Tumour-stroma ratio to predict pathological response to neo-adjuvant treatment in rectal cancer.

INTRODUCTION: Management of rectal cancer has advanced, with an increasing use of neoadjuvant chemoradiotherapy (nCRT). This opens options for organ preserving treatment for those with a major response to nCRT. However, the degree of clinical response, based on MRI and post-treatment biopsies, only poorly matches the degree of actual pathological response. In order to select patients with major pathological response without surgical resection, it is of importance to define tumour markers predicting the degree of pathological response to nCRT. The intra-tumoural tumour-stroma ratio (TSR) might be this marker. METHODS: TSR in pre-treatment biopsies was estimated according to the method described by van Pelt et al. The degree of pathological response was assessed on the tumour resection according to tumour regression grading (TRG) by Mandard. The primary endpoint of this study was the difference in pathological response to nCRT between TSR-high and TSR-low groups. RESULTS: We found that 26.2% of patients with major response was classified as TSR-high, while 73.8% of patients were classified as TSR-low. A high TSR in pre-treatment biopsies was associated with a lower chance of major-response to nCRT (OR = 0.37, 95%CI; 0.19-0.73), p = 0.004), independent of tumour stage and time between nCRT and surgery. CONCLUSION: In rectal cancer, TSR in pre-treatment biopsies predicts pathologic response to nCRT, with a high TSR bringing twice the risk of poor to no response compared to low TSR. In future, assessment of TSR may fulfil a role in a therapeutic algorithm identifying patients who will or will not respond to nCRT prior to treatment initiation.

A high tumour-stroma ratio (TSR) in colon tumours and its metastatic lymph nodes predicts poor cancer-free survival and chemo resistance.

PURPOSE: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance. METHODS: Two independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 µm haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (≤ 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan-Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I-II, TNM-stage III and patients with an indication for chemotherapy separately. RESULTS: We found that high TSR was associated with poor cancer-free survival in TNM-stage I-II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II-III colon tumour. CONCLUSION: In colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance.

Is pre-operative heart rate variability a prognostic indicator for overall survival and cancer recurrence in patients with primary colorectal cancer?

BACKGROUND: Heart Rate Variability (HRV) represents efferent vagus nerve activity which is suggested to be inversely related to fundamental mechanisms of tumorigenesis and to be a predictor of prognosis in various types of cancer. HRV is also believed to predict the occurrence and severity of post-operative complications. We aimed to determine the role of pre-operative HRV as a prognostic factor in overall and cancer free survival in patients with colorectal cancer. METHODS: Retrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January 2010 and December 2016, who underwent curative surgical treatment. HRV was measured as time-domain parameters (SDNN (Standard Deviation of NN-intervals) and RMSSD (Root Mean Square of Successive Differences)) based on pre-operative 10 second ECGs. Groups were created by baseline HRV: Low HRV (SDNN <20ms or RMSSD <19ms) and normal HRV (SDNN ≥20ms or RMSSD ≥19ms). Primary endpoints were overall and cancer free survival. RESULTS: A total of 428 patients were included in this study. HRV was not significantly associated with overall survival (SDNN <20ms vs SDNN ≥20ms:24.4% vs 22.8%, adjusted HR = 0.952 (0.607-1.493), p = 0.829; RMSSD <19ms vs RMSSD ≥19ms:27.0% vs 19.5%, adjusted HR = 1.321 (0.802-2.178), p = 0.274) or cancer recurrence (SDNN <20ms vs ≥20ms:20.1% vs 18.7%, adjusted HR = 0.976 (0.599-1.592), p = 0.924; RMSSD <19ms vs ≥19ms, 21.5% vs 16.9%, adjusted HR = 1.192 (0.706-2.011), p = 0.511). There was no significant association between HRV and CEA-level at one year follow-up, or between HRV and occurrence of a post-operative complication or the severity of post-operative complications. CONCLUSIONS: Heart rate variability was not associated with overall or cancer free survival in patients with primary colorectal cancer who underwent curative surgical treatment. These results do not align with results found in studies including only patients with advanced cancer, which suggests that there is only an association in the other direction, cancer causing low HRV.

Factors influencing the density of aerobic granular sludge.

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1-5 %) strongly increased the granular density and thereby the settling velocity. For nitritation-anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.

Full-scale granular sludge Anammox process.

The start-up of the first full scale Anammox reactor is complete. The reactor shows stable operation, even at loading rates of 10 kg N/m3.d. This performance is the result of the formation of Anammox granules, which have a high density and settling velocities exceeding 100 m/h. With this performance, the Anammox granular sludge technology has been proven on full scale.

Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria.

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.

Treatment of nitrogen-rich wastewater using partial nitrification and anammox in the CANON process.

Partial nitrification combined with Anammox in a single reactor (the CANON process) is an energy-efficient N-removal technology that could substantially lower the N-load of a WWTP by separate treatment of nitrogen-rich side streams, preventing the need for extensive expansion and reducing the total energy requirement. This study looks at the enrichment of Anammox from activated sludge and its application in the CANON process on lab-scale. The aim was to identify the critical process control parameters necessary for successful operation of CANON. An Anammox culture capable of removing 0.6 kg N/m3/d was enriched in 14 weeks in a sequencing batch reactor. Nitrifying biomass was inoculated into the Anammox reactor (10% v/v) together with limited oxygen supply (< 8 mL/min) to initiate the CANON process in continuous culture. The small flocs formed by the biomass (< 1000 microm) were sensitive to low O2 concentrations (< 0.1 mg/L) which prevented simultaneous nitrification and Anammox. Operation with 20 min aerobiosis and 30 min anaerobiosis was necessary to achieve sustained, completely autotrophic N-removal for an extended period at a rate of 0.08 kg N/m3/d. Essential process control parameters for stable CANON operation were the nitrite concentration, oxygen concentration, pH and the temperature.

Enrichment of anammox from activated sludge and its application in the CANON process.

A microbial culture capable of actively oxidizing ammonium to dinitrogen gas in the absence of oxygen, using nitrite as the electron acceptor, was enriched from local activated sludge (Western Australia) in <14 weeks. The maximum anaerobic ammonium oxidation (i.e., anammox) activity achieved by the anaerobic culture was 0.26 mmol NH (4) (+) (g biomass)(-1) h(-1) (0.58 kg total-N m(-3) day(-1)). Qualitative FISH analysis (fluorescence in situ hybridization) confirmed the phylogenetic position of the enriched microorganism as belonging to the order Planctomycetales, in which all currently identified anammox strains fall. Preliminary FISH analysis suggests the anammox strain belongs to the same phylogenetic group as the Candidatus 'Brocadia anammoxidans' strain discovered in the Netherlands. However, there are quite a few differences in the target sites for the more specific probes of these organisms and it is therefore likely to represent a new species of anammox bacteria. A small amount of aerobic ammonium-oxidizing biomass was inoculated into the anammox reactor (10% v/v) to initiate completely autotrophic nitrogen removal over nitrite (the CANON process) in chemostat culture. The culture was always under oxygen limitation and no organic carbon was added. The CANON reactor was operated as an intermittently aerated system with 20 min aerobiosis and 30 min anaerobiosis, during which aerobic and anaerobic ammonium oxidation were performed in sequential fashion, respectively. Anammox was not inhibited by repeated intermittent exposure to oxygen, allowing sustained, completely autotrophic ammonium removal (0.08 kg N m(-3) day(-1)) for an extended period of time.

1994-2004: 10 years of research on the anaerobic oxidation of ammonium.

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Complete conversion of nitrate into dinitrogen gas in co-cultures of denitrifying bacteria.

In the past 10 years many molecular aspects of microbial nitrate reduction have been elucidated, but the ecophysiology of this process is hardly understood. In this contribution, our efforts to study the complex microbial communities and interactions involved in the reduction of nitrate to dinitrogen gas are summarized. The initial work concentrated on emission of the greenhouse gas nitrous oxide during incomplete denitrification by Alcaligenes faecalis. As more research methods became available, the fitness of A. faecalis could be tested in mixed cultures with other denitrifying bacteria, most notably with the nitrate-reducing bacterium Pseudomonas G9. Finally, the advancement of molecular diagnostic tools made it possible to survey complex microbial communities using specific primer sets for/and antibodies raised against the various NO(x) reductases. Given the enormous complexity of substrates and environmental conditions, it is evident that mixed cultures rather than single species are responsible for denitrification in man-made and natural ecosystems. However, it is surprising that even for the breakdown of a single compound, such as acetate, mixed cultures are responsible, and that the consecutive denitrification steps are commonly performed by mutualistic co-operating species. Our observations also indicate that we seldom know the identity of the major key players in the nitrogen cycle of these ecosystems.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Completely autotrophic nitrogen removal over nitrite in one single reactor.

The microbiology and the feasibility of a new, single-stage, reactor for completely autotrophic ammonia removal were investigated. The reactor was started anoxically after inoculation with biomass from a reactor performing anaerobic ammonia oxidation (Anammox). Subsequently, oxygen was supplied to the reactor and a nitrifying population developed. Oxygen was kept as the limiting factor. The development of a nitrifying population was monitored by Fluorescence In Situ Hybridization and off-line activity measurements. These methods also showed that during steady state, anaerobic ammonium-oxidizing bacteria remained present and active. In the reactor, no aerobic nitrite-oxidizers were detected. The denitrifying potential of the biomass was below the detection limit. Ammonia was mainly converted to N2 (85%) and the remainder (15%) was recovered as NO3-. N2O production was negligible (less than 0.1%). Addition of an external carbon source was not needed to realize the autotrophic denitrification to N2.

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell.

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces maris and "Candidatus Brocadia anammoxidans" was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellula marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces maris is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising ("anammox" process) chemoautotroph "Candidatus Brocadia anammoxidans" the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anammoxosome. In Gemmata obscuriglobus, the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

Microbiology and application of the anaerobic ammonium oxidation ('anammox') process.

Ten years ago, an anaerobic ammonium oxidation ('anammox') process was discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community was obtained, dominated by a single deep-branching planctomycete, Candidatus Brocadia anammoxidans. Phylogenetic inventories of different wastewater treatment plants with anammox activity have suggested that at least two genera in Planctomycetales can catalyse the anammox process. Electron microscopy of the ultrastructure of B. anammoxidans has shown that several membrane-bounded compartments are present inside the cytoplasm. Hydroxylamine oxidoreductase, a key anammox enzyme, is found exclusively inside one of these compartments, tentatively named the 'anammoxosome'.

Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation.

Recently, a bacterium capable to oxidize ammonium anaerobically at a high rate was identified as novel member of the Planctomycetales (Strous, M., Fuersi, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K. T., Webb, R. I., Kufnen, J. G., and Jetten, M. S. M.: Nature 400, 446-449, 1999). Here we investigated the microbial community structure of a trickling filter biofilm with a high anaerobic ammonium oxidation activity. Fluorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium oxidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic ammonium oxidizers a full-cycle 16S rDNA approach was performed by using a Planctomycetales-specific forward primer for PCR amplification. Of the twenty-five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, nine were affiliated to the Planctomycetales. Comparative analysis showed that these sequences were more than 98.9% similar to each other but only distantly related to the previously recognized anaerobic ammonium oxidizer (below 91% similarity) and all other organisms represented in public 16S rRNA databases (similarities of below 79%). The retrieved sequences and the previously recognized anaerobic ammonium oxidizer represent two well-separated groups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel bacterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. In situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobacteria were also present, albeit in significant smaller amounts, within the anoxic biofilm. Comparative sequence analysis of a stretch of the gene encoding ammonia-monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers within the biofilm.

Missing lithotroph identified as new planctomycete.

With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation ('anammox') is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as "lithotrophs missing from nature". Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

The anaerobic oxidation of ammonium.

From recent research it has become clear that at least two different possibilities for anaerobic ammonium oxidation exist in nature. 'Aerobic' ammonium oxidizers like Nitrosomonas eutropha were observed to reduce nitrite or nitrogen dioxide with hydroxylamine or ammonium as electron donor under anoxic conditions. The maximum rate for anaerobic ammonium oxidation was about 2 nmol NH4+ min-1 (mg protein)-1 using nitrogen dioxide as electron acceptor. This reaction, which may involve NO as an intermediate, is thought to generate energy sufficient for survival under anoxic conditions, but not for growth. A novel obligately anaerobic ammonium oxidation (Anammox) process was recently discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community with one dominating peculiar autotrophic organism was obtained. With nitrite as electron acceptor a maximum specific oxidation rate of 55 nmol NH4+ min-1 (mg protein)-1 was determined. Although this reaction is 25-fold faster than in Nitrosomonas, it allowed growth at a rate of only 0.003 h-1 (doubling time 11 days). 15N labeling studies showed that hydroxylamine and hydrazine were important intermediates in this new process. A novel type of hydroxylamine oxidoreductase containing an unusual P468 cytochrome has been purified from the Anammox culture. Microsensor studies have shown that at the oxic/anoxic interface of many ecosystems nitrite and ammonia occur in the absence of oxygen. In addition, the number of reports on unaccounted high nitrogen losses in wastewater treatment is gradually increasing, indicating that anaerobic ammonium oxidation may be more widespread than previously assumed. The recently developed nitrification systems in which oxidation of nitrite to nitrate is prevented form an ideal partner for the Anammox process. The combination of these partial nitrification and Anammox processes remains a challenge for future application in the removal of ammonium from wastewater with high ammonium concentrations.

Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (anammox) sludge.

The anaerobic ammonium oxidation (Anammox) process is a promising novel option for removing nitrogen from wastewater. In this study it was shown that the Anammox process was inhibited reversibly by the presence of oxygen. Furthermore, aerobic nitrifiers were shown not to play an important role in the Anammox process.