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CHEMO CAUSES MUTATIONS?

Cancer drugs shown to cause mutations in mice offspring

January 31st, 2012 in Research 

 

(Medical Xpress) -- For many years, most of the studies done to see what effects cancer treatment has on the offspring of survivors, has involved radiation. This is because radiation is known to cause mutations in cells. Not so well studied have been the generational effects of chemicals used to treat cancer. Now, research by Colin Glen and Yuri Dubrova at the University of Leicester in the UK, shows that male rats given chemotherapy drugs sire pups that have twice as many mutations in a part of their DNA as do their fathers. They have published their results in the Proceedings of the National Academy of Sciences.

Lead researcher Dubrova, a geneticist, has been studying the effects of cancer therapies on animals for the past decade, mostly focused on the aftereffects of radiation treatment. Recently however, he began looking into the possible genetic effects chemicals might be having on offspring, or even subsequent generations. Such drugs are given systemically whereas body parts not being treated by radiation are covered, potentially making the outcome worse for chemotherapy. To find out, he and his colleague subjected male mice to three of the most common types of chemotherapy drugs at dosages relative to their size. They then studied a section of the mouse genome in the male offspring produced after mating with non-treated females and found twice as many mutations as in either parent.

The authors suggest their findings should not be cause for alarm however, as most cancer patients are too old to conceive children, or become sterile as a result of treatment, leaving children who receive treatment as the one group at possible risk. But they say, their results need to be taken in proper context. The mice used in the study only live for a couple of years, thus they reproduce much sooner after receiving the chemicals than children would as they take much longer to reach child bearing age. To find out if a time lag might allow for repair of the damaged genes before reproducing, more research will need to be done with animals that live much longer.

Another interesting find in the research was that DNA in the offspring that was contributed by the mother, who was not given any of the drugs, was affected in much the say way as that from the treated fathers, a result that they say has been seen before in other studies, but is still not understood.

More information: Exposure to anticancer drugs can result in transgenerational genomic instability in mice, PNAS, Published online before print January 30, 2012, doi: 10.1073/pnas.1119396109

Abstract 
The genetic effects of human exposure to anticancer drugs remain poorly understood. To establish whether exposure to anticancer drugs can result not only in mutation induction in the germ line of treated animals, but also in altered mutation rates in their offspring, we evaluated mutation rates in the offspring of male mice treated with three commonly used chemotherapeutic agents: cyclophosphamide, mitomycin C, and procarbazine. The doses of paternal exposure were approximately equivalent to those used clinically. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat locus Ms6-hm was established in DNA samples extracted from sperm and bone marrow of the offspring of treated males. After paternal exposure to any one of these three drugs, expanded simple tandem repeat mutation frequencies were significantly elevated in the germ line (sperm) and bone marrow of their offspring. This observed transgenerational instability was attributed to elevated mutation rates at the alleles derived from both the exposed fathers and from the nonexposed mothers, thus implying a genome-wide destabilization. Our results suggest that paternal exposure to a wide variety of mutagens can result in transgenerational instability manifesting in their offspring. Our data also raise important issues concerning delayed transgenerational effects in the children of survivors of anticancer therapy.

© 2011 PhysOrg.com

"Cancer drugs shown to cause mutations in mice offspring." January 31st, 2012. http://medicalxpress.com/news/2012-01-cancer-drugs-shown-mutations-mice.html

 

AVASTIN INCREASES CANCER STEM CELLS!!!

Avastin, Sutent increase breast cancer stem cells, study shows

January 25th, 2012 in Cancer 

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This is Max S. Wicha, M.D. Credit: University of Michigan Comprehensive Cancer Center

 

Cancer treatments designed to block the growth of blood vessels were found to increase the number of cancer stem cells in breast tumors in mice, suggesting a possible explanation for why these drugs don't lead to longer survival, according to a new study by researchers at the University of Michigan Comprehensive Cancer Center.

The drugs Avastin and Sutent have been looked at as potential breast cancer treatments. But while they do shrink tumors and slow the time till the cancer progresses, the effect does not last, and the cancer eventually regrows and spreads.

"This study provides an explanation for the clinical trial results demonstrating that in women with breast cancer antiangiogenic agents such as Avastin delay the time to tumor recurrence but do not affect patient survival. If our results apply to the clinic, it suggests that in order to be effective, these agents will need to be combined with cancer stem cell inhibitors, an approach now being explored in the laboratory," says study author Max S. Wicha, M.D., director of the U-M Comprehensive Cancer Center.

The researchers treated mice with breast cancer using Avastin (bevacizumab) and Sutent (sunitinib), both of which work by stopping the growth and formation of blood vessels, a process called angiogenesis. The researchers found that tumors treated with these drugs developed more cancer stem cells, the small number of cells within a tumor that fuel a cancer's growth and spread and that are often resistant to standard treatment. Both the number of cancer stem cells and the percentage of cancer stem cells that make up the tumor increased after being treated with each of these therapies.

The researchers found that the cancer stem cells increased because of a cellular response to low oxygen, a condition called hypoxia. And they were able to determine the specific pathways involved in hypoxia that activate the cancer stem cells.

Results of the study appear online in the Proceedings of the National Academy of Sciences Early Edition.

The U.S. Food and Drug Administration recently revoked approval of Avastin for treating breast cancer, although the drug is approved for use in other types of cancer. The reversal was in response to clinical trials showing that the drug's benefit was short-lived, with breast cancer patients quickly relapsing and the cancer becoming more invasive and spreading further throughout the body. Overall, the drug did not help patients live any longer.

The current study suggests the possibility of combining anti-angiogenesis drugs with a cancer stem cell inhibitor to enhance the benefit of this treatment. The researchers are testing this approach in mice and preliminary data looks promising.

Breast cancer statistics: 209,060 Americans will be diagnosed with breast cancer this year and 40,230 will die from the disease, according to the American Cancer Society

More information: Proceedings of the National Academy of Sciences Early Edition, DOI: 10.1073/pnas.1018866109

Provided by University of Michigan Health System

"Avastin, Sutent increase breast cancer stem cells, study shows." January 25th, 2012. http://medicalxpress.com/news/2012-01-avastin-sutent-breast-cancer-stem.html

 

EUROPEAN MUCOUS ADENOCARCINOMA STUDY

Mucinous adenocarcinomas: Poor prognosis in metastatic colorectal cancer

Leonie J.M. Mekenkamp, Karin J. Heesterbeek, Miriam Koopman, Jolien Tol, Steven Teerenstra, Sabine Venderbosch, Cornelis J.A. Punt, Iris D. Nagtegaal

published online 06 January 2012

European Journal of Cancer

Abstract 

Purpose

Mucinous histology of metastatic colorectal cancer (CRC) has been associated with poor prognosis, however this has never been assessed in large well-defined study populations treated with the current used systemic agents. We investigated the prognostic value of mucinous histology in two large phase III studies in metastatic CRC.

Patients and methods

The study population included 1010 metastatic CRC patients who were treated with chemotherapy and targeted therapies in two phase III studies. Patients were classified according to the histology of the primary tumour in mucinous adenocarcinomas (MC) and non-mucinous adenocarcinomas (AC).

Results

Patients with MC (n=99) were older, had more often a normal serum lactate dehydrogenase (LDH), extrahepatic localisation of metastases, larger primary tumour diameter and a higher T classification compared to patients with AC (n=911). A deficient mismatch repair system and BRAF mutations were observed in 17% and 22% of patients with MC, compared to 3% and 7% in patients with AC, respectively. Clinical outcome was investigated in both studies separately, showing a worse overall survival (OS), progression free survival and overall response rate in patients with MC compared to patients with AC. Patients with MC received less cycles of treatment compared to AC, but did not suffer from a higher incidence of grade 3/4 toxicity. In multivariate analysis, mucinous histology was as an independent negative prognostic factor for OS, resulting in a combined hazard ratio of 1.78 (95%confidence interval (CI) 1.35–2.35).

Conclusions

Patients with metastatic mucinous CRC have distinct clinicopathological features and poor response to chemotherapy and targeted agents. The strong negative prognostic value of MC warrants the use of this pathological feature as a stratification factor for clinical trials in metastatic CRC.

Keywords: Chemotherapy, Metastatic colorectal cancer, Mucinous adenocarcinomas, Prognostic marker, Targeted therapy

 

 

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1. Introduction 

Mucinous adenocarcinomas (MC) are a histological subtype of colorectal cancer (CRC), in which the tumour cells secrete abundant extracellular mucin involving more than 50% of the tumour volume. They account for 5–15% of all primary colorectal carcinomas,1 and MC are clinically, morphologically and molecularly different from adenocarcinomas (AC). MC are more often correlated with an advanced stage at presentation,2, 3 a larger diameter,4 localisation in the proximal colon,2, 4 and peritoneal dissemination.5, 6, 7 Furthermore, MC affect younger patients and are associated with the Lynch syndrome.8, 9 Mucinous tumours exhibit a distinct molecular profile compared to AC, involving a higher incidence of KRAS and BRAF mutations,10, 11 diploidy,12 deficient mismatch repair system (dMMR),13 and CpG island hypermethylation.14

Approximately 50% of CRC patients develop metastatic disease and in case of irresectable metastases there is no curative treatment available. However, survival can be improved by using cytotoxic regimens (fluoropyrimidines, oxaliplatin, irinotecan) in combination with targeted therapy (vascular endothelial growth factor (VEGF)- and epidermal growth factor receptor (EGFR) antibodies). Two studies investigated the response to chemotherapy in advanced CRC patients with MC versus AC. Mucinous tumours appeared to be less responsive to fluoropyrimidines,6 irinotecan and oxaliplatin-based chemotherapy.7 The response of tumours with mucinous histology to chemotherapy plus targeted agents has not been reported.

The prognostic value of mucinous histology remains controversial. Several studies report that MC are associated with poor prognosis,6, 7, 15 which however is not confirmed by others.2, 16 This may be attributed to the small number of patients with MC in these studies. It should be noted that several known prognostic factors, like tumour node metastasis (TNM) classification and BRAF mutation status,17 were not included in most of these studies.

Therefore, the aim of this retrospective analysis was to assess the prognostic value of MC in metastatic CRC in more detail.

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2. Patients and methods 

2.1. Study population 

All patients included in our analysis participated in two phase III randomised clinical trials from the Dutch Colorectal Cancer Group (DCCG). In the CAIRO study18 (CKTO 2002–07, ClinicalTrials.gov; NCT00312000) 820 metastatic CRC patients were randomised between first-line sequential or combination treatment with capecitabine, irinotecan and oxaliplatin. The primary end-point was overall survival (OS), and secondary end-points included progression free survival (PFS) and response rate. Median OS was 16.3months (95%confidence interval (CI) 14.3–18.1) for the sequential treatment group and 17.4months (95%CI 15.2–19.2) for the combination group (p=0.33). As expected, combination therapy was associated with a prolonged first-line PFS compared with sequential therapy (7.8 versus 5.8months; p=0.0002). The CAIRO2 study19 (CKTO 2005–02, ClinicalTrials.gov; NCT00208546) included 755 metastatic CRC patients, who were randomly assigned to receive first-line treatment with capecitabine, oxaliplatin and bevacizumab, or the same schedule with the addition of cetuximab. The primary end-point of this study was PFS, and secondary end-points were OS and response rate. The addition of cetuximab significantly decreased the median PFS (9.6months (95%CI 8.4–10.5) versus 10.7months (95%CI 9.7–12.3); p=0.01). The median OS was 20.3months (95%CI 17.8–24.7) in the patients treated without cetuximab and 19.4months (95%CI 17.5–21.4) in the patients treated with cetuximab (p=0.16). In both studies, treatment cycles were given every 3weeks. Assessment of the tumour response was performed every 3 cycles (9weeks) using computerized tomography (CT) imaging and evaluated according to the Response Evaluation Criteria for Solid Tumours (RECIST).20 The written informed consent required for all patients before study entry also included translational research on tumour tissue.

In our analysis, we included eligible patients with a resection of the primary tumour of which formalin-fixed paraffin-embedded (FFPE) tissue was available. The patients were classified based on the histology of the primary tumour according to the guidelines of the World Health Organization (WHO).21 Tumours were considered MC if more than 50% of their volume consisted of mucin. AC were defined as tumours without extracellular mucin. Other histological subtypes were excluded from our analysis.

2.2. Clinicopathological features 

The following clinical parameters were collected for each patient: age, gender, Eastern Cooperative Oncology Group (ECOG) performance status, serum lactate dehydrogenase (LDH), site of the primary tumour, prior adjuvant therapy, number and sites of metastatic disease, metachronous (>6months after initial diagnosis) or synchronous (⩽6months of initial diagnosis) presentation of metastases, and regimen used as first-line treatment.

Histopathological review was performed on haematoxylin and eosin-stained coupes by two independent observers, including an experienced pathologist (I.D.N). The TNM classification of malignant tumours was used to describe the extent of cancer spread in terms of invasion depth and lymph node stage.22 The maximum diameter, median number of detected and involved lymph nodes were recorded.

2.3. Microsatellite instability 

Immunohistochemistry (IHC) was performed on tissue microarrays (TMA’s) of FFPE material of the primary tumour. To determine the expression of the four mismatch repair (MMR) proteins (MLH1, MSH2, MSH6 and PMS2) the slides were stained with antibodies against these proteins, as described before.23 Tumours were considered positive for MMR expression if nuclear staining was present in at least one tumour cell and negative if there was complete absence of nuclear staining.

Analysis of microsatellite instability (MSI) was performed in all tumours of which at least one staining for the MMR proteins was negative or the IHC staining was not interpretable, using a validated protocol.23 Two microsatellite markers (BAT 25 en BAT 26) were used, and if only one of these markers showed instability, the analysis was extended with four other markers (BAT 40, D2S123, D5S346, D17S250). A tumour was defined MSI if at least two of the six markers showed instability.

2.4. Hypermethylation of MLH1 promoter 

The DNA methylation status of the MLH1 promoter regions was determined after bisulphite treatment of the DNA using the EZ DNA methylation KIT, ZYMO Research (Orange, CA, United States of America (USA)) as described before.24

2.5. KRAS and BRAF mutation analysis 

Genomic DNA was isolated from 4–8 microdissected 50μm sections of FFPE primary tumour tissue as previously described.25 The KRAS and BRAF mutation status25, 26 were assessed in duplicate by sequencing analysis in patients of the CAIRO2 study.

2.6. Statistical analysis 

The comparison in baseline clinicopathological features between MC and AC was done regardless of study treatment, using the χ2-test, Fisher’s Exact Test or Wilcoxon rank sum test where appropriate. Clinical outcome of patients with MC and AC was investigated in both studies separate, due to an improvement of clinical outcome caused by the addition of bevacizumab to first-line chemotherapy in the CAIRO2 study. OS was calculated as the interval from the date of randomisation until death from any cause or until the date of last follow-up. PFS for first-line treatment was calculated from the date of randomisation to the first observation of disease progression, death from any cause or last follow-up date. OS and PFS curves were estimated using the Kaplan–Meier method and compared with the log-rank test. Overall response was defined as partial response or complete response. Disease control was defined by stable disease with a duration of more than four months or partial response or complete response. Differences in response and disease control rates were tested with a χ2-test. Multivariate analysis of OS was performed by means of a Cox proportional hazard model to determine if mucinous histology was an independent prognostic factor for survival after correction for; age, gender, localisation of metastases, performance status, serum LDH, site of primary tumour, prior adjuvant therapy, metastatic sites involved, onset of metastases, treatment arm, invasion depth and lymph node status. In the CAIRO2 study, KRAS and BRAF mutation status were also included in the multivariate analysis. The adjusted hazard ratios of mucinous histology in the CAIRO and CAIRO2 study were checked for heterogeneity. If no heterogeneity was present, the hazard ratios were combined on a log scale, using a DerSimonian-Laird random effects meta-analysis with inverse variance weighting. p values below 0.05 were considered as statistically significant. The analyses were performed using SAS 8.2 software.

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3. Results 

3.1. Study population 

A total of 1099 eligible patients, 552 from the CAIRO and 547 from the CAIRO2 study were available for our analysis. In the CAIRO study, 50 patients (9%) were classified with MC, 435 patients (79%) with AC and 67 patients (12%) with other histological subtypes, including adenocarcinomas with a mucinous component of less than 50%. In the CAIRO2 study, 49 patients (9%) were diagnosed with MC, 476 patients (87%) with AC and 22 patients (4%) with other histological subtypes of CRC. Patients with other histological subtypes were excluded from our analysis.

3.2. Patient characteristics 

The median age of patients with MC (n=99) was 67years compared to 63years for patients with AC (n=911) (p=0.005). Patients with MC more often had a normal serum LDH (p<0.0001) and extrahepatic localisation of metastases (p=0.02) compared to patients with AC. A trend was observed for a decreased incidence of MC versus AC in rectum carcinomas (p=0.08). In the CAIRO2 study, a lower percentage of patients with MC was treated with cetuximab (p=0.03) (Table 1).

Table 1. Baseline characteristics of patients with MC and AC treated in the CAIRO and CAIRO2 study.

		MC (n=99)	AC (n=911)	p-Value
Age	Median (range)	67 (36–84)	63 (28–81)	0.005
Gender	Male	58 (59%)	548 (60%)	0.83
	Female	41 (41%)	363 (40%)
Localisation metastases	Hepatic	19 (20%)	303 (34%)	0.02
	Extrahepatic	40 (41%)	281 (31%)
	Hepatic+extrahepatic	38 (39%)	312 (34%)
	Locally advanced	0	7 (1%)
	Unknown	2	8
WHO performance status	0	66 (67%)	594 (65%)	0.61
	1	30 (30%)	301 (33%)
	2	3 (3%)	16 (2%)
Serum LDH	Normal	80 (82%)	557 (62%)	<0.0001
	>ULN	18 (18%)	347 (38%)
	Unknown	1	7
Site of primary tumour	Colon	52 (53%)	418 (46%)	0.08
	Rectosigmoid	30 (30%)	240 (26%)
	Rectum	17 (17%)	252 (28%)
	Unknown	0	1
Prior adjuvant therapy	No	84 (85%)	758 (83%)	0.69
	Yes	15 (15%)	152 (17%)
	Unknown	0	1
Metastatic sites involved	1	41 (42%)	420 (47%)	0.47
	2	34 (35%)	316 (35%)
	>2	22 (23%)	160 (18%)
	Unknown	2	15
Metastases onset	Metachronous	46 (46%)	411 (45%)	1.00
	Synchronous	53 (54%)	500 (55%)
Treatment arm CAIRO study	Sequential	26 (52%)	212 (49%)	0.77
	Combination	24 (48%)	223 (51%)
Treatment arm CAIRO2 study	CAPOX+Bev	32 (65%)	231 (49%)	0.03
	CAPOX+Bev+Cet	17 (35%)	245 (51%)

MC, mucinous adenocarcinomas; AC, adenocarcinomas; ULN, upper limit of normal; CAPOX, capecitabine and oxaliplatin; Bev, bevacizumab; Cet, cetuximab.

 

 

3.3. Primary tumour characteristics 

The primary tumour diameter of patients with MC (n=99) was significantly higher compared to primary tumours of AC patients (n=911) (p<0.0001). Tumours with mucinous histology had a higher T classification in comparison with AC (p=0.03). Furthermore, a trend was observed towards a higher median number of positive lymph nodes in patients with MC compared to AC (p=0.06). dMMR was observed in 17% of the mucinous primary tumours compared to 3% in AC (p<0.0001). Hypermethylation of the MLH1 promoter was the cause of dMMR in 83% of the tumours with mucinous histology. In the CAIRO2 study, BRAF mutations were demonstrated in 22% of the MC patients compared to 7% in AC patients (p=0.002) (Table 2).

Table 2. Primary tumour characteristics of patients with MC and AC treated in the CAIRO and CAIRO2 study.

		MC (n=99)	AC (n=911)	p-Value
Diameter (mm)	Median (range)	50 (20–140)	40 (3–135)	<0.0001
Invasion depth	T 1–2	3 (3%)	71 (8%)	0.03
	T 3	64 (67%)	632 (72%)
	T 4	28 (30%)	172 (20%)
	Unknown	4	36
Lymph node status	N 0	24 (26%)	246 (29%)	0.20
	N 1	27 (30%)	307 (36%)
	N 2	40 (44%)	294 (35%)
	Unknown	8	64
Number lymph nodes	Median (range)	9 (1–55)	8 (0–44)	0.23
Number positive lymph nodes	Median (range)	3 (0–54)	2 (0–41)	0.06
MMR status	pMMR	82 (83%)	884 (97%)	<0.0001
	dMMR	17 (17%)	27 (3%)
KRAS mutation status (CAIRO2 study)	Wild type	27 (59%)	275 (61%)	0.75
	Mutation	19 (41%)	176 (39%)
	Unknown	3	25
BRAF mutation status (CAIRO2 study)	Wild type	35 (78%)	421 (93%)	0.002
	Mutation	10 (22%)	30 (7%)
	Unknown	4	25

MC, mucinous adenocarcinomas; AC, adenocarcinomas; MMR, mismatch repair system; pMMR, proficient MMR; dMMR, deficient MMR.

 

 

3.4. Outcome of treatment with chemotherapy (CAIRO study) 

Patients in the CAIRO study were treated with first-line sequential or combination chemotherapy containing capecitabine, irinotecan and oxaliplatin. In univariate analysis, the median OS for patients with MC was 13.2 (95%CI 10.2–17.9) compared to 19.2months (95%CI 17.9–20.6) in the group with AC (p=0.03). A trend of decreased PFS was observed in patients with MC versus AC (p=0.09). These differences in OS and PFS were observed in both treatment arms. The overall response rate in the 437 patients who were evaluated was 12% in the MC group and 37% in the group patients with AC (p=0.0006). Disease control was observed in 79% of the patients in the MC group and 87% of those in the AC group (p=0.17). The median number of the overall treatment cycles was significantly different between patients with MC versus AC (6 versus 12, respectively, p=0.004). The reasons for treatment discontinuation were not significantly different between both patients groups. The incidence of any grade 3 or 4 adverse event was 72% in the patients with mucinous histology and 66% in the AC patients (p=0.43) (Table 3).

Table 3. Efficacy of the CAIRO study treatment (capecitabine, oxaliplatin and irinotecan) in patients with MC and AC.

		MC (n=50)	AC (n=435)	p-Value
Median OS	Months (95%CI)	13.2 (10.2–17.9)	19.2 (17.9–20.6)	0.03
Median PFS1	Months (95%CI)	5.3 (3.9–6.6)	7.2 (6.6–8.1)	0.09
Overall response rate 1	CR+PR	5/43 (12%)	144/394 (37%)	0.0006
Disease control rate 1	CR+PR+SD	34/4 (79%)	341/394 (87%)	0.17
Median cycle number	Median (range)	6 (1–42)	12 (0–55)	0.004
Overall toxicity	Grade >3	36 (72%)	287 (66%)	0.43

MC, mucinous adenocarcinomas; AC, adenocarcinomas; OS, overall survival; PFS, progression free survival; CR, complete response; PR, partial response; SD, stable disease; CI, confidence interval.

 

 

3.5. Outcome of treatment with chemotherapy plus targeted agents (CAIRO2 study) 

In the CAIRO2 study patients received first-line capecitabine, oxaliplatin and bevacizumab with or without cetuximab. The median OS was significantly lower for patients with MC compared to patients with AC (median 13.1 (95%CI 9.9–20.2) versus 21.5months (95%CI 19.8–22.9); p=0.009). Furthermore, the median PFS in the MC group was 7.2months (95%CI 5.7–8.6) compared to 10.6months (95%CI 10.1–11.4) in patients with AC (p<0.0001). These differences in OS and PFS were observed in both treatment arms. The overall response rate for the patients with MC was 10% and 54% for the group with AC (p<0.0001). In 85% of the MC patients disease control was observed compared to 94% of the patients with AC (p=0.003). Patients with MC received less cycles of treatment compared to patients with AC (6 versus 9 cycles, p=0.006). No significant differences were observed in the causes for discontinuation of treatment between the group with MC and the group with AC. The incidence of any grade 3 or 4 adverse event was 71% versus 79% in the MC and AC patients (p=0.27) (Table 4).

Table 4. Efficacy of the CAIRO2 study treatment (capecitabine, oxaliplatin, bevacizumab with or without cetuximab) in patients with MC and AC.

		MC (n=49)	AC (n=476)	p-Value
Median OS	Months (95%CI)	13.1 (9.9–20.2)	21.5 (19.8–22.9)	0.009
Median PFS	Months (95%CI)	7.2 (5.7–8.6)	10.6 (10.1–11.4)	<0.0001
Overall response rate	CR+PR	4/41 (10%)	222/411 (54%)	<0.0001
Disease control rate	CR+PR+SD	35/41 (85%)	387/411 (94%)	0.03
Median cycle number	Median (range)	6 (1–36)	9 (0–48)	0.006
Overall toxicity	Grade >3	35 (71%)	376 (79%)	0.27

MC, mucinous adenocarcinomas; AC, adenocarcinomas; OS, overall survival; PFS, progression free survival; CR, complete response; PR, partial response; SD, stable disease; CI, confidence interval.

 

 

3.6. The independent prognostic role of mucinous histology 

In multivariate Cox regression analysis, mucinous histology was a strong predictor of OS in the CAIRO study (hazard ratio (HR) 1.80; 95%CI 1.24–2.62; p=0.003). Other independent negative predictors for OS in metastatic CRC patients were male gender (HR 1.32; 95%CI 1.05–1.64; p=0.01), primary tumour localisation in the colon (HR 1.55; 95%CI 1.19–2.01; p=0.01), WHO performance status 2 (HR 1.92; 95%CI 1.16–3.23; p=0.01), abnormal serum LDH (HR 1.85; 95%CI 1.48–2.31; p<0.0001), more than 2 metastatic sites involved (HR 2.53; 95%CI 1.89–3.38; p<0.0001), T4 classification (HR 1.59; 95%CI 0.99–2.56; p=0.04) and a poor differentiation grade (HR 1.53; 95%CI 0.89–2.64; p=0.006).

In the CAIRO2 study, mucinous histology remained also strongly associated with OS in multivariate analysis (HR 1.76; 95%CI 1.16–2.67; p=0.008). Other clinicopathological features associated with worse survival in this multivariate model were; WHO performance status 1 (HR 1.36; 95%CI 1.06–1.75; p=0.02), abnormal serum LDH (HR 1.65; 95%CI 1.28–2.12; p=0.0001), N2 classification (HR 1.45; 95%CI 1.05–2.01; p=0.05) and BRAF mutations (HR 2.61; 95%CI 1.57–4.32; p=0.0002).

The test for heterogeneity (p=0.93) showed that the adjusted hazard ratios for MC versus AC were similar in the CAIRO (1.80; 95%CI 1.24–2.62) and CAIRO2 study (1.76; 95%CI 1.16–2.67). Therefore these hazard ratios were combined in a meta-analysis which resulted in an overall hazard ratio of 1.78 (95%CI 1.35–2.35) for mucinous histology in advanced CRC patients treated with systemic therapy (Fig. 1).

 

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            Fig. 1. 
Meta-analysis of both studies estimating the prognostic value of mucinous histology in metastatic colorectal cancer (CRC) treated with systemic therapy.

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4. Discussion 

This is the largest retrospective analysis in mucinous metastatic CRC patients treated with the current standard systemic treatments. We demonstrated that MC are less responsive to fluoropyrimidine-based chemotherapy and targeted agents compared to AC. Furthermore, in a multivariate analysis mucinous histology was shown to be a strong negative prognostic factor for survival.

It is recognised that MC are a different entity in patients with CRC. Specific clinical and pathological features are associated with mucinous histology, of which a larger diameter, higher T classification and extrahepatic localisation of metastases were confirmed in our analysis. Patients with MC treated in the CAIRO studies were older compared to the patients with AC, which has not been previously described. In stage II and III CRC, patients with MC were significantly younger. Mucinous histology is associated with Lynch syndrome,27 however, the incidence of MSI in metastatic CRC is low.23 Therefore, it is likely that the association between mucinous histology and age depends on the presence and cause of a dMMR system.

Our data confirm previous results on differences in molecular signatures between MC and AC in respect to MMR10, 28, 29 and BRAF mutation status,14, 30 supporting the hypothesis that MC are a distinct biologic entity. In previous studies, the incidence of KRAS mutations between MC and AC showed opposite results.30, 31 However, these data were derived from a small subset of patients and could not be confirmed in our larger patient cohort. Further identification of specific genetic changes in the mucinous phenotype, i.e. using gene expression profiling, are necessary and useful to understand the molecular aetiology of these tumours.

Fluoropyrimidines, irinotecan and oxaliplatin are the effective chemotherapeutic agents used in metastatic CRC. Negri et al.6 first reported about the reduced response to 5-FU based chemotherapy of metastatic mucinous CRC. These results were confirmed in a similar subset of patients that received irinotecan and oxaliplatin in addition to fluoropyrimidines as first-line chemotherapy.7 Our analysis also showed a highly significant worse response to these chemotherapeutic agents for MC compared to AC. Bevacizumab, a humanised monoclonal antibody against VEGF, combined with 5-FU based chemotherapy is nowadays the standard first-line treatment for metastatic CRC. Our study is first reporting the outcome of chemotherapy plus targeted agents in MC versus AC. Since the CAIRO2 study19 had a negative outcome, possibly due to a negative interaction between the study drugs,32 the responsiveness of metastatic mucinous CRC to cetuximab cannot be assessed in our series.

The mechanisms for the difference in treatment sensitivity of MC compared to AC remains unclear, but there are several possible explanations. First, differences in molecular features may partly explain the unresponsiveness of tumours with mucinous histology. dMMR is more frequently observed in MC compared to AC, and associated with a reduced response to adjuvant 5-FU treatment in stage II and III CRC patients.33, 34 However, the predictive value of dMMR in response to systemic therapy in metastatic CRC is difficult to assess due to its low incidence in metastatic CRC patients.23 Other molecular markers, such as thymidylate synthase (TYMS) and GSTP1, are also overexpressed in mucinous CRC35 and possibly correlated with resistance to chemotherapy. We did not investigate these or other chemotherapy pathway markers in our analysis, because they have not been validated for clinical use in metastatic CRC.36 No predictive markers for response to bevacizumab have yet been identified.

Secondly, the mucins themselves may play a role in the ability of tumour cells to escape the effect of systemic therapy. It has been established that mucins play a role in the processes of tumour progression, invasion, survival and protection against the host immune response.37 MUC2 is a colonic mucin that is overexpressed in the mucinous subtype of CRC38 and correlated with resistance to 5-FU in vitro.39 The mucin lakes may also be a physical barrier for the delivery of targeted therapy to the tumours cells, however this has not been investigated.

A third explanation could be that the evaluation of the response to treatment is inadequate in MC patients. In the CAIRO studies we used CT scanning and evaluated response according to RECIST criteria. If neoplastic cells in MC respond to systemic therapy, the total tumour volume is probably more affected by the unresponsive mucin lakes, which could result in false negative conclusions. Our observation that the difference in disease control rate between MC and AC was less compared to the difference in objective response rate supports this hypothesis. If confirmed, the RECIST criteria may not be the optimal instrument to evaluate the objective response rate to treatment in the mucinous subtype of CRC.

The prognostic value of MC is highly controversial, which may be attributed to a large amount of heterogeneous studies with small subsets of patients. Most studies suggested that MC are associated with poor prognosis,6, 7, 15 while others found no correlation between histological subtype and clinical outcome.2, 16 Our study differs from the published literature in one important aspect, in that only patients with a previous resection of the primary tumour were included, for the obvious reason of tissue availability. Our group has shown that patients with a resection of the primary tumour may have a better prognosis compared to patients without resection.40 Since the prognostic value of resection of the primary tumour in metastatic CRC patients has not been clearly established, the possible influence of this parameter cannot be assessed. Therefore we assessed the prognostic value of mucinous histology in metastatic patients with a resection of the primary tumour and we observed a significant shorter OS in patients with MC compared to AC. As mentioned before, MC present with distinct clinicopathological features and most of these features are correlated with a worse prognosis. For example, BRAF mutations were more frequently observed in MC compared to AC, which is a strong negative prognostic marker in metastatic CRC.17 However, even with BRAF mutations included in a multivariate analysis, mucinous histology remained a strong independent negative prognostic factor. The hazard ratio for mucinous histology is equal or even higher compared to well known stratification factors, such as serum LDH, WHO performance status and number of metastatic sites involved. Appropriate stratification facilitates the interpretation of study results and prevents heterogeneity in response and survival rates. Our findings suggest that mucinous histology should be one of the stratification factors in metastatic CRC trials. Due to the considerable inconsistency in reporting clinicopathological features and use of stratification factors, we believe there is an urgent need for re-establishing the most important prognostic factors in metastatic CRC.

In conclusion, MC are a distinct entity of CRC with specific clinicopathological and genetic features. Mucinous histology is an independent negative prognostic factor for OS in 1010 metastatic CRC patients treated with chemotherapy and targeted agents, which may be explained by resistance to this systemic treatment. We recommend including mucinous histology as a prognostic factor for patients with metastatic CRC.

COMBO ANTI-INFLAMMATORY & ANTI-METHYLATION DRUGS WORK?

Inflammatory mediator promotes colorectal cancer by stifling protective genes

January 22nd, 2012 in Cancer 

 

Chronic inflammation combines with DNA methylation, a process that shuts down cancer-fighting genes, to promote development of colorectal cancer, scientists at The University of Texas MD Anderson Cancer Center report today in the advance online publication of the journal Nature Medicine.

The team's connection of these two separate influences eventually may lead to better combination therapies for treating and preventing colorectal cancer.

In animal experiments, researchers found that prostaglandin E2, a chemical that promotes inflammation, accelerates the development of colorectal cancer by shutting down genes that suppress tumors and repair damaged DNA. They also found that while either an anti-inflammatory drug or a demethylating agent reduced the size and number of tumors in mice with colorectal cancer, the most powerful response occurred when both drugs were used together.

"We've known that chronic inflammation increases the risk of developing cancer and progression of disease," said senior author Raymond DuBois, M.D., Ph.D., provost and executive vice president at MD Anderson. "We've also known that tumor-suppressing genes are silenced in human colorectal cancer. However, nobody had made a molecular connection between these inflammatory mediators and changes in gene expression or silencing of genes through affects on DNA methylation."

Cancer prevention potential

The two drugs used in the animal experiments - the anti-inflammatory agent celecoxib (known commercially as Celebrex) and the demethylating agent azacitidine (Vidaza) - are both approved for human use.

"One potential application of our research would be a clinical trial for patients who are at extremely high risk for developing colorectal cancer, such as those with a genetic predisposition, to see if treatment with these agents would decrease their risk," DuBois said.

Prostaglandin E2 and methylation

Prostaglandin E2 (PGE2) is a lipid mediator found at high levels at sites of inflammation where immune cells are aggregating. DuBois and colleagues looked for correlations between levels of PGE2 and a class of enzymes called DNA methyltransferases, which attach methyl groups (one carbon atom joined to three hydrogen atoms) to the promoter region of genes, blocking gene expression.

"We found that levels of PGE2 correlate with levels of two methyltransferases, DNMT1 and DNMT3, in human colorectal cancer specimens," DuBois said.

Subsequent experiments showed PGE2: 

• Directly increased levels of both methylating enzymes in three human colorectal cancer cell lines;
• Increased the silencing by methylation of the tumor-suppressor gene CNR1 and the DNA repair gene MGMT;
• Also expanded methylation of a variety of other DNA repair genes, most importantly silencing CDKN2B and MLH1, which repairs DNA mismatches.
• PGE2 silences protective genes in mice

Treating mice that are genetically altered to develop colon tumors with PGE2 increased: 

• Levels of the methyltransferase gene expression in tumor cells;
• Methylation of the four tumor-suppressing genes, which reduced the expression of their corresponding messenger RNA and protein levels in tumor cells; and
• Size and number of precancerous polyps.

Giving those mice the demethylating agent azacitidine reversed the effect of PGE2 on tumor growth and on the silencing of tumor-suppressing and DNA repair genes.

 

Mice treated with azacitidine alone experienced a 60 percent reduction in tumors, and those treated with celecoxib alone, a 77 percent tumor reduction. Treatment with both drugs in tandem cut the number of tumors by 93 percent. All three regimens also reduced the average size of tumors; however, the combination therapy led to the greatest decrease, cutting the size of tumors by half.

Same correlations evident in human colorectal cancers

The researchers found that various processes observed in mice - such as inflammation promotion through PGE2 and another inflammatory agent called PTGS2, methlytransferases DNMT1 and DNMT3B, and the methylation of CNR1, MGMT and MLH1 - are all positively associated in human colorectal cancer, as well.

"These mouse studies make us optimistic that we can extrapolate our data to help treat humans," DuBois said. "Improved understanding of PGE2's roles in cancer progression and the regulation of DNA methylation may provide the basis for developing combination therapy to treat targeted groups of patients, and to prevent cancer from occurring or recurring in high-risk groups."

Provided by University of Texas M. D. Anderson Cancer Center

 

"Inflammatory mediator promotes colorectal cancer by stifling protective genes." January 22nd, 2012. http://medicalxpress.com/news/2012-01-inflammatory-colorectal-cancer-stifling-genes.html