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Hepatocellular carcinoma (HCC) located in the caudate lobe (caudate HCC) is rare; however, patients with this type of tumour have poorer prognoses than those with HCC in other segments. Despite many published reports on the clinical usefulness of proton beam therapy (PBT) for HCC, data on the clinical outcomes of patients undergoing PBT for caudate HCC remain scarce. Therefore, the present study aimed to investigate the outcomes of this group of patients. Thirty patients with caudate HCC who underwent definitive PBT between February 2002 and February 2014 were retrospectively analysed. The total irradiation doses ranged from 55 to 77 (median 72.6) Gy relative biological dose. The median follow-up period was 37.5 (range, 3.0–152.0) months. The overall survival (OS) rates at one, three and five years were 86.6%, 62.8% and 46.1%, respectively. According to univariate and multivariate analyses, Child-Pugh A (P

Canine mammary tumours represent a hard-prognostic task for veterinary clinicians. TNM staging and grading systems refer to a single tumour. Significant limits come to light when these systems are applied to multiple mammary tumours due to the arbitrary criterion in determining which single tumour is representative of the patient's prognosis. This study explored some clinical features of 50 dogs affected by at least one malignant mammary tumour. Clinical features and staging, together with histological classification and grading, have been related to disease-free survival (DFS) with the purpose to evaluate their impact on prognosis. The prognosis was worse in 10-11-year-old dogs (P 0.05), in dogs affected by complex carcinoma (P 0.05), and in patients assigned to Peña grade I (P 0.05). The bodyweight was not linearly related to DFS (P 0.01), and patients with a low number of neoformations (n ≤ 2) showed a better prognosis than dogs with 3-5 tumours (P 0.05). Both the average and the total size of malignant tumours were related to DFS (P 0.05). Dogs assigned with stage I had the best DFS (P 0.05). In conclusion, the Peña grade I alone would not seem to guarantee a favourable prognosis when applied to mammary tumours in dogs affected by multiple simultaneous presentations. Different characteristics, besides tumour grading, such as tumour immunophenotype and expression of hormonal receptors, could in the future, contribute to elucidate the clinical behaviour of multiple canine mammary tumours.

Radiation therapy plays a vital role in the treatment of tumours. In particular, the occurrence of the "abscopal effect" brings about a favourable turn for the treatment of patients with advanced metastatic malignant tumours. Because of the abscopal effect, non-irradiated areas are also treated. However, the abscopal effect occurs by chance, not through seeking. Although the abscopal effect has been studied enthusiastically, the desired result does not appear to be achieved. Moreover, its combination with immunotherapy appears to be overwhelming. There is an opinion that abscopal effect is difficult to achieve by irradiation of a single tumour, and irradiation of multiple or total lesions is advocated to increase the possibility of obtaining clinically meaningful outcomes. Obviously, there are still questions about the mechanism, condition and possibility underlying the occurrence of the abscopal effect. Can the abscopal effect truly change the future treatment strategy as the researchers expect? What are the current problems? This article reviewed the research in recent years to explore the progress and controversy surrounding the abscopal effect of radiation therapy.

Great success has been achieved in recent years in the development of synthetic or assembled nanobiomaterials. Among these, biomolecule-based nanoarchitectures with special optical property are of particular interest. Here, we demonstrate that vitamin B2 nanocrystals assembled as nanorods can be obtained with precise control. Excitingly, such one-dimensional nanostructures not only exhibit intrinsic optical waveguiding properties but also the ability to sensitize oxygen to produce reactive oxygen species. With these properties, we applied the obtained vitamin B2 nanorods under remotely localized light illumination into single tumour cells in vitro for anticancer photodynamic therapy. Further, vitamin B2 nanorods were explored for in vivo photodynamic therapy by using a tumour model. With such bionanostructures, new features and functions of vitamin B2 and its derivatives have been developed.

Contains fulltext : 232944.pdf (Publisher’s version ) (Closed access) Tumour budding in colorectal cancer, defined as single tumour cells or small clusters containing four or fewer tumour cells, is a robust and independent biomarker of aggressive tumour biology. On the basis of published data in the literature, the evidence is certainly in favour of reporting tumour budding in routine practice. One important aspect of implementing tumour budding has been to establish a standardised and evidence-based scoring method, as was recommended by the International Tumour Budding Consensus Conference (ITBCC) in 2016. Further developments have aimed at establishing methods for automated tumour budding assessment. A digital approach to scoring tumour buds has great potential to assist in performing an objective budding count but, like the manual consensus method, must be validated and standardised. The aim of the present review is to present general considerations behind the ITBCC scoring method, and a broad overview of the current situation and challenges regarding automated tumour budding detection methods.

Genetic heterogeneity displayed by tumour cells (intratumoural heterogeneity, ITH) represents a diagnostic challenge when assessing tumour mutational profile. In oral squamous cell carcinoma (OSCC), ITH may be found both in tumour cells and in adjacent mucosa. Genetic heterogeneity of the adjacent mucosa can be interpreted as evidence of the field cancerization (field heterogeneity, FH). The aim of the study was to investigate the impact of intratumoural and intrafield heterogeneity on locoregional control. Ten OSCC patients (5 recurrent and 5 nonrecurrent) were studied. Multiple areas were sampled from the bulk of the tumour and the adjacent nonneoplastic mucosa. A panel of 10 tumour-specific OSCC driver genes was analysed for each sample and was used to calculate heterogeneity. Values were compared among recurrent and nonrecurrent OSCC. Mutational analysis highlighted that a single tumour sample has limited accuracy in assessing the genetic profiles of tumours. High values of ITH considering shared mutations between specimens were found in both recurrent and non-recurrent OSCC (p = 0.095). On the contrary, the intrafield genetic heterogeneity was significantly less frequently in the non-recurrent OSCC group (p = 0.032). Heterogeneity within each specimen calculated with variant allele frequency confirmed that there was better discrimination between recurrent and nonrecurrent groups using nonneoplastic adjacent mucosa than tumour tissue (p value 0.0006 and 0.0048 respectively). In agreement with the theory of field cancerization, intrafield genetic heterogeneity correlates with a higher risk of developing loco-regional recurrences and second primaries. In order to reduce the ITH effects, analysis of multiple tumour areas should be encouraged.

Breast cancer (BC) displays striking clinical, morphological, and behavioural diversity within a single tumour and between tumours. Currently, mounting evidence indicates that the morphological heterogeneity of BC reflects an underlying spectrum of genetic and epigenetic portraits that control BC behaviour. Further understanding of BC heterogeneity will have an impact, not only on the routine diagnostic practices but also on patients' management decisions. Phenomena like diagnostic inconsistencies and therapeutic resistance, both primary and acquired, could be attributed, at least in part, to tumour heterogeneity within the same cancer and between the primary disease and subsequent recurrences. From a practical standpoint, and to minimise the impact of BC intratumoral heterogeneity, pragmatic approaches for adequate tumour sampling have been suggested in translational biomarker discovery and validation research studies and in the clinical setting. Here, we provide a brief overview of BC heterogeneity, with an emphasis on the clinical consequences of intratumoral heterogeneity.

Introduction: In patients with metastatic melanoma, progression of a single tumour lesion (solitary progression) after response to immune checkpoint inhibition (ICI) is increas

Background Response Evaluation Criteria in Solid Tumours (RECIST)-based tumour burden measurements involve observer variability, the extent of which ought to be determined. Methods A literature search identified studies on observer variability during manual measurements of tumour burdens via computed tomography according to the RECIST guideline. The 95% limit of agreement (LOA) values of relative measurement difference (RMD) were pooled using a random-effects model. Results Twelve studies were included. Pooled 95% LOAs of RMD in measuring unidimensional longest diameters of single lesions ranged from −22.1% (95% confidence interval [CI], −30.3% to −14.0%) to 25.4% (95% CI, 17.2% to 33.5%) between observers and −17.8% (95% CI, −23.6% to −11.9%) to 16.1% (95% CI, 10.1% to 21.8%) for a single observer. Pooled 95% LOAs of RMD in measuring the sum of multiple lesions ranged from −19.2% (95% CI, −23.7% to −14.9%) to 19.5% (95% CI, 15.2% to 23.9%) between observers, and −9.8% (95% CI, −19.0% to −0.3%) to 13.1% (95% CI, 3.6% to 22.6%) for a single observer. Pooled 95% LOA of RMD in calculating the interval change of tumour burden with a single lesion ranged from −31.3% (95% CI, −46.0% to −16.5%) to 30.3% (95% CI, 15.3% to 44.8%) between observers. Studies on calculating the interval change of tumour burden for a single observer or with multiple lesions were lacking. Conclusion Interobserver RMD in measuring single tumour burden and calculating its interval change may exceed the 20% cut-off for progression. Variability decreased when tumour burden was measured by a single observer or assessed by the sum of multiple lesions.

Intratumor heterogeneity (ITH), referring to coexistence of different cell subpopulations in a single tumor, has been a major puzzle in cancer research for almost half a century. The lack of understanding of the underlying mechanism of ITH hinders progress in developing effective therapies for cancers. Based on the findings in a recent quantitative experiment on pancreatic cancer, we developed a general evolutionary model for one type of cancer, accounting for interactions between different cell populations through paracrine or juxtacrine factors. We show that the emergence of a stable heterogeneous state in a tumor requires an unequal allocation of paracrine growth factors ("public goods") between cells that produce them and those that merely consume them. Our model provides a quantitative explanation of recent {\it in vitro} experimental studies in pancreatic cancer in which insulin growth factor (IGF-II) plays the role of public goods. The calculated phase diagrams as a function of exogenous resources and fraction of growth factor producing cells show ITH persists only in a narrow range of concentration of exogenous IGF-II. Remarkably, maintenance of ITH requires cooperation among tumor cell subpopulations in harsh conditions, specified by lack of exogenous IGF-II, whereas surplus exogenous IGF-II elicits competition. Our theory also quantitatively accounts for measured {\it in vivo} tumor growth in glioblastoma multiforme (GBM). The predictions for GBM tumor growth as a function of the fraction of tumor cells are amenable to experimental tests. The mechanism for ITH also provides hints for devising efficacious therapies., Comment: 27 pages, 7 figures

In this podcast, a new biological insight in brain tumours is discussed. The author's group has identified the existence of a tumour cell network in incurable gliomas which facilitates multicellular communication and exchange of small molecules between single tumour cells. The tumour cells that are integrated in this network, around 50% of cells according to studies in mouse models and patient samples, appear to be protected from the effects of radiotherapy and possibly also chemotherapy, which may explain how such tumours develop resistance to therapies and why patients relapse after treatment. An overview of ideas that are being investigated preclinically to therapeutically target this network of tumour cells is given. These include approaches to disrupt the network, such as obstructing cellular communication with gap junction blockers and targeting the neurodevelopmental pathways required to form the networks. Conversely, methods to exploit the network through the local application of gap junction-permeable drugs that specifically target the integrated tumour cells could also being studied. This new discovery may result in the development of therapeutic strategies which the author hopes will reach the clinic in the next few years.

Cytoplasmic viscosity (μc) is a key biomechanical parameter for evaluating the status of cellular cytoskeletons. Previous studies focused on white blood cells, but the data of cytoplasmic viscosity for tumour cells were missing. Tumour cells (H1299, A549 and drug-treated H1299 with compromised cytoskeletons) were aspirated continuously through a micropipette at a pressure of −10 or −5 kPa where aspiration lengths as a function of time were obtained and translated to cytoplasmic viscosity based on a theoretical Newtonian fluid model. Quartile coefficients of dispersion were quantified to evaluate the distributions of cytoplasmic viscosity within the same cell type while neural network-based pattern recognitions were used to classify different cell types based on cytoplasmic viscosity. The single-cell cytoplasmic viscosity with three quartiles and the quartile coefficient of dispersion were quantified as 16.7 Pa s, 42.1 Pa s, 110.3 Pa s and 74% for H1299 cells at −10 kPa (ncell= 652); 144.8 Pa s, 489.8 Pa s, 1390.7 Pa s, and 81% for A549 cells at −10 kPa (ncell= 785); 7.1 Pa s, 13.7 Pa s, 31.5 Pa s, and 63% for CD-treated H1299 cells at −10 kPa (ncell= 651); and 16.9 Pa s, 48.2 Pa s, 150.2 Pa s, and 80% for H1299 cells at −5 kPa (ncell= 600), respectively. Neural network-based pattern recognition produced successful classification rates of 76.7% for H1299 versus A549, 67.0% for H1299 versus drug-treated H1299 and 50.3% for H1299 at −5 and −10 kPa. Variations of cytoplasmic viscosity were observed within the same cell type and among different cell types, suggesting the potential role of cytoplasmic viscosity in cell status evaluation and cell type classification.

Introduction Studies of breast carcinoma indicated the presence of type I and III collagens localization at the invasion front of the tumour. Mesenchymal invasion accompanied by proteolysis, in particular collagens, and remodelling of tissue structures by matrix metalloprotases. Possible marker of this process is expression of Cross Linked C-telopeptide of Type III Collagen (CTX-III). The project is aimed to study frequency of CTX-III expression around different of single tumour cells subtypes of invasive breast carcinoma of no specific type. Material and methods Fourteen patients with invasive breast carcinoma of no specific type (IC NST, all molecular subtypes, T1-3N0-3M0) were enrolled in study. CTX-III expression around different subtypes of single tumour cells were analysed in FFPE tumour samples using confocal microscope LSM780 (Carl Zeiss, Germany). Results and discussions CTX-III expression around single tumour cells was observed at 21% of breast cancer patients. In positive cases only 5% of single tumour cells were express CTX-III and these cells have features of stemness, EMT or both. Conclusion Only small part of single tumour cells showed signs of a mesenchymal type of invasion, and it didn’t depend on the state of stem and EMT. The study was supported by RSF (#16-15-10221).

For a genomically unstable cancer, a single tumour biopsy will often contain a mixture of competing tumour clones. These tumour clones frequently differ with respect to their genomic content (copy number of each chromosome segment) and structure (order/adjacency of segments on tumour chromosomes). Whole genome sequencing mixes the signals of tumour clones and contaminating normal cells. The ability to unmix these signals and infer divergent genome structure and content is relevant to current avenues of cancer research. We propose a method to unmix tumour and contaminating normal signals and jointly predict genome structure and content of each tumour clone.

Background Analysis of tumour samples for mutations is becoming increasingly important in driving personalised therapy in cancer. As more targeted therapies are developed, options to survey mutations in multiple genes in a single tumour sample will become ever more attractive and are expected to become the mainstay of molecular diagnosis in non-small cell lung cancer (NSCLC) in the future. Materials and Methods 238 non-small cell lung cancer (NSCLC) tumour samples were analysed using a custom panel of 82 mutation assays across 14 oncogenes including KRAS and EGFR using Sequenom iPlex Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF). We compared the data generated for KRAS mutations to those detected by Amplification Refractory Mutation System (ARMS) based DxS TheraScreen K-RAS Mutation Kit. Results The ARMS detected mutations in 46/238 tumour samples. For samples with mutations detected by both approaches, 99.1% overall agreement was observed. The MALDI-TOF method detected an additional 6 samples as KRAS mutation positive and also provided data on concomitant mutations including PIK3CA and TP53. Conclusions The Sequenom MALDI-TOF method provides a sensitive panel-based approach which makes efficient use of patient diagnostic samples. This technology could provide an opportunity to deliver comprehensive screening of relevant biomarkers to the clinic earlier in disease management, without the need for repeat biopsy and allow for additional downstream analysis in NSCLC where available tissue may have been exhausted.

Intratumour heterogeneity (ITH) may foster tumour adaptation and compromise the efficacy of personalized medicine approaches. The scale of heterogeneity within a tumour (intratumour heterogeneity) relative to genetic differences between tumours (intertumour heterogeneity) is unknown. To address this, we obtained 48 biopsies from eight stage III and IV clear cell renal cell carcinomas (ccRCCs) and used DNA copy-number analyses to compare biopsies from the same tumour with 440 single tumour biopsies from the Cancer Genome Atlas (TCGA). Unsupervised hierarchical clustering of TCGA and multi-region ccRCC samples revealed segregation of samples from the same tumour into unrelated clusters; 25% of multi-region samples appeared more similar to unrelated samples than to any other sample originating from the same tumour. We found that the majority of recurrent DNA copy number driver aberrations in single biopsies were not present ubiquitously in late-stage ccRCCs and were likely to represent subclonal events acquired during tumour progression. Such heterogeneous subclonal genetic alterations within individual tumours may impair the identification of robust ccRCC molecular subtypes classified by distinct copy number alterations and clinical outcomes. The co-existence of distinct subclonal copy number events in different regions of individual tumours reflects the diversification of individual ccRCCs through multiple evolutionary routes and may contribute to tumour sampling bias and impact upon tumour progression and clinical outcome.

Intra-tumoural heterogeneity of proliferation has been assessed by taking multiple biopsies from 30 colorectal cancers. Following in vivo IUDR labelling, dual parameter flow cytometry was used to measure tumour DNA index (DI) and labelling index (LI) and to derive DNA synthesis time (Ts) and potential doubling time (Tpot). Heterogeneity was seen for all parameters under investigation. Overall coefficients of variation (CV) and logarithmic transformation of Ts and Tpot (due to their non-gaussian distributions) indicate that LI (CV 25%) was the most variable parameter. Intra-tumoral heterogeneity in Tpot (lnTpot CV = 22%) was less than inter-individual variation (CV = 63%), suggesting that this variation should not be a limitation to the possible usefulness of this technique as an independent prognostic indicator. Correlations of Tpot values were examined between the shortest, the median and the value for a pooled homogenate sample from a single tumour. Using an homogenate, it was possible to accurately predict classification of tumour Tpot values as being below the median ('fast tumours') in 15 of 19 cases (79%). The data suggest that assaying an homogenate may allow a more rapid analysis of a multiply sampled tumour.

To report on the clinical features of eccrine hidrocystoma involving the eyelid. Data on a series of consecutive patients with histopathologically confirmed diagnosis were reviewed. Among 34 patients, 69 tumours were identified. The mean age at diagnosis was 59 years (range 39–91 years). The majority (71%) of patients had only a single tumour. The tumours appeared as a small (median size=1 mm) clear cystic lesion with 87% located near the eyelid margin. The eccrine hidrocystoma is a benign small cystic tumour that characteristically occurs close to but does not involve the eyelid margin.

In an immunohistochemical study of 25 canine chemodectomas, 17 tumours were stained with antisera to neurone specific enolase and the same number were stained for synaptophysin; a single tumour was stained for S100. Staining for Ki-67 occurred in 18 cases; the Ki-67-labelling index and the intensity of immunostaining was increased in more pleomorphic and malignant tumours, as assessed on histological grounds. Immunohistochemistry did not aid in recognition of less well-differentiated tumours.

Summary A rare case of a poorly-differentiated acinic cell carcinoma with primary presentation in a hyperplastic intraparotid lymph node, is reported. As the tumour mainly consisted of ductular and undifferentiated cells growing in solid cords, diagnosis was rendered difficult. Typical acinic cells were only rarely observed. In the multiple sections reviewed a single tumour nodulus occurred in salivary gland tissue outside the lymph node capsule. The possibility of a microscopic clinically occult primary acinic cell carcinoma that metastasized and presented primarly in an intraparotid lymph node, is mentioned. A multifocal origin in salivary tissue within and outside the intraglandular lymph node is another assumption, which is discussed. Differential diagnoses are mentioned and results of immuno- and histochemical studies are reported.

As discussed briefly in Chapter 1, there is a fundamental difference between unicellular and multicellular organisms in the factors which determine their rate of cell growth and division. Growth of unicellular organisms is usually limited by the availability of nutrients, and in rich medium they divide exponentially. By contrast, cells in a multicellular organism are normally bathed continually in a nutrient-rich medium (the extracellular fluid), yet their division must be strictly controlled. In healthy animals, cells tend to grow in organized layers or sheets, and cell proliferation generally occurs at well-defined locations (e.g. at the base of the dermis in skin) where there are stem cells capable of proliferation. In adult animals, proliferation generally only occurs to replace cells which have a limited lifetime (e.g. skin, gut and blood), and to repair wounds. Clearly there has to be a balance between production of new cells and loss of cells due to death or terminal differentiation. Cancer is caused by a breakdown in the systems which control this balance. A tumour is thought to be usually derived from a single aberrant stem cell, and it is interesting to note that a 1 g tumour, containing perhaps 109 cells, could result from only 30 division cycles. If it stops at that stage, a single tumour may be harmless or benign. However, malignant tumours go a stage further and metastasize: cells become detached and invade other parts of the body, where they form new tumours, with devastating results. Leukaemias are caused by uncontrolled division of leukocytes (white blood cells), and are also very serious.

Summary Cytofluorometric nuclear DNA analyses were performed on single tumour cells from eight argyrophilic gastric carcinoids. Three tumours displayed grossly diploid DNA values, with a fraction of S-phase cells of 1ߝ8%. Four other tumours were also mainly diploid, but showed an increased number of tetraploid cells, and one tumour was aneuploid with a stem-cell line of triploid cells. These and previous results indicate that gastric carcinoids are relatively heterogeneous with respect to their morphology, nuclear DNA profiles and biological behaviour, in contrast to most intestinal classical" carcinoids. Furthermore, the results are in accordance with the suggestion that gastric carcinoids may represent a highly differentiated variety within a spectrum of neuroendocrine neoplasia analogous to the corresponding variety observed in the bronchus (carcinoid — small cell, undifferentiated carcinoma).

A series of 87 patients with Wilms' tumour seen during the period 1960-73 included 11 (13%) with bilateral tumours. 6 patients presented with simultaneous bilateral tumours, 2 had tumours in each side of a horseshoe kidney, and 3 later developed a tumour in the remaining kidney. There was no reported familial incidence of Wilms' tumour. Maternal age at birth of the patients with simultaneous bilateral tumours was over 30 years in 7/8 cases. The average of patients with bilateral tumours was 15 months, whereas that of patients with unilateral tumours was 31/2 years. All the simultaneously occurring bilateral tumours and those within a horseshoe kidney were multifocal, whilst the sequentially occurring bilateral tumours and the unilateral tumours all developed as a single tumour mass within the affected kidney. Associated congenital anomalies were found in 5 (45%) of 11 patients with bilateral tumours, several of whom had more than one defect. Of 76 patients with a unilateral tumour, only 3 (4%) had congenital anomalies.

The ability of acutely hypoxic tumour cells to recover from sublethal damage following irradiation in vivo and in vitro has been measured using a single tumour system. The methods of assay were tumour growth delay, local tumour control and tumour cell survival in vitro following treatment in vivo or in vitro. Tumours in vivo or cells in vitro rendered acutely hypoxic during irradiation were irradiated with either single doses or two doses 24 hours apart. Cells left in situ had a greater capacity for recovery than those treated either in vivo or in vitro and then assayed in vitro. It is suggested that tumours may not show a systematically reduced capacity for recovery relative to normal tissues, unless chronically hypoxic tumour cells have a reduced capacity for recovery and determine the response. However, the results imply that deductions as to the ability of tumour cells to recover from sublethal damage (whether chronically hypoxic or not), which rely on in vitro assays, may underestimate the extent of recovery.

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.

Out of 960 human brain neoplasms seven primary lymphomas were cultured and grown up to twenty-eight days. The monolayers had common cytological characteristics; /i/ immediately after explantation a high density of uniform cells was observ. d. /ii/ many cells were lost during subsequent medium changes; /iii/ the monolayers contained lymphocyte-like cells in different numbers. According to their indiv. d. al characteristics the cultures could be classified into three categories: 1./ In the cultures of three tumours lymphocyte-like cells predominated. These tumours had low proliferative capacity in vitro. 2./ Cultures of three other tumours consisting mainly of tissue macrophages had a high proliferative capacity in vitro. 3./ Cultures of a single tumour showed the combined features of the former two categories: both lymphocyte-like cells, and tissue macrophages were present. These cultures showed the highest proliferative activity.

The problem of the use of the appropriate dose distribution is most important in radiotherapeutic treatment of every tumour. The best solution of course, would be to be able to use an appropriate distribution for each single tumour situation; that is to say the distribution judged by the radiologist as giving the best results. Can this objective be reached? It is possible in certain cases, but in the majority of cases we have to be content with approximations; and then again other problems arise, for which a possible solution is extremely hard to find. The aim of the following papers (1.3.2. to 1.3.9.) is to indicate and discuss such possibilities, in the light of the means at our disposal today.

IT has been suggested1,2 that the quantity of deoxypentosenucleic acid is constant for different somatic cells in the same species. On this assumption, the deoxypentosenucleic acid content of different tumour cells can be compared with that of normal cells from the same species. Many efforts have been made to compare the deoxypentosenucleic acid content of homologous normal and neoplastic tissues (see reviews in refs. 3 and 4). The results are rather divergent, several authors having reported an increase in deoxypentosenucleic acid in tumour cells, whereas others failed to observe any difference. We have found the Ehrlich ascites tumour of mice to be a good material for determinations of the average nucleic acid content of a single tumour cell5,6 and have found the deoxypentosenucleic acid content of such cells to be about twice as great as that of normal mouse cells6,7.

SummaryAn assay system is described in which a number of single tumour cells are injected intravenously into recipient mice and grow in the lungs of these mice to give macroscopic tumour nodules in 16 to 20 days. The relationship between the number of tumour nodules observed and the number of cells injected has been found to be linear up to a mean of 50 colonies/lung for both non-irradiated and irradiated cells, and the inter-experiment consistency has been shown to be good. The number of tumour nodules observed has been found to be affected by the simultaneous injection of a large number of heavily-irradiated cells, provided they are injected intravenously. An aerobic radiation survival-curve has been established and agrees very well with one obtained for the same tumour using the end-point dilution assay method. The assay has the advantage that the time required for completion is short, compared with that required for end-point dilution assays.

Background Comparing survival of patients with a single tumour and patients with multiple primaries poses different methodological problems. In population based studies, where we cannot rely on detailed clinical information, the issue is disentangling the share of survival probability from the first and second cancer, and their compounded effect. We examined three hypotheses: A) the survival probability since the first tumour does not change with the occurrence of a second tumour; B) the probability of surviving a tumour does not change with the presence of a previous primary; C) the probabilities of surviving two subsequent primary tumours are independent (additivity hypothesis on mortality rates). Methods We studied the survival probabilities modelling mortality rates according to hypotheses A), B) and C). Mortality rates were calculated using Aalen-Johansen estimators which allowed to discount for the lag-time survival before developing a second tumour. We applied this approach to a cohort of 436 women with breast cancer (BC) and a subsequent tumour in the resident population of Turin, Italy, between 1985 and 2002. Results We presented our results in term of a Standardised Mortality Ratio calculated (SMRAJ) after 10 years of follow-up. For hypothesis A we observed a significant excess mortality of 2.21 (95% C.I. 1.94 – 2.45). Concerning hypothesis B we found a not significant SMRAJ of 0.98 (95% C.I. 0.87 – 1.10). The additivity hypothesis (C) was not confirmed as it overestimated the risk of death, in fact SMRsAJ were all below 1: 0.75 (95% C.I. 0.66 – 0.84) for BC and all subsequent cancers, 0.72 (95% C.I. 0.55 – 0.94) for BC and colon-rectum cancer, 0.76 (95% C.I. 0.48 – 1.14) for BC and corpus uteri cancer (not significant). Conclusion This method proved to be useful in disentangling the effect of different subsequent cancers on mortality. In our application it shows a worse long-term mortality for women with two cancers than that with BC only. However, the increase in mortality was lower than expected under the additivity assumption.