Use of total and unbound imatinib and metabolite LC-MS/MS assay to understand individual responses in CML and GIST patients.

OBJECTIVES: Trough total imatinib (t-IM) concentrations have been reported to be associated with therapeutic and toxic responses in patients with chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST). Little is known about the relationships between effects and concentrations of either unbound imatinib (f-IM) or imatinib's major metabolite, N-desmethyl imatinib (NDI). In part, this is because of the lack of a single, validated, well-described clinically useful assay for these measurements. The authors report the development and application of such an assay. MATERIALS AND METHODS: A single liquid-chromatography tandem-mass-spectrometry assay was used to monitor t-IM, f-IM, and t-NDI concentrations in CML and GIST patients treated at a tertiary German teaching hospital. The assay was also validated for measuring other kinase inhibitors, including t-nilotinib, sunitinib, and erlotinib. Ultrafiltration assays were validated and used to measure f-IM and to compare free fractions to plasma α1-acid glycoprotein concentrations (AGP). RESULTS: The assays were linear over a working range (in micrograms per liter) of 8.4-8370, 8.3-4165, and 1.0-250 and had within- and between-run coefficient of variance of <7%, <12%, and <9% for t-IM, t-NDI, and f-IM, respectively. The f-IM assay was reproducible despite high (25.2%-31.6%) but concentration-independent binding to ultrafiltration devices. Clinically relevant results, such as nondetectable (ND) t-IM (<8.4 µg/L) in non-responders and >1500 µg/L in patients with major toxicity, were found. Of 156 total samples from 68 adult CML patients and 127 total samples from 42 adult GIST, only 48 samples from 22 CML patients and 40 samples from 20 GIST patients were trough samples with adequate dosing and collection information. More than half (27 of 48 CML and 24 of 40 GIST) had t-IM concentrations ≥10% below recommended target concentrations (1002 µg/L for CML and 1100 µg/L for GIST). Concentrations >50% over targets were also found in 6 of 48 CML and 4 of 40 GIST samples. Wide variations in concentrations of t-IM (range, ND to 2973 µg/L), t-NDI (range, ND to 659 µg/L), f-IM (range, 8.3-262 µg/L), and t-IM:f-IM ratios (range, 2.6%-14%) were found both between and within patients. A statistically significant association (Spearman correlation coefficient and P value for all samples, r = 0.290 and P = 0.023; for trough only, r = -0.585 and P = 0.028) was found between AGP and f-IM concentrations but wide interpatient and intrapatient variations made individual predictions unreliable. CONCLUSIONS: The liquid-chromatography tandem-mass-spectrometry methods developed provided information useful to understand individual responses to therapy even though necessary sampling and dosing information was often not available. Wide unpredictable variations in t-IM, t-NDI, and f-IM were found. Clinical outcome trials are needed to examine whether f-IM or NDI monitoring can improve the ability to predict individual responses.

Analysis of a case with disappearance of the primary gastrointestinal stromal tumor and progressive liver metastases under long-term treatment with tyrosine kinase inhibitors.

The response of gastrointestinal stromal tumors (GISTs) to tyrosine kinase receptor inhibitors (TKR-I) has been a breakthrough for small molecular therapy. We report here on the very different long-term outcome of a synchronous metastatic GIST with complete remission of the primary tumor and progressive liver metastases under TKR-I therapy. In 2003, a 52-year-old patient was diagnosed with gastric GIST and synchronous multiple liver metastases. Therapy with imatinib, 400 mg daily, was started immediately. Fifteen months later, the primary was no longer detectable by endoscopy. In 2006, progression of the liver metastases was observed. Mutation analysis of the initial biopsy specimen from the primary, as well as the biopsy from the three main liver metastases after 3 years of imatinib treatment, revealed the common KIT exon 11 deletion (W557_K558del) in all tumor samples. Two of the metastases had a separate secondary mutation in KIT exon 14 and 17, respectively, while the largest cystic metastatic lesion had no other mutation. Imatinib was then increased to a daily dose of 800 mg, and in April 2007 the treatment was changed to sunitinib. Fifty-two months after initial diagnosis, the patient died of liver failure. At no time point, relapse of the gastric primary tumor was observed. Whilst TKR-Is are commonly very effective in treating GISTs, the present case illustrates their varying effects regarding the clinical behavior and genetic variations within different tumors of the same patient after long-term treatment.

Response of the primary tumor in symptomatic and asymptomatic stage IV colorectal cancer to combined interventional endoscopy and palliative chemotherapy.

BACKGROUND: The treatment of the primary tumor in advanced metastatic colorectal cancer (CRC) is still a matter of discussion. Little attention has thus far been paid to the endoscopically observable changes of the primary in non-curatively resectable stage IV disease. METHODS: 20 patients [14 men, 6 women, median age 67 (39-82) years] were observed after initial diagnosis of non-curatively resectable metastasized symptomatic (83%) or asymptomatic (17%) CRC, from June 2002 to April 2009. If necessary, endoscopic tumor debulking was performed. 5-FU based chemotherapy was given immediately thereafter. In 10 patients, chemotherapy was combined with antibody therapy. RESULTS: Response of the primary was observed in all patients. Local symptoms were treated endoscopically whenever necessary (obstruction or bleeding), and further improved after chemotherapy was started: Four patients showed initial complete endoscopic disappearance of the primary. In an additional 6 patients, only adenomatous tissue was histologically detected. In both these groups, two patients revealed local tumor relapse after interruption of therapy. Local tumor regression or stable disease was achieved in the remaining 10 patients. 15 patients died during the observation time. In 13 cases, death was related to metastatic disease progression. The mean overall survival time was 19.6 (3-71) months. No complications due to the primary were observed. CONCLUSION: This study shows that modern anti-cancer drugs combined with endoscopic therapy are an effective and safe treatment of the symptomatic primary and ameliorate local complaints without the need for surgical intervention in advanced UICC stage IV CRC.

Does imatinib turn recurrent and/or metastasized gastrointestinal stromal tumors into a chronic disease? - single center experience.

BACKGROUND: Gastrointestinal stromal tumors (GIST) are mesenchymal tumors of the gastrointestinal tract supposed to arise from the cells of Cajal because of gain-of-function mutations of the tyrosine receptor kinases c-kit or platelet-derived growth factor receptor A. Imatinib selectively inhibits the kinase activity of both receptors. Despite this breakthrough in the treatment of GIST, resistance against imatinib has been reported to be as high as 50% after the first 2 years of treatment. AIM: Outcome of 13 consecutive patients with relapsed or metastasized GIST who were treated with imatinib was analyzed. RESULTS: Mean duration of treatment was 53.5 months. Four patients developed progressive disease and died after a mean treatment time of 31 months in spite of increase of imatinib dosages to 800 mg daily. Two patients (23%) developed a progressive disease after 46 months or 52 months of treatment. Two patients had a stable disease and five had a partial response. The overall progression rate was 46%, the mean survival time since primary diagnosis was 85.8 months. CONCLUSION: From our experience, frequency of resistance development to imatinib may be below that given in the literature (50% after 2 years). Individual treatment in specialized centers may improve compliance.

Immune cells in primary gastrointestinal stromal tumors.

INTRODUCTION: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. They are regarded as having relatively uniform histology, although their potential for malignant behavior varies. Despite a strong promoting role of tumor-infiltrating innate immune cells in neoplastic progression, the presence of immune cells in GISTs has not yet been studied. METHODS: A total of 47 untreated, c-kit-positive primary GISTs were immunohistochemically analyzed to distinguish histiocytic and dendritic cells (DCs) (KIM-1P, fascin, and CD68) from cells of lymphoplasmacellular origin (CD3, CD20, and CD56). Furthermore, the gene expression of proinflammatory cytokines was characterized by real-time, reverse transcription-PCR analysis of total RNA extracted from frozen tissue samples. RESULTS: KIM-1P+ cells were the dominant immune cells (851+/-295 cells/mm2) and were scattered among the tumor cells. Most of the KIM-1P+ cells showed cellular projections characteristic of DCs. Fascin positivity identified a subgroup of DCs. In comparison to KIM-1P+ cells, there were significantly fewer CD68+ macrophages (196+/-217 cells/mm2). CD3+ T cells were the dominant lymphocytes (201+/-331 cells/mm2), whereas B cells (60+/-126 cells/mm2) were few. On transcriptional level, a concomitant gene expression of cytokines for the classical acute phase cytokines TNF-alpha and IL-6 was missing, thus supporting the rather innate status of immune cells. CONCLUSION: GISTs contain, beside T lymphocytes, a high number of monocyte-derived cells, which we suggest are, at least in part, immature DCs. Together with the lack of gene expression of inflammatory cytokines in tumor tissue our results point to a possible 'symbiotic relationship' between the tumor and the local immune cells.

Chemotherapy-induced suppression to adenoma or complete suppression of the primary in patients with stage IV colorectal cancer: report of four cases.

BACKGROUND: Although modern chemotherapy of stage IV advanced colorectal cancer (CRC) has impressively improved overall survival, the response of the primary tumor has not been studied because surgical resection of the primary continues to be the standard procedure in stage IV CRC. AIM: Long-term follow-up of the primary in patients with stage IV CRC under chemotherapy. METHODS AND RESULTS: Here we report on the histological changes in the primary tumor in four patients suffering from stage IV CRC. Systemic chemotherapy was started immediately after endoscopic tumor debulking in three cases. In one case no endoscopic intervention was performed before chemotherapy. Neither macroscopic nor histological evidence for malignant tumor growth was found at the former site of the primary after 6, 23, 26 or 48 months, respectively. Two patients had a complete suppression of the primary, two patients had an adenoma at the former site of the primary. To date, three patients have died because of progression of liver metastases and one patient is still alive with no signs of tumor growth. CONCLUSION: The four cases illustrate that today's chemotherapy may effectively induces suppression of the primary in CRC. The development of CRC may follow different pathways.

Site-dependent differential KIT and PDGFRA expression in gastric and intestinal gastrointestinal stromal tumors.

In gastrointestinal stromal tumors (GISTs), mutually exclusive gain-of-function mutations of KIT and PDGFRA are associated with different mutation-dependent clinical behavior. Taking into account the well-known different clinical behavior of GISTs from the stomach or the intestine, the aim of the current study is to evaluate the mutation- and site-dependent effects on mRNA and protein expression of KIT and PDGFRA in a large series of primary GISTs. Fresh-frozen tissue of 53 primary GISTs from gastric (75%) or intestinal (25%) sites were analyzed for mutation of KIT or PDGFRA using direct sequencing. Furthermore, KIT and PDGFRA mRNA and protein expression were determined using quantitative RT-PCR and quantitative densitometric evaluation of Western blot data. Each tumor either had a mutation of KIT (79%) or PDGFRA (21%). All GISTs with PDGFRA mutation were from gastric sites. Mutation-dependently, GISTs with KIT mutation had a significantly higher expression of KIT and at the same time a significantly lower expression of PDGFRA compared to GISTs with PDGFRA mutation. Site-dependently, gastric GISTs had a significantly higher expression of PDGFRA and a significantly lower expression of KIT compared to intestinal GISTs. Additionally, even if the KIT-mutated GISTs alone were considered, a significantly higher expression of PDGFRA could be observed in gastric than in intestinal tumors. We also found a significant correlation between a higher protein expression of PDGFRA and longer disease-free survival. The correlation of gastric site and PDGFRA mutation with higher PDGFRA expression and longer disease-free survival suggests different regulatory roles of KIT and PDGFRA gene expression on the control of cell proliferation, and, thereby on clinical behavior. The higher PDGFRA expression in gastric GISTs possibly contributes to the well-known site-dependent clinical behavior.

Multicentric sporadic gastrointestinal stromal tumors (GISTs) of the stomach with distinct clonal origin: differential diagnosis to familial and syndromal GIST variants and peritoneal metastasis.

Most sporadic gastrointestinal stromal tumors (GISTs) occur solitary, whereas a multicentric appearance is suspicious for a familial or syndromal setting such as with germline mutations of proto-oncogene tyrosine protein kinase Kit (KIT) or platelet derived growth factor receptor alpha (PDGFRA), or even for metastases. The aim of this study was to evaluate whether multicentric sporadic GISTs are of clonal origin. Four patients with 1 clinically apparent tumor (mean size 5.6 cm) and 1 to 3 further small incidental tumors (mean size 0.7 cm) were analysed by mutation analysis and comparative genomic hybridization for mutations of KIT and PDGFRA and chromosomal imbalances in their tumors. No clinicopathologic features have been found being indicative of one of the established familial or syndromal GIST variants. Each of the small GISTs were localized in the muscularis propria, and were visible from the serosal but not from the mucosal side. Different mutations of KIT and PDGFRA were present among individual tumors of each patient, and germline mutation of KIT and PDGFRA could be excluded. Comparative genomic hybridization revealed a mean count of 7 chromosomal imbalances in the clinically apparent tumors compared with a mean count of 0.3 in the small incidental counterparts. Sporadic GISTs can appear multicentric by coincidence. They are an important differential diagnosis to familial and syndromal GIST variants, or even to peritoneal metastases. Different mutations of KIT and PDGFRA among individual tumors in 1 patient refer to different clonal origin of multicentric sporadic GISTs. The type of mutation of KIT and PDGFRA was independent of tumor size, whereas small GISTs <1 cm rarely had genomic imbalances.

Colorectal tumors with complete obstruction--endoscopic recovery of passage replacing emergency surgery? A report of two cases.

BACKGROUND: Incomplete or complete obstructive ileus due to colorectal cancer is generally treated by emergency surgery that has higher morbidity and mortality than elective surgery. CASE PRESENTATION: Here we describe an endoscopic technique by which a safe bowel decompression was performed instead of emergency surgery in two patients with complete tumorous obstruction of the colon. By means of a polypectomy snare, a soft wire, an ERCP catheter, a set of endoscopes with different diameters (baby endoscope, gastroscope) and of argon plasma coagulation the tumor mass was reduced and the tumor stenosis was passed. The patients recovered from symptoms of colon obstruction, no procedure-associated complications were observed. One patient had surgery of the sigmoid tumor one week later (UICC-stage III), the other patient (UICC-stage IV) received systemic chemotherapy starting one week after endoscopic decompression. CONCLUSION: Complete tumorous obstruction of the colon may be managed by endoscopic tumor debulking avoiding high risk emergency surgery and allowing immediate medical treatment of the primary tumor and of metastases.

Hepatic tumorigenesis in acute hepatic failure.

We report on a 59-year-old woman and a 31-year-old man with no previous medical history of liver disease presenting with acute liver failure probably caused by drug toxicity. High urgency liver transplantation was performed 30 and 51 days after the onset of symptoms, respectively. Histomorphological evaluation of the explanted livers revealed incidental dysplastic nodules and hepatocellular carcinoma of up to 8 mm in diameter. Up to now only a few cases of metastatic liver disease and even fewer cases of primary liver cancer presenting as acute liver failure have been described. Our cases indicate hepatic tumorigenesis not as a cause of hepatic failure but either as an event taking place in parallel or as a process being induced by progressive liver failure.

Expression of osteoactivin in rat and human liver and isolated rat liver cells.

BACKGROUND/AIMS: The transmembrane glycoprotein osteoactivin is expressed in osteoblasts, dendritic cells and tumor cells. It is suggested to influence osteoblast maturation, cell adhesion and migration. We studied osteoactivin expression within the liver. METHODS: Expression of osteoactivin was analysed by RT-PCR, Northern blotting, in situ hybridisation (ISH) and immunohistochemistry (IHC) comparing normal and acutely injured rat liver [induced by carbon tetrachloride (CCl(4)) administration], liver cell populations, and normal or diseased human liver. RESULTS: By ISH osteoactivin expression was detected in sinusoid-lining cells found by IHC to be positive for the common mononuclear phagocyte marker antibody anti-CD68. While total liver contained only traces, isolated Kupffer cells expressed abundant amounts of osteoactivin mRNA further increasing during culture. In acutely injured rat liver osteoactivin expression was strongly increased reaching maximum of expression 48h after CCl(4). By ISH osteoactivin expression was localised in pericentral inflammatory cells and sinusoid-lining cells again anti-CD68 positive. Dexamethasone and lipopolysaccharide decreased osteoactivin expression in cultured mononuclear phagocytes of normal as well as of acutely injured liver. In human liver osteoactivin was increased in fulminant hepatitis and paracetamol intoxication. CONCLUSIONS: Osteoactivin is expressed at high levels in normal and inflammatory liver macrophages suggesting a significant role in acute liver injury.

Induction of Mx-2 in rat liver by toxic injury.

BACKGROUND/AIMS: Mx proteins are supposed to be strictly regulated by viruses or interferon-alpha (IFN-alpha). We used a non-viral model of acute liver injury to study Mx expression. METHODS: We induced toxic liver injury by CCl(4), and studied the expression of IFN-alpha, IFN-gamma, and IFN-inducible antiviral genes (Mx-2; 2'-5' oligoadenylate synthetase, 2-5 A; double-stranded RNA-activated protein kinase, PKR). RESULTS: Similar to 2-5 A and PKR, Mx-2 gene expression was biphasically induced after CCl(4) administration with a maximum at 24 h, and a second peak at 72 h. On protein level, Mx-2 only was up-regulated. IFN-alpha remained constant for the first 24 h while IFN-gamma peaked at 6 h. Thereafter, IFN-alpha increased to a maximum at 72 h while IFN-gamma decreased to 77+/-4%. Small monocyte-like liver macrophages, but not large macrophages, expressed Mx-2 constitutively. In vitro, IFN-alpha but not IFN-gamma induced Mx-2 in different liver cell populations. IFN-gamma, instead, reduced the susceptibility of liver macrophages to the actions of IFN-alpha. CONCLUSIONS: Our data suggest that Mx expression does not invariably result from the presence of a viral particle or IFN-alpha synthesis but may represent an innate defensive armamentarium that may be up-regulated without antigen specificity upon liver injury.

Successful treatment of hepatocellular carcinoma with the tyrosine kinase inhibitor imatinib in a patient with liver cirrhosis.

Several mechanisms of development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis have been discussed. One hypothesis suggests that the somatic stem cells of the liver, the so-called oval cells, may undergo malignant transformation. Oval cells are derived from the biliary cells of the canal of Hering and are characterized by c-kit-positivity, the transmembrane receptor of stem cell factor. Constitutively activated tyrosine kinases have been identified as major pathogenetic mechanisms in the development of malignant diseases like gastrointestinal stromal tumors (c-kit) and chronic myelogenous leukemia (bcr-abl). The prognosis of these diseases improved enormously since the drug imatinib, a tyrosine kinase inhibitor of c-kit and bcr-abl, was introduced. Here we report the successful cure of a patient with liver cancer by this tyrosine kinase inhibitor.

Modulation of fibronectin gene expression in inflammatory mononuclear phagocytes of rat liver after acute liver injury.

BACKGROUND/AIMS: In acute liver injury, fibronectin (FN) is deposited at the site of hepatocellular necrosis. We have previously shown that liver inflammatory mononuclear phagocytes (MNP), in contrast to quiescent hepatic macrophages, synthesize abundant amounts of FN. We now analyzed effects of agents known to influence macrophage functions to better understand liver damage and repair. METHODS: Acute rat liver injury was induced by CCl(4). Liver cellular FN (cFN) expression was analyzed by in situ-hybridization. Liver MNP were isolated and characterized immunocytochemically. Protein synthesis was studied by biosynthetic labeling, immunoprecipitation, and SDS-PAGE. RNA was analyzed by Northern Blotting. RESULTS: cFN gene expression was localized by in situ-hybridization and immunohistochemistry within the pericentral inflammatory infiltrate. Treatment of inflammatory MNP with dexamethasone, or interferon-gamma, or lipopolysaccharide induced a dose-dependent decrease in cFN gene expression, whereas transforming growth factor-beta increased cFN gene expression. CONCLUSIONS: 1. Inflammatory MNP express cFN. 2. Downregulation of cFN expression by dexamethasone in inflammatory MNP may explain delayed wound healing after corticosteroid therapy. Interferon-gamma and lipopolysaccharide could also delay the repair process in the liver. Transforming growth factor-beta may promote liver wound healing after acute liver injury by increasing local cFN synthesis in inflammatory mononuclear phagocytes of the inflammatory infiltrate.

Quality of life and outcome of ultrasound-guided laser interstitial thermo-therapy for non-resectable liver metastases of colorectal cancer.

OBJECTIVE: Patients with non-resectable liver metastases of colorectal cancer have poor prognosis and are mainly treated by palliative chemotherapy. Laser interstitial thermo-therapy is an innovative minimal invasive procedure for local tumour destruction within solid organs. The aim of the study was to investigate quality of life and outcome of ultrasound-guided laser interstitial thermo-therapy (US-LITT) in patients with liver metastases of colorectal cancer. METHODS: In this prospective non-randomized study, 45 patients with liver metastases of colorectal cancer were palliatively treated by US-LITT. Patient survival was analysed by the Kaplan-Meier method and the quality of life by questionnaire C30 of the European Organisation for Research and Treatment of Cancer before, and 1 week, 1 month, and 6 months after initiation of US-LITT. RESULTS: Median survival after initiation of US-LITT was 8.5 +/- 0.7 months with a range of 1.5-18 months. Body weight was constant 1 month after US-LITT. In the multivariate analyses, quality-of-life symptoms and functioning scales did not deteriorate in patients alive at 6 months after initiation of US-LITT. Univariate analyses outlined a significant increase of the pain subscale before and at 1 week after US-LITT. CONCLUSIONS: This study first describes the quality of life in patients with liver metastases of colorectal cancer treated by US-LITT. Potential benefits of the minimal invasive procedure could be prolonged survival time by preserved quality of life, but this first impression needs to be verified in a comparative study.

Early gene expression of hepatocyte growth factor in mononuclear phagocytes of rat liver after administration of carbon tetrachloride.

BACKGROUND: Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen supposed to be a main stimulant of hepatocyte replication during liver regeneration. During acute liver injury, HGF has been detected in nonparenchymal cells of the liver. METHODS: We performed in situ hybridization of HGF in rat livers after administration of carbon tetrachloride (CCl4). Mononuclear phagocytes (MNP) were isolated from normal and injured livers and HGF expression was analyzed by Northern blotting, in situ hybridization, and immunoprecipitation of 35S-labeled proteins. RESULTS: In situ hybridization of normal liver revealed few HGF positive cells within hepatic sinusoids. In injured livers, the number of cells containing HGF transcripts was increased at 6-24 h after CCl4. Hepatocyte growth factor transcripts in MNP from normal liver were detectable in trace amounts, but became clearly detectable at 6 h and persisted up to 24 h after CCl4 administration. In situ hybridization of MNP isolated from normal liver did not reveal positive cells. Mononuclear phagocytes became HGF-positive when isolated 6 h after CCl4. Hepatocyte growth factor protein was detected in MNP isolated 24 h after CCl4. CONCLUSIONS: Hepatocyte growth factor in MNP is not directly induced by interferon-alpha, interferon-gamma or tumour necrosis factor-alpha (TNF-alpha). Stimulated resident mononuclear phagocytes may play a significant role in the increase of HGF expression in liver regeneration after acute liver injury.