Patterns of hepatotoxicity after chemotherapy for colorectal cancer liver metastases: Effect of Bevacizumab

M. Klingerº , S. Kandutsch¹, S. Hackerº , B. Gruenberger2, T. Gruenbergerº ºDept. of Surgery, Hepatobiliary Service, ¹Clinical Institute of Pathology, Medical University of Vienna;

2Department of Oncology, Rudolfstiftung Hospital Vienna, Austria

Results

Sinusoidal Damage and Bevacizumab:Sinusoidal damage is the result of an injury pattern with sinusoidal dilatation, perisinusoidal fibrosis and hepatocellular necrosis as histomorphologic signs. In the table, the dispersion of sinusoidal dilatation grades among the non-bevacizumab and the bevacizumab group is depicted. The incidence of liver specimen positive for sinusoidal dilatation (Grade 1-3) is 42.3% in the bevacizumab and 52.3% in the non-bevacizumab group, respectively. Bevacizumab reduces the sinusoidal dilatation grade significantly (p< 0.05). Futhermore bevacizumab significantly reduces the markedness of perisinusoidal fibrosis and hepatocellular necrosis as shown in the table. In a multivariate regression model bevacizumab showes an independent effect on dilatation grade after adjustment for age, sex, diabetes and BMI (p<0.05).

Hepatic Steatosis, Fibrosis and Bevacizumab:Tissue samples are analyzed for steatosis and steatohepatitis employing the NAFLD activity score (NAS). Among the non-bevacizumab and bevacizumab group there is no difference in the distribution of NAS (p= 0.49). In the table, the absolute counts and percentages of the two groups are outlined. Hepatocyte ballooning is significantly less distinct in the bevacizumab group (p<0.05). A multivariate regression model shows no influence of parameters employed (age, sex, BMI, age, diabetes, bevacizumab) on NAS. The bevacizumab and the non-bevacizumab group do not show a difference with respect to liver fibrosis.

Conclusion

Bevacizumab protects against sinusoidal dilatation and its associated pathohistological patterns.

Bevacizumab has no impact on hepatic steatosis and fibrosis.

This analysis provides the histological background for the save use of bevacizumab before liver resection.

BACKGROUNDCombining bevacizumab with XELOX/ FOLFOX4 is inreasingly being recognized as state of the art in the neoadjuvant treatment of colorectal cancer liver metastases. Our group has recently shown that bevacizumab does not increase perioperative morbidity after surgical resection of colorectal cancer liver metastases. Furthermore, we have shown recently that XELOX/FOLFOX4 causes profound morphological changes in liver parenchyma with sinusoidal dilatation and liverfibrosis as its pathohistological correlate. The aim of this study was to determine if the addition of bevacizumab does change the injury pattern of XELOX/ FOLFOX4.PATIENTS, METHODSLiver specimen of 56 patients receiving biweekly bevacizumab plus capecitabine and oxaliplatin for six cycles ( the sixth cycle of therapy did not including bevacizumab) and liver specimen of 50 patients solely receiving capecitabine and oxaliplatin (XELOX) or 5-fluorouracil, leucovorin plus oxaliplatin (FOLFOX4) for six cycles were analyzed.RESULTSSteatosis, fibrosis, concomitant necroinflammation as well as sinusoidal injury were evaluated according to widely accepted pathohistological scores.We found no difference in grading of steatohepatitis among the two groups. The incidence of sinusoidal dilatation of any grade was significantly lower in patients treated with bevacizumab. Furthermore, the incidence of moderate or severe sinusoidal dilatation was significantly higher in patients treated without bevacizumab.CONCLUSIONWe conclude that the addition of bevacizumab to XELOX/FOLFOX4 therapy has a protective effect with respect to sinusoidal injury.

Introduction:

Bevacizumab (Avastin®), a monoclonal humanized antibody directed against vascular- endothelial derived factor (VEGF) is being increasingly used in the treatment of mCRC. Bevacizumab is considered being only effective in combination with cytotoxic drugs and may be given in combination with 5-fluorouracil based chemtherapy plus oxaliplatin (5-FU/ oxaliplatin) [1]. The neoadjuvant use of oxalplatin is associated with the occurence of a distinctive type of hepatic injury, namely sinusoidal dilatation [2,3]. For 5-fluorouracil and irinotecan, an association with hepatic steatosis has been shown. For this type of hepatic steatosis belonging to the family of non-alcoholic fatty liver disease (NAFLD) the term CASH (chemotherapy-associated steatohepatitis) has been introduced. For bevacizumab, impaired wound healing and increased transfusion requirements in liver surgery have been reported. However, we recently reported the save use of bevacizumab before liver surgery [4]. In this study, we compared for non tumor bearing liver histology between patients treated with 5-FU/ oxaliplatin and patients treated with 5-FU/ oxaliplatin plus bevacizumab.

Patients and Chemotherapy:

Two phase II trials of our institution including patients with resectable colorectal cancer liver metastases were analysed retrospectively for pathohistology of the non tumor bearing liver. One trial affiliated 56 patients treated neoadjuvantly with capecitabine, oxaliplatin and bevacizumab (bevacizumab group). The primary objective of this study was to assess the feasibility of bevacizumab therapy before surgery [4]. The second trial analysed 50 patients treated with 5-flourouracil based chemotherapy (30 patients received capecitabine, 20 patients received 5-flourouracil plus leucovorin) and oxaliplatin before surgery with response rate, postoperative morbidity and recurrence-free survival being evaluated (non-bevacizumab group)[5]. Treatment, as described in detail in [4,5] was administered for six cycles, the sixth cycle of therapy did not include bevacizumab. Liver resection was performed 2 to 5 weeks after the last administration of chemotherapy.

Histopathological Examination:

Tissue samples were obtained from 99 patients, samples of seven patients (4 out of the bevacizumab and 3 out of the non-bevacizumab group) were inconclusive because of a too small volume of the non tumor bearing liver volume. Non tumor bearing liver parenchyma of the resected liver was fixed in 4% neutral buffered formaldehyde and embedded in paraffin blocks. The morphological analyses were based on hematoxilin-eosin (HE) and chromotrop-anilinblue trichrome staining (CAB). Examination of the slides was done by two pathologists blinded for the chemotherapeutic regime. Sinusoidal injury was graded comprising sinusoidal dilatation, centrilobular vein fibrosis, nodular hepatic regeneration and hepatocellular necrosis. Sinusoidal dilatation was graded as follows: 0, absent; 1, mild (centrilobular involvement limited to one-third of the lobular surface); 2, moderate (centrilobular involvement extending to two-thirds of the lobular surface); 3, severe (complete lobular involvement). Steatosis, which was graded separately for micro- and macrovesicular steatosis, was estimated as the percentage of involved hepatocytes: 0, < 5%; 1, 5-33%; 2, 33-66%; 3, >66%. Lobular Inflammation was graded separately for neutrophile granulocytes and leucocytes as follows: 0, 0 foci; 1, <2 foci/200x; 2, 2-4 foci/200x; 3, >4 foci/200x. Hepatocyte ballooning was scored: 0, none; 1, few balloon cells; 2, many cells/prominent ballooning. Fibrosis stage was scored with: 0, none; 1 perisinusoidal or periportal; 2, perisinusoidal and portal/periportal; 3, bridging fibrosis and 4, cirrhosis. Finally all slides were graded according the NAFLD Activity Score (NAS) [6] by following the guidelines of the Non Alcoholic Steatohepatitis Clinical Research Network (http//tpis.upmc.edu). The total NAS represents the sum of scores of steatosis, lobular inflammation and hepatocyte ballooning, and ranges from 0-8. For steatosis and lobular inflammation, the higher score (macro vs. microvesicular and neutrophil vs. monocyte) was employed for grading,

 Low power magnification (10×) of chromotrop–anilinblue trichrome stain (A,C) of the liver and HE stain (B,D) with mild (A,B) and severe dilatation (C;D). The chromotrop–anilinblue trichrome stain stains the collagen fibers in parenchymal tissue and was used here to better visualise fibrosis. The filled triangle indicates perisinusoidal fibrosis; the arrow points to an area of severe sinusoidal dilatation (grade 3) with erythrocyte congestion. The asterisk marks a centrilobular vein, the crosses indicate portal fields. In panel B, an area with erytrocyte congestion is indicated.

Literature

1. Nordlinger B, Sorbye H, Glimelius B, et al.: Perioperative chemotherapy with FOLFOX4 and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC Intergroup trial 40983): a randomised controlled trial. Lancet 2008;371:1007-1016.

2. Rubbia-Brandt L, Mentha G, Terris B: Sinusoidal obstruction syndrome is a major feature of hepatic lesions associated with oxaliplatin neoadjuvant chemotherapy for liver colorectal metastases. J Am Coll Surg 2006;202:199-200.

3. Kandutsch S, Klinger M, Hacker S, et al.: Patterns of hepatotoxicity after chemotherapy for colorectal cancer liver metastases. Eur J Surg Oncol 2008.

4. Gruenberger B, Tamandl D, Schueller J, et al.: Bevacizumab, capecitabine, and oxaliplatin as neoadjuvant therapy for patients with potentially curable metastatic colorectal cancer. J Clin Oncol 2008;26:1830-1835.

5. Gruenberger B, Scheithauer W, Punzengruber R, et al.: Importance of response to neoadjuvant chemotherapy in potentially curable colorectal cancer liver metastases. BMC Cancer 2008;8:120.

6. Kleiner DE, Brunt EM, Van Natta M, et al.: Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313-1321.

ParameterAll patients (n= 99)

Non-bevacizumab group (n= 47)

Bevacizumab group (n= 52)

P

n % n % N %

Sinusoidal dilatation

Any grade 98* 100 46* 100 52 100

p< 0.05

Grade 0 52 53.1 22 47.8 30 57.7

Grade 1 25 25.5 8 17.4 17 32.7

Grade 2 9 9.2 5 10.9 4 7.7

Grade 3 12 12.2 11 23.9 1 1.9

Perisinusoidal Fibrosis

26/98* 26.517/46

*37.0 9/52 17.3 p< 0.05

Hepatocellular Necrosis

33/98* 33.723/46

*50.0 10/52 19.2 p< 0.05

NAS grading

Any grade 99 100 47 100 52 100

p=0.49

Grade 0 19 19.2 7 14.9 12 23.1

Grade 1 29 29.3 14 29.8 15 28.8

Grade 2 22 22.2 12 25.5 10 19.2

Grade 3 11 11.1 5 10.6 6 11.5

Grade 4 9 9.1 6 12.8 3 5.8

Grade 5 7 7.1 1 2.1 6 11.5

Grade 6 2 2 2 4.3 0 0

Grade 7 0 0 0 0 0 0

Grade 8 0 0 0 0 0 0

Hepatoyte ballooning

Any grade 99 100 47 100 52 100

p< 0.05Grade 0 52 52.5 15 31.9 37 71.2

Grade 1 45 45.5 32 68.1 13 25.0

Grade 2 2 2.0 0 0 2 3.8

Fibrous staging

Any stage 99 100 47 100 52 100

p=0.30

Grade 0 27 27.3 12 25.5 15 28.8

Grade 1 49 49.5 21 44.7 28 53.8

Grade 2 10 10.1 6 12.8 4 7.7

Grade 3 13 13.1 8 17.0 5 9.6

Grade 4 0 0 0 0 0 0

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* In one patient, sinusoidal dilation was not evaluable