Clinical classification of cancer cachexia: phenotypic correlates in human skeletal muscle

Abstract

Background: Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system.

Methods: 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation.

Findings: Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (p = 0.022) and with >10%WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups.

Conclusion: Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.

Figure 1. Fibre type cross sectional area (FCSA) according to different definitions of cachexia.

(A) Mean (± SEM) fibre size for both MyHC1 and MyHCIIa. A comparison is made between patients with the proposed cachexia definition absent (dark grey) and those with the proposed cachexia definition present (light grey) for the four definitions set out in Methods (I–IV). (*, P<0.05 and **, P<0.01, by Student's t test). (B) Immunohistological sections of muscle for a healthy control, patient with weight loss alone (10.1%) (Group II), and patient with low muscularity and >2% weight loss (Group IV). Laminin is shown in green, MyHC1 shown in red, and MyHCIIa is shown in blue.

Figure 2. Variations in protein and nucleic acid content according to the different definitions of cancer cachexia.

A comparison is made between patients with the proposed cachexia definition absent (dark grey) and those with the proposed cachexia definition present (light grey) for the four definitions set out in the methods (I–IV). (A) Mean (± SEM) wet weight protein content. (B) Mean (± SEM) RNA content. (C) Mean (± SEM) DNA content. (C) Mean (± SEM) RNA/DNA ratio. (*, P<0.05 by Student's t test).

Figure 3. Skeletal muscle Beclin and ATG5 protein levels in patients with or without >10% weight loss (Group II).

Western blot analysis with indicated antibodies, α-tubulin was used as a loading control. Graph shows the mean (± SEM) protein level represented in arbitrary units (A.U). (*, P<0.05 and **, P<0.01, by Student's t test).

Figure 4. Total SMAD3, phospho-SMAD3 and ratio of phospho-SMAD3/SMAD3 in patients with or without >5% WL (Group I) levels.

Western blot analysis with indicated antibodies, α-tubulin was used as a loading control. Graph shows the mean (± SEM) protein level represented in arbitrary units (A.U). (*, P<0.05 by Student's t test).

Figure 5. Inflammatory pathways in patients with (CRP >10 mg/L) and without (CRP ≤10 mg/L) systemic inflammation.

(A) Western blot analysis in the presence or absence of systemic inflammation with indicated antibodies, α-tubulin was used as a loading control. (B) Graph shows the mean (± SEM) protein level of phospho-NF-κB, represented in arbitrary units (A.U). (C) Representative immunohistochemistry and nuclei count of phospho-STAT3 (area shown is representative of field) of a patient with or without systemic inflammation. (D) Graph shows the staining density of phospho-STAT3 nuclei (A.U.) (± SEM) in the presence or absence of systemic inflammation.