Zhong Lu
James Doery
George Grigoriadis
Jake Shortt
George Streitberg
Healthscope
Mirette Saad
Alfred Hospital
Christina Trambas
RCPA Chemical Pathology QAP