• Lack of a relationship between colony-forming efficiency and surviving fraction at 2 Gy.

      West, Catharine M L; Davidson, Susan E; Pool, Claire; James, Roger D; Schofield, Philip F; Cancer Research Campaign Department of Radiobiology, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1991-05)
      The relationship between in vitro radiation sensitivity and colony-forming efficiency has been examined for primary human tumors of the cervix, colorectum, and lymph gland. Tumors were cultured using the Courtenay-Mills soft agar clonogenic assay and radiosensitivity was assessed as surviving fraction at 2 Gy. There was no correlation between colony-forming efficiency and surviving fraction at 2 Gy, giving confidence to the use of surviving fraction at 2 Gy as a predictor of clinical outcome.
    • Lack of association between in vitro clonogenic growth of human cervical carcinoma and tumour stage, differentiation, patient age, host cell infiltration or patient survival.

      Davidson, Susan E; West, Catharine M L; Hunter, Robin D; Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Manchester, UK. (1992-01-02)
      Biopsies from 117 patients with cervical carcinoma were studied using a clonogenic assay to assess in vitro growth. Successful colony growth was achieved in 84 tumours (72%) with a mean colony-forming efficiency (CFE), based on total viable nucleated cell counts, of 0.18 +/- 0.49% (+/- 1 standard deviation). There was a wide range of values, from 0.003-4.28%, with a coefficient of variation of 272%. The relationship between clinical features of cervical carcinoma and in vitro colony formation was investigated. No significant association was demonstrated between in vitro growth and either clinical stage (r = 0.02), tumour differentiation (r = -0.08) or patient age (r = -0.12). There was no significant difference in tumour grade between the group of tumours which failed to grow in culture and those which grew well (p = 0.70). In addition, there was no correlation between CFE and the degree of macrophage (r = 0.001), lymphocyte (r = 0.12), or granulocyte (r = 0.08) infiltration. There was no significant difference between CFEs of tumours from patients who had died and from those who were alive and well after a minimum of 2 years' follow-up after radiotherapy (p = 0.51). Ability to form colonies in agar was not associated with a worse prognosis (p = 0.49). Although CFE is an independent biological parameter, our results suggest that, for cervical carcinoma, it is not useful as a univariate prognostic factor.
    • Lack of association of HLA polymorphisms with human papillomavirus-related cervical cancer.

      Glew, Susan S; Duggan-Keen, Margaret F; Ghosh, Anna K; Ivinson, A; Sinnott, P; Davidson, J; Dyer, P A; Stern, Peter L; Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, England. (1993-07)
      An association of HLA-DQ3 with SCC of the cervix has been reported by researchers in Germany and Norway. This article documents a similar-sized study with patients and controls from northwest England. We report in detail on serologically determined HLA polymorphism in SCC patients with respect to HPV 16 infection, MHC class II expression within the tumor, serologic response to HPV, and other relevant clinical variables. We have also extended our studies to include DNA-based analysis using PCR and SSO probes for HLA-DQ. No significant association of any HLA-A, -B, -C, -DR, or -DQ antigen with SCC patients was found. While a possible explanation of the differences among studies could be a reflection of disease heterogeneity, the several tumor and clinical factors examined do not account for the observed differences from previous reports. Further studies are needed for a greater understanding of the interaction of HPV and HLA type in the development of cervical neoplasia.
    • Lack of consensus identifies important areas for future clinical research: Advanced Prostate Cancer Consensus Conference (APCCC) 2019 findings

      Vogl, U. M.; Beer, T. M.; Davis, I. D.; Shore, N. D.; Sweeney, C. J.; Ost, P.; Attard, G.; Bossi, A.; de Bono, J.; Drake, C. G.; et al. (2021)
    • Lack of correlation between differentiation status and response to radiation of three murine squamous cell carcinomas.

      Moore, James V; Moses, R; Cowie, V; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1985)
      The gross growth rate, histology, cellular kinetics, and in situ radiobiological response have been measured for three murine, keratinising squamous cell carcinomas that differed in their degree of differentiation. Growth rate was fastest in the least-differentiated tumour, slowest in the best-differentiated. However, the kinetics of the compartment of undifferentiated cells that are likely to be radiotherapeutically important, were the same for the three lines. There was no correlation between degree of differentiation and intrinsic or apparent radiosensitivity as measured by the growth delay assay. The radiobiologically best-oxygenated tumour was that which has the largest stromal component and this was not the best-differentiated tumour.
    • Lack of correlation between peripheral blood lymphokine-activated killer (LAK) cell function and clinical response in patients with advanced malignant melanoma receiving recombinant interleukin 2.

      Ghosh, Anna K; Dazzi, H; Thatcher, Nick; Moore, M; Cancer Research Campaign, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1989-03-15)
      A phase-I/II study of recombinant interleukin 2 (rIL-2) was performed in 31 melanoma patients. The first dose of rIL-2 was given intrasplenically followed 4 hr later by an i.v. dose and 3 further i.v. doses on alternate days. Three courses of treatment were planned at 3-week intervals. The maximum tolerated single dose was 11 x 10(6) Cetus U/m2. Haematological and immunological data were available on 20 patients. Post-treatment response to rIL-2 therapy was evident from (i) a rapid depletion of peripheral blood lymphocytes (PBL) with a rebound at 4-7 days (2 times pre-treatment values); (ii) an increase in the number of IL-2 receptor-positive lymphocytes (4-15 times pre-treatment values); (iii) an increase in the number of "positive" patients with cytotoxic (anti-K562) peripheral blood mononuclear cells (PBMC) from 30% to 80%; (iv) amplified killing of K562 by positive patients in relation to pre-treatment values; and (v) the induction of PBMC cytotoxicity (in 45% of patients) against the NK-resistant, LAK-sensitive target, Mel I. Partial clinical responses to rIL-2 treatment were observed in 4 patients, but these were not reflected in the PBMC LAK activity or the other parameters examined.
    • Lack of correlation between residual radiation-induced DNA damage, in keratinocytes assayed directly from skin, and late radiotherapy reactions in breast cancer patients.

      Kiltie, Anne E; Barber, James B P; Swindell, Ric; Ryan, Anderson J; West, Catharine M L; Hendry, Jolyon H; Magee, Brian; CRC Section of Genome Damage and Repair, Christie Hospital NHS Trust, Manchester, United Kingdom. (1999-02-01)
      PURPOSE: To study the relationship between the severity of late reactions to radiotherapy in breast cancer patients, and the extent of residual radiation-induced DNA damage, using a rapid assay of keratinocytes obtained directly from skin biopsies. METHODS AND MATERIALS: A review was made of 32 patients with breast cancer, treated uniformly by radiotherapy between 1983 and 1988, following breast-conserving surgery. Their late radiotherapy reactions were scored (9-14 years post-radiotherapy) using a modified LENT SOMA scale, and a 5-mm buttock skin punch biopsy was obtained. Intact skin was irradiated at room temperature, and after allowing 24 h for repair, the tissue was disaggregated and the cells processed for pulsed field gel electrophoresis (PFGE). Residual DNA damage was expressed as the fraction of DNA released (FDR) following 150 Gy. RESULTS: Studies using flow cytometry on disaggregated breast skin showed that over 90% of the cells were keratinocytes. The PFGE assay was robust with low background FDRs in unirradiated skin samples (mean 3.2%) and a wide range of FDRs following irradiation from 11.5% to 26.6%. No correlation was found between the FDR at 150 Gy (FDR 150) and any of the late reaction scores or retrospective acute reaction scores. There was, however, a borderline significant correlation for family history and FDR 150 (p = 0.059). CONCLUSION: Rapid measurement of residual DNA damage in irradiated differentiated keratinocytes, the predominant cell population in skin biopsies, showed no correlation with the severity of symptomatic early or documented late reactions in a retrospectively studied group of 32 breast cancer patients.
    • The lack of correlation between toxicity and free radical formation of two diaziridinyl benzoquinones.

      Lea, J S; Garner, H J; Butler, John; Hoey, Brigid M; Ward, Timothy H; Paterson Institute for Cancer Research, Christie Hospital, Holt Radium Institute, Manchester, U.K. (1988-05-15)
      L1210 and K562 leukaemic cells have been used to study the relationship between cytotoxicity and free radical production by two aziridinyl benzoquinones, 2,5-bis(carboethoxyamino)3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ). BZQ showed a high level of toxicity in both cell lines, but no ESR signal was detectable, while AZQ readily produced an ESR signal but much lower cytotoxicity was observed, particularly in L1210 cells. The rate of superoxide formation was measured for each drug. The results demonstrate that cell killing and free radical production do not necessarily concur.
    • Lack of differential sparing of late ischaemic atrophy and early epidermal healing, after dose fractionation of mouse tails down to 2.6 Gy per fraction.

      Hendry, Jolyon H; Department of Radiobiology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, U.K. (1987-02)
      Long-term atrophy of irradiated mouse tails began after about 5 months, and the incidence rose steadily to the end of the lifespan. The major associated histological change was atherosclerosis in the single tail artery. The incidence of the ischaemic atrophy was dependent on the size of the irradiated volume. The probability of ischaemic atrophy assessed at 3 years after irradiation was little dependent on the dose. The fractionation effect was described by alpha/beta congruent to 30 Gy, which was not lower than the range of values applicable for healing of the early epidermal reactions on the tail. Hence the general finding of a sparing of late effects in tissues using low doses per fraction was not observed in these experiments using dose fractions down to 2.6 Gy and the present endpoints.
    • Lack of effect of a granulocyte proliferation inhibitor or their committed precursor cells.

      Lord, Brian I; Testa, Nydia G; Wright, Eric G; Banerjee, R; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1977-05)
      Using the agar culture technique, we have measured the effect of granulocyte extracts GCE (and of erythrocyte-RCE and lymph node extracts-LNE) on the growth and proliferation of the committed granulocytic precursor cells, CFU-C. In addition we have determined their effects on the proliferation of the developing colony cells and on the ultimate cell production in the colonies. The results show that GCE has no effect on the growth or proliferative activity on the CFU-C. It does, however, reduce both the autoradiographic labelling indices of the developing colony cells and the net colony cellularities, acting as a cell cycle modulator. These are effects specific to the GCE since at the dose levels used, neither RCE nor LNE affected these measurements.
    • Lack of late skin necrosis in man after high-dose irradiation using small field sizes: experiences of grid therapy.

      Shirato, H; Gupta, Nirmal K; Jordan, Thomas J; Hendry, Jolyon H; Department of Radiotherapy, Christie Hospital and Holt Radium Institute, Manchester, UK. (1990-11)
      Out of a total of 437 patients with superior vena caval syndrome or advanced malignancy, given single-dose grid radiotherapy, four survived to 7 years. The dose to the skin under each of the 77 holes in the grid was approximately 58 Gy. The lack of skin necrosis in the total of 308 skin circles of 1 cm diameter among these survivors, compared with known necrosis rates in larger irradiated areas, implies that there is a marked field-size effect for late necrosis in small areas of irradiated skin.
    • Lack of response of bone marrow, in vitro, to growth factors in congenital neutropenia.

      Chang, James; Craft, A W; Reid, M M; Coutinho, Lucia H; Dexter, T Michael; Department of Haematology, Christie Hospital, Manchester, United Kingdom. (1990-10)
      Severe congenital neutropenia has a poor outlook. In vitro clonogenic assays using recombinant growth factors may improve understanding of the underlying pathogenetic mechanisms and identify those in whom growth factors might be clinically useful. Marrow from a boy with congenital neutropenia was cultured with a variety of recombinant growth factors. The results show that the neutropenia did not result from a lack of myeloid progenitors but that these progenitors could not produce mature neutrophils. Bone marrow transplantation is being considered as the most likely approach to correct neutropenia.
    • Lapatinib inhibits stem/progenitor proliferation in preclinical in vitro models of ductal carcinoma in situ (DCIS).

      Farnie, Gillian; Johnson, R; Williams, Kathryn E; Clarke, Robert B; Bundred, N (2013-02-01)
      Breast-conserving surgery for ductal carcinoma in situ (DCIS) is often combined with irradiation, reducing recurrence rates to 20% within 10 years; however, there is no change in overall survival. Evidence in the invasive breast indicates that breast cancer stem cells (CSCs) are radiotherapy-resistant and are capable of re-initiating a tumor recurrence; hence, targeting CSCs in high risk DCIS patient may improve survival. HER2 is overexpressed in 20% of DCIS and is known to be highly active in breast CSCs; we therefore investigated the effect of Lapatinib on DCIS CSC activity using 2 in vitro culture systems. Two DCIS cell lines DCIS.com (HER2 normal) and SUM225 (HER2 overexpressed) as well as DCIS cells from patient samples (n = 18) were cultured as mammospheres to assess CSC activity and in differentiated 3D-matrigel culture to determine effects within the non-CSCs. Mammosphere formation was reduced regardless of HER2 status, although this was more marked within the HER2-positive samples. When grown as differentiated DCIS acini in 3D-matrigel culture, Lapatinib only reduced acini size in the HER2-positive samples via decreased proliferation. Further investigation revealed lapatinib did not reduce self-renewal activity in the CSC population, but their proliferation was decreased regardless of HER2 status. In conclusion we show Lapatinib can reduce DCIS CSC activity, suggesting that the use of Lapatinib in high-risk DCIS patients has the potential to reduce recurrence and the progression of DCIS to invasive disease.
    • Lapatinib versus hormone therapy in patients with advanced renal cell carcinoma: a randomized phase III clinical trial.

      Ravaud, Alain; Hawkins, Robert E; Gardner, Jason P; Von der Maase, Hans; Zantl, Niko; Harper, P; Rolland, Frédéric; Audhuy, Bruno; Machiels, Jean-Pascal; Pétavy, Frank; et al. (2008-05-10)
      PURPOSE: Lapatinib is an orally reversible inhibitor of epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER-2) tyrosine kinases with demonstrated activity in patients with HER-2-positive breast cancer. In the current phase III open-label trial, lapatinib was compared with hormone therapy (HT) in patients with advanced renal cell carcinoma (RCC) that express EGFR and/or HER-2. PATIENTS AND METHODS: Patients with advanced RCC who had experienced disease progression through first-line cytokine therapy--stratified by Karnofsky performance status and number of metastatic sites--were randomly assigned to lapatinib 1,250 mg daily or HT. The primary end point was time to progression (TTP); secondary end points included overall survival (OS), safety, and biomarker analyses. RESULTS: Four hundred sixteen patients were enrolled onto the study. Median TTP was 15.3 weeks for lapatinib versus 15.4 weeks for HT (hazard ratio [HR] = 0.94; P = .60), and median OS was 46.9 weeks for lapatinib versus 43.1 weeks for HT (HR = 0.88; P = .29). In a biomarker analysis of patients with EGFR-overexpressed tumors (3+ by immunohistochemistry [IHC]; n = 241) median TTP was 15.1 weeks for lapatinib versus 10.9 weeks for HT (HR = 0.76; P = .06), and median OS was 46.0 weeks for lapatinib versus 37.9 weeks for HT (HR = 0.69; P = .02). These results were confirmed by Cox regression analysis. No unexpected toxicities were observed; the most commonly reported drug-related adverse events (all grades) for lapatinib were rash (44%) and diarrhea (40%). CONCLUSION: Lapatinib was well tolerated with equivalent overall efficacy to HT in advanced RCC patients who had experienced disease progression while receiving cytokines, and the study supports that lapatinib prolonged OS relative to HT in patients with 3+ EGFR status determined by IHC.
    • Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

      Johnson, Suzanne M; Banyard, Antonia; Smith, Christopher; Mironov, A.; McCabe, Martin; Children's Cancer Group, Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology Medicine and Health, University of Manchester, Oglesby Cancer Research Building, Manchester Academic Health Science Centre, Manchester Cancer Research Centre, Manchester M20 4GJ, UK. (2020)
      Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.
    • The Large Scale Synthesis and Chromatographic and Spectral Properties of a Series of Alkylated Thymine and Uracil-Containing Nucleosides. O2-, 3- and O4-Alkylpyrimidine Derivatives

      Saffhill, Roy; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom (1987)
    • Large-scale immunoprecipitation from fission yeast cell extracts.

      Grallert, Agnes; Hagan, Iain M; CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom (2017-02-01)
      We outline immunoprecipitation (IP) procedures to isolate the large quantities of a molecule of interest that are required to identify posttranslational modifications (PTMs) in subsequent targeted mass spectrometry analysis. In situ denaturation by trichloroacetic acid precipitation inhibits the activities of modifying enzymes that could alter the PTM profile to preserve the PTMs on a target of interest throughout the precipitation step. In contrast, isolation of the same molecule with the nondenaturing variation on this IP procedure can maintain associations with partner molecules whose PTMs can also be mapped, albeit with the caveat that modifications could have occurred during the extended IP period.
    • Laser flash photolysis of rhodopsin at room temperature

      Bensasson, R V; Land, Edward J; Truscott, T G (2011-08-08)
    • Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection.

      Weekes, M; Tan, S; Poole, E; Talbot, S; Antrobus, R; Smith, Duncan L; Montag, C; Gygi, S; Sinclair, J; Lehner, P; et al. (2013-04-12)
      The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance-associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14(+) and CD34(+) progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.
    • Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA.

      Stacey, Simon N; Jordan, Deborah; Williamson, Andrew J K; Brown, Michael D; Coote, Joanna H; Arrand, John R; Cancer Research Campaign, Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 4BX, United Kingdom. sstacey@picr.man.ac.uk (2000-08)
      Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.