Manganese-enhanced MRI predicts the histological grade of hepatocellular carcinoma in potential surgical candidates

Materials and methods The inclusion criteria were patients with HCC who underwent Mn-MRI prior to transplantation or resection from 2001–2008. T1-weighted MRI images were acquired at 0 and 24 h after manganese dipyridoxal diphosphate (MnDPDP) intravenous contrast medium and reviewed prospectively. Manganese retention at 24 h was correlated with tumour differentiation and disease-free survival. Results Eighty-six patients underwent Mn-MRI (transplantation 60, resection 26); 114/125 lesions (91%) that were arterialised as evidenced at computed tomography (CT) and had manganese uptake on MRI were HCC. There were 11 false positives (9%) that were regenerative nodules. Ten of fourteen non-manganese-retaining HCC (71%) were poorly differentiated, compared with only 13/114 manganese-retaining HCC (11%) ( p < 0.0001). Sensitivity, specificity, positive and negative predictive values of non-retention of MnDPDP in predicting poorly differentiated tumours were 0.43, 0.96, 0.71 and 0.88. Median disease-free survival of patients with non-manganese-retaining HCC was less than for patients with manganese-retaining HCC (14 ± 5 months versus 39 ± 3 months, log rank p = 0.025). Conclusion Non-manganese-retaining HCCs are likely to be poorly differentiated and have a poor prognosis. Manganese-enhanced MRI appears to have a role in preoperative assessment of HCC and warrants further evaluation. Introduction Hepatocellular carcinoma (HCC) is the third commonest cause of cancer deaths worldwide, and usually develops against a background of cirrhosis of any aetiology or chronic hepatitis B infection. 1,2 Patients with localized intrahepatic disease are potentially cured by either liver transplantation or resection, depending on the tumour extent and the presence of underlying liver disease. 3,4 Cross-sectional imaging by biphasic computed tomography (CT) and/or magnetic resonance imaging (MRI) are necessary to screen for extrahepatic disease and to stage the disease by evaluating tumour size and multiplicity prior to considering surgery. 5,6 The majority of centres select patients for liver transplantation on the basis of either Milan or University of California, San Francisco (UCSF) criteria, which incorporate tumour size and number but do not take tumour biology into account. 3,7 Additional information about tumour biology, including tumour grade and the presence of vascular invasion may be obtained from histological evaluation of tumour tissue. Histological grade and microscopic vascular invasion are both independent predictors of postoperative recurrence, 8–12 but are not routinely evaluated preoperatively due to the perceived risk of tumour seeding along the biopsy tract. 13,14 There is an urgent need to develop a diagnostic test that can accurately predict the biological behaviour of HCC. 15 Recent improvements in the sensitivity of cross-sectional imaging and development of newer contrast agents may provide additional insight into the biological behaviour of HCC in the near future. Manganese dipyridoxal diphosphate (MnDPDP) is a hepatocyte-specific contrast agent that has been shown to improve the discriminatory value of MRI in the diagnosis of benign and malignant hepatocellular neoplasms. 16,17 Previous reports have also suggested that retention of manganese within HCC may correlate with histological grade, 18–21 but this finding has not been replicated by others. 17 The aim of the present study was to correlate manganese retention with histological grade in patients undergoing liver transplantation or resection for HCC. Materials and methods This was a retrospective study of patients who underwent liver transplantation or resection for HCC at our institution between January 2001 and December 2008. Patients were identified from an electronic database. During the study period, all potential surgical candidates were staged using arterial and venous phase CT. MnDPDP-enhanced MRI (Mn-MRI) was used in patients who were either within or close to the limits of transplant criteria on CT (Milan criteria: single tumour less than 5 cm in diameter; three tumours or fewer less than 3 cm; no extrahepatic disease) or candidates for resection. Tumours were considered for resection if there was no underlying liver disease or if the patient had well-compensated cirrhosis (Child–Pugh class A or B). Since 2005, Mn-MRI has been increasingly used on a routine basis for all potential transplant candidates. MRI was performed on a 1.5 T MRI machine (Siemens Avanto 1.5T, Siemens Medical, Erlangen, Germany) using phased array multicoils. For MnDPDP-enhanced images, MnDPDP (Teslascan ® GE Healthcare, Amersham, UK) was administered by slow intravenous injection. Breath-hold, T1-weighted, spoiled gradient echo (GRE) in-phase and out-of-phase axial images were obtained pre- and immediately post-MnDPDP administration [repetition time (TR) 81 ms; echo time (TE) 2.38/4.76 ms, flip angle 70°). T1-weighted GRE water-excited axial and coronal images were obtained 24 h post-MnDPDP injection (TR 101 ms, TE 4.76 ms, flip angle 70°). Patients were included if they underwent preoperative Mn-MRI. Exclusion criteria were (1) incidental tumours found in explanted livers after transplantation; (2) fibrolamellar variants of HCC; and (3) lack of histological data due to tumour necrosis (secondary to pre-transplant loco-regional therapy). Preoperative clinical, laboratory and radiological data were obtained by review of a prospective database and electronic patient records. Preoperative Mn-MRI images were reviewed prospectively by two radiologists (D.L. and J.B.K.) to confirm the presence or absence of manganese retention at 24 h within each tumour. A tumour that retained any amount of contrast medium at 24 h was considered to be manganese-retaining. The histological grade of each tumour was classified as moderate to well differentiated or poorly differentiated. 22 The presence of microscopic vascular invasion and satellite lesions was determined for each tumour by review of postoperative histopathology reports. The presence of manganese retention was correlated with postoperative tumour histology. Data are presented as means ± standard deviation unless otherwise stated. Univariate statistical analysis was determined using the Mann–Whitney test for means of continuous variables and Fisher’s exact test for categorical variables. Kaplan–Meier survival analysis was used to determine actuarial disease-free survival (differences between groups calculated using log rank test). Statistics were performed using SPSS version 12 (Chicago, IL, USA). A p -value less than 0.05 was considered to be statistically significant. Results Preoperative data Between January 2001 and December 2008, 194 patients underwent liver transplantation ( n = 156) or liver resection ( n = 38) for HCC at our institution. One hundred and eight patients were excluded from the study due to lack of preoperative MRI ( n = 64), incidental tumours ( n = 30), fibrolamellar variant ( n = 2), or lack of histological data ( n = 12). Histological data were not available in 12 patients as a result of complete tumour necrosis secondary to preoperative chemo-embolization ( n = 11) or radiofrequency ablation ( n = 1). The remaining 86 patients formed the study group. The mean age of the study group was 58 ± 11 years. Seventy patients were men (81%). Seventy-six patients (88%) had underlying liver disease. The aetiology of liver disease was viral hepatitis ( n = 52), alcohol ( n = 11), haemochromatosis ( n = 7), primary biliary cirrhosis ( n = 2), non-alcoholic steatohepatitis ( n = 1), autoimmune ( n = 1), and cryptogenic ( n = 2). Mean preoperative unmodified Model for End-Stage Liver Disease score was 9 ± 6. An elevated serum alpha-fetoprotein (median 11 kU/l, range 7–71,800) was noted in 55 patients (64%). Preoperative CT detected 154 lesions (median one lesion per patient, range 1–4), of which 130 were arterialised. The mean size of the largest lesion detected by CT was 38 ± 25 mm (range 10–170 mm). Forty-eight out of 60 patients (80%) who underwent liver transplant were within Milan criteria on preoperative CT. Of the 12 patients who were outside Milan criteria, three had solitary tumours (diameters 60, 65, and 67 mm), and nine had multiple, small tumours (number 2–4; maximum diameter 20–45 mm). Mn-MRI was performed at a median of 106 days before surgery (range 1–483 days). Preoperative Mn-MRI detected 148 lesions (median one tumour per patient, range 1–4). The mean size of the largest lesion detected by MRI was 40 ± 28 mm (range 5–175 mm). Eighteen lesions (12%) in 12 patients failed to retain manganese at 24 h. One hundred and twenty-five out of 148 lesions (84%) detected by MRI were arterialised on CT. CT and MRI were concordant in 66 patients (77%). Operative data and tumour pathology Sixty patients underwent liver transplant and 26 patients underwent liver resection. Preoperative data for each group are summarized in Table 1 . In the transplant group, the types of graft used were whole ( n = 54), right lobe ( n = 5), and domino ( n = 1). Liver resection was anatomical in 13 patients, extended in five patients and non-anatomical in eight patients. In the resection group, the resection margins were negative in 23 out of 26 patients (88%). Fifty-nine explanted livers in 60 transplant recipients (98%) were cirrhotic. One patient was transplanted for bilobar HCCs on the background of chronic hepatitis B without cirrhosis. Of 26 resection specimens, six (23%) also had underlying cirrhosis due to hepatitis C ( n = 4), haemochromatosis ( n = 1), and cryptogenic cirrhosis ( n = 1). Ten patients (38%) in the resection group had pre-cirrhotic chronic liver disease (hepatitis B, n = 5; hepatitis C, n = 2; haemochromatosis, n = 2; alcohol, n = 1) and 10 patients (38%) had no identifiable underlying liver disease. One hundred and seventy-eight HCCs were identified in 86 patients. One hundred and forty-three tumours in 66 patients (77%) were moderate to well differentiated, and 35 tumours in 20 patients (23%) were poorly differentiated. Microscopic vascular invasion was present in 92 tumours in 42 patients (49%). Satellite lesions were present in 32 tumours in 13 patients (15%). Correlation between radiological and pathological findings Of 130 arterialised lesions on CT, 119 (92%) were HCCs on histological analysis. The remaining 11 arterialised lesions (8%) were found to be regenerative nodules. Of the 125 lesions that were arterialised on CT and detected by MRI, 114 (91%) were HCCs, and 11 were regenerative nodules. All false-positive lesions were detected in patients with viral liver disease ( n = 7; hepatitis B 4; hepatitis C 3). Of 23 lesions that were detected by MRI but did not arterialize on CT, 14 (61%) were HCCs. No lesions detected by either CT or MRI were found to be dysplastic nodules on histological analysis. The sensitivity and positive predictive value of Mn-MRI for detecting HCC were 0.72 and 0.86, respectively. MRI characteristics were correlated with histological features in 128 histologically proven HCCs ( Table 2 ). The mean size of moderate to well-differentiated and poorly differentiated tumours was similar (31 ± 26 mm versus 41 ± 28 mm, p = 0.09, Mann–Whitney test). Tumours greater than 5 cm in diameter were more likely to be poorly differentiated (32% versus 16%, p = 0.1) and exhibit vascular invasion (74% versus 39%, p = 0.01) than smaller tumours, but this did not reach statistical significance for tumour grade. There was a significant association between microscopic vascular invasion and poorly differentiated tumours. Of 57 tumours with vascular invasion, 17 (30%) were poorly differentiated, compared to only six out of 71 tumours (8%) without vascular invasion ( p = 0.002). There was no association between tumour size and the presence of satellite lesions (>5 cm versus <5 cm: 11% versus 17%, p = 0.74). Of 14 tumours (71%) that did not retain manganese after 24 h, 10 (71%) were poorly differentiated, compared with only 13 out of 114 tumours (11%) that retained manganese ( p < 0.0001, Fisher’s exact test). Of the 14 HCC that did not exhibit arterial enhancement on CT, three tumours failed to retain manganese and were all poorly differentiated. The sensitivity and specificity of manganese retention in predicting poor differentiation were 0.43 (95% CI 0.26–0.63) and 0.96 (95% CI 0.91–0.99), respectively. The positive and negative predictive values were 71 and 89%, respectively. Positive and negative likelihood ratios were 11.4 (95% CI 3.9–33.2) and 0.6 (95% CI 0.4–0.8). There was no significant correlation between manganese retention and tumour size, vascular invasion or presence of satellite lesions (see Table 2 ). Clinical follow-up Of 60 patients who underwent liver transplant, eight (13%) have developed tumour recurrence (systemic metastases, n = 5; liver and systemic, n = 3) at a median follow-up of 12 months (range 3–60 months). Fifteen out of 26 patients (58%) who underwent liver resection also developed tumour recurrence (liver, n = 13; lung, n = 2) at a median follow-up of 12 months (range 4–57 months). For transplant recipients, there was no difference in the incidence of recurrence in patients who were within or outside Milan or UCSF criteria ( Table 3 ). Recurrence was more likely after resection compared with after transplantation (58% versus 13%, p = 0.0001). Patients with tumours exhibiting vascular invasion or poor differentiation were at increased risk of recurrence, and had significantly reduced actuarial disease-free survival ( Fig 1 ). Patients with tumours that failed to retain manganese also had significantly reduced actuarial disease-free survival compared with patients with managanese-retaining HCC (14 ± 5 months versus 39 ± 3 months, log rank p = 0.023, Fig 1 b). The effect of tumour differentiation on disease-free survival was also significant for subgroups of patients according to treatment group (transplantation, log rank p = 0.001; resection, p = 0.004). Due to small sample size, the effect of manganese retention by tumours was not significant when patients were analysed according to treatment. Discussion The standard treatment for patients with HCC confined to the liver is surgical resection or liver transplantation. 23 Despite widespread adoption of the Milan criteria by most transplant centres, postoperative tumour recurrence develops in up to 30% of patients, and is a major cause of morbidity and mortality. 7,9,24,25 Recurrence indicates the presence of microscopic residual disease, which may progress rapidly in an immunocompromised transplant recipient. The risk factors for recurrence after either resection or transplantation are well established, and principally relate to tumour volume (size, multiplicity, and resection margin) and tumour biology (grade, vascular invasion, satellite lesions). 7,12,23,25–27 Triple-phase CT provides information about tumour size, number, and vascularity, but does not give any indication of tumour biology (except for portal venous invasion), and understages a significant proportion of patients who are submitted to liver transplantation. 3 MRI appears to be more accurate than CT in terms of intrahepatic staging, and has become a useful adjunct in preoperative assessment, particularly in the transplant setting. 28,29 The diagnostic performance of MRI has been enhanced by the use of hepatocyte-specific contrast agents, such as Gd-EOB-DTPA and gadobenate dimeglumine, 30,31 although to date, neither agent has been shown to correlate with tumour grade. Availability of a non-invasive diagnostic test to assess tumour biology without the need for biopsy would be an advantage in the preoperative staging of HCC. It may be possible to minimize inappropriate use of scarce donor organs, and to identify those patients with larger tumours with favourable biology who might benefit from transplantation. 10 The present study provides convincing data to support a role for manganese-enhanced MRI in preoperative assessment of tumour biology in patients being considered for either liver transplantation or resection. MnDPDP is an anionic manganese chelate that readily yields free Mn 2+ ions, which are taken up by hepatocytes and excreted in bile. 32 Mn-MRI is capable of distinguishing between hepatocellular and non-hepatocellular liver tumours. 17,33 Previous studies have also indicated that the degree of enhancement of HCCs during delayed-phase images may correlate with histological differentiation. 18,19 Correlation between Mn-MRI and tumour differentiation was first observed in an animal model of HCC in 1993, 18 and subsequently in humans in 1996. 19 However, other studies have failed to corroborate this finding, 17,20,21,34 which may be due to a combination of small sample sizes, different time intervals between administration of MnDPDP and image acquisition (range 0–24 h), and incomplete histological data. Timing of image acquisition varies between centres and appears to play an important role in the diagnostic capability of Mn-MRI. The present data have shown that manganese retention after 24 h is a predictor of tumour differentiation. In other studies, histological data have been derived primarily from core biopsies, which have been shown to correlate poorly with postoperative histology. 35 A recent study reported on 37 resection specimens, and demonstrated a weak association between manganese uptake and tumour differentiation. 21 Our report, which includes complete radiological and histological correlation for 128 HCCs, is the largest series to date, and shows a highly significant relationship between manganese uptake at 24 h and both tumour differentiation and disease-free survival ( Table 2 ). This relationship appears to be true for all patients, irrespective of the aetiology of liver disease (viral or non-viral), presence/absence of cirrhosis and mode of treatment (resection or transplantation). However, due to small numbers in the subgroup of patients with viral liver disease and those without cirrhosis, this did not reach statistical significance ( Table 2 ). The different patterns of manganese retention by HCC are illustrated in Fig 2 . Although the present results indicate that larger tumours are more likely to be poorly differentiated, this was not statistically significant. Sixty-eight percent of tumours greater than 5 cm were moderately to well differentiated, of which the majority (86%) retained manganese. There was no association between tumour size and manganese retention, which concurs with previous data. 21 In addition, 10 out of 12 transplant recipients (83%) who were outside Milan criteria on CT had manganese-retaining tumours on MRI. Eight (80%) of these patients had well or moderately well-differentiated tumours on histology, and may have been excluded from transplantation if the Milan criteria had been strictly applied. In addition, five out of 48 transplant recipients (10%) who were within Milan criteria had lesions that did not retain manganese, including four patients with poorly differentiated tumours. Critically, this observation correlated with clinical outcome, as there was a significant reduction in median disease-free survival in patients with manganese non-retaining HCC. Although it is probable that Mn-MRI correlates with tumour grade in patients with a lower tumour burden, the present data cannot be extrapolated back, as the criteria for using Mn-MRI in our unit during the study period was selective. This is a limitation of this retrospective study, but could be addressed by prospective evaluation in a consecutive series of patients with HCC. In conclusion, manganese retention at 24 h is highly predictive of the histological grade of HCCs. 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