Correction: Safety and utility of image-guided research biopsies in relapsed high-grade serous ovarian carcinoma-experience of the BriTROC consortium.

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Safety and utility of image-guided research biopsies in relapsed high-grade serous ovarian carcinoma-experience of the BriTROC consortium.

BACKGROUND: Investigating tumour evolution and acquired chemotherapy resistance requires analysis of sequential tumour material. We describe the feasibility of obtaining research biopsies in women with relapsed ovarian high-grade serous carcinoma (HGSC). METHODS: Women with relapsed ovarian HGSC underwent either image-guided biopsy or intra-operative biopsy during secondary debulking, and samples were fixed in methanol-based fixative. Tagged-amplicon sequencing was performed on biopsy DNA. RESULTS: We screened 519 patients in order to enrol 220. Two hundred and two patients underwent successful biopsy, 118 of which were image-guided. There were 22 study-related adverse events (AE) in the image-guided biopsies, all grades 1 and 2; pain was the commonest AE. There were pre-specified significant AE in 3/118 biopsies (2.5%). 87% biopsies were fit-for-purpose for genomic analyses. Median DNA yield was 2.87 µg, and was higher in biopsies utilising 14 G or 16 G needles compared to 18 G. TP53 mutations were identified in 94.4% patients. CONCLUSIONS: Obtaining tumour biopsies for research in relapsed HGSC is safe and feasible. Adverse events are rare. The large majority of biopsies yield sufficient DNA for genomic analyses-we recommend use of larger gauge needles and methanol fixation for such biopsies, as DNA yields are higher but with no increase in AEs.

Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line.

The association between T3 and the T cell antigen receptor was examined using the T3 bearing T cell leukemic line Jurkat. A monoclonal antibody, C305, was produced, which reacted with idiotypic-like determinants expressed on Jurkat. The molecule with which this antibody reacted was a disulfide-linked heterodimer of 90 kD, composed of polypeptides of 42 and 54 kD. Thus, C305 reacted with a molecule with characteristics of the putative T cell antigen receptor described by others. A series of mutants of Jurkat, induced with ethyl methane sulfonate or radiation, was selected for T3 or antigen receptor negativity. In every instance, there was a concomitant loss of both T3 and the antigen receptor as assessed by quantitative absorption, indirect immunofluorescence, and antibody plus complement-mediated cytotoxicity. The absence of antigen receptor molecules was confirmed on diagonal gels, excluding the possibility that conformational changes of the antigen receptor on such T3-negative mutants were responsible for the failure of such mutants to react with C305. Moreover, in a mutant that expressed a marked decrease in the level of T3 expression, there was a comparable decrease in the expression of antigen receptor determinants. These results suggest that there is an obligate requirement for the coexpression of T3 and the T cell antigen receptor. Furthermore, attempts to activate such mutants with the lectin phytohemagglutinin suggested that the expression of T3 and/or the antigen receptor was required for activation of these cells.

Specificity and function of a human autologous reactive T cell.

Normal human peripheral blood contains a population of T cells (autologous reactive cells [ARC]) capable of proliferating in response to signals from autologous B cells and monocytes. Selective suicide of proliferating ARC with 5-bromo-2-deoxyuridine and light demonstrated that this ARC was responsive to signals coded for by genes more closely linked to the HLA-DR, than to the HLA-A, or HLA-B, loci. Density-gradient fractionation of T cells indicated that populations enriched in ARC reactivity were also enriched for helper influences required for Ig synthesis by autologous B cells. In contrast, populations negatively selected for proliferating ARC were deficient in helper activity. These studies indicate that the ARC is responsive, at least in part, to products of genes closely linked to the HLA-DR locus and can function as a helper cell.

Heterogeneity of murine regulatory T cells. I. Subpopulations of amplifier and suppressor T cells.

Immunization of C3H/HeJ mice with 4 X 10(9) SRBC yields a whole splenic T-cell population which can, upon transfer, specifically suppress recipient direct and indirect plaque-forming cells (PFC) responses to sheep erythrocytes (SRBC). Discontinuous bovine serum albumin density gradient fractionation of these T cells demonstrated a population of low density T cells which augmented and a population of high density T cells which suppressed recipient responses irrespective of the number of T cells transferred. Moreover, infusion of admixtures of low and high density cells resulted in intermediate regulatory functions which could be predicted by knowing the regulatory capacity of each population alone. In addition to heterogeneity existing among regulatory T cells as regards amplification and suppression, it appeared that heterogeneity existed within the suppressor T population. Thus, T cells capable of inhibiting direct PFC could be distinguished from those suppressing indirect PFC by their differential localization in peripheral lymphoid tissue, differences in the dissipation of suppressive influences during incubation at 37 degrees C, and by differences in the possible requirement for adherent cell populations. While the relative frequency of both low density amplifier and high density suppressor cells increased with the dose of SRBC used for their induction, it appeared that suppressor cells might be generated in response to feedback signals from amplifier cells. These studies indicate that further delineation of heterogeneity existing within suppressor populations may be helpful in defining mechanisms required for the induction and manifestation of suppressive regulatory forces.

Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores.

Antibodies against the T3-antigen receptor complex can activate the human T cell line, Jurkat, to produce interleukin 2 (2-5). This activation is initiated by a receptor-mediated increase in the concentration of free cytoplasmic calcium ions [Ca2+]i (3, 4). In this communication, we investigate the mechanism by which the receptor complex increases [Ca2+ )i in Jurkat cells. The initial receptor-mediated change in [Ca2+]i can occur when extracellular Ca2+ is depleted by EGTA. Perturbation of the T cell antigen receptor, therefore, generates a signal which mobilizes Ca2+ from intracellular stores. As inositol trisphosphate appears to function as such a signal for certain hormone receptors, we measured the levels of inositol trisphosphate and of the other inositol phosphate compounds in Jurkat. Antibodies to either the antigen receptor heterodimer or T3 determinants result in marked elevations of all three inositol phosphates. These changes in inositol phosphates are not secondary to the receptor-mediated increases in [Ca2+]i as demonstrated by the inability of the Ca2+ ionophore, ionomycin, to affect the levels of any of these compounds. In concentrations between 0.1 and 1 microM, purified inositol trisphosphate releases Ca2+ from permeabilized Jurkat cells. Taken together, these data indicate that, during activation, perturbation of the T3-antigen receptor complex generates inositol trisphosphate. This compound functions as an intracellular signal to release Ca2+ from intracellular stores, leading to increases in [Ca2+]i.

Macrophage heterogeneity in man. A subpopulation of HLA-DR-bearing macrophages required for antigen-induced T cell activation also contains stimulators for autologous-reactive T cells.

Utilizing somatic cell hybridization, we have developed a monoclonal antibody that interacts only with cells of the monocyte/macrophage (M phi) line and not with other myeloid or lymphoid cells. This antibody detects a 120,000-dalton determinant present on 37 +/- 2.8% of the peripheral blood M phi from several (HLA-DR)-disparate individuals and only depicts a subpopulation (approximately 30%) of HLA-DR-bearing M phi from any single subject. Cytolytic removal of this subpopulation of HLA-DR-bearing cells markedly diminished antigen-induced T cell reactivity, a deficiency that can be reconstituted with autologous M phi but not with either their soluble products containing lymphocyte-activating factor or with intact HLA-DR-disparate M phi. Whereas M phi bearing both the 120,000-dalton determinant and HLA-DR serve as effective stimulators for autologous mixed lymphocyte reactions. M phi bearing only HLA-DR determinants do not. However, this latter population of M phi can stimulate proliferation among alloreactive T cells. These studies indicate that the Mac-120 monoclonal antibody detects a subpopulation of HLA-DR-bearing M phi that is required for the genetically restricted presentation of conventional antigen to reactive T cells. Within the M phi population, these Mac-120+ cells constitute the most effective stimulators for autologous mixed lymphocyte reactions.

Antigen-reactive T cells can be activated buy autologous macrophages in the absence of added antigen.

T cells responsive to macrophages (M phi) in the autologous mixed lymphocyte reaction (AMLR) contain those cells that can be induced to proliferate by soluble antigens. Negative solution (5-bromo-2-deoxyuridine and light) of T cells activated by autologous M phi also removed those cells required for reactivity to Candida albicans and purified protein derivative. Positive selection of T cells responsive to autologous M phi yields a population that is simultaneously enriched in antigen reactivity. Some patients demonstrating cutaneous anergy and diminished in vitro blast transformation in response to soluble antigen also lack T cells responsive to the AMLR to M phi. When considered in conjunction with previously reported data, these findings indicate the AMLR occurring between T cells and M phi in the absence of soluble antigen represents self recognition occurring between antigen-reactive T cells and antigen-presenting M phi.

Functional heterogeneity of murine lymphoid cells. V. Lymphocytes lacking detectable surface theta or immunoglobulin determinants.

An appreciable percent (3-14%) of the lymphocyte-like cells of the mouse spleen lack both the theta-isoantigen and sufficient surface immunoglobulin to be detected by conventional immunofluorescence or autoradiographic procedures. These theta(-),Ig(-) cells are increased in frequency after treatment of mice with antithymocyte serum or in mice that have been thymectomized, irradiated (850 R), and reconstituted with bone marrow cells. Moreover, in chimeric C57BL/6 mice in which the T cells are derived from (BALB/c x C57BL/6)F(1) donors, theta(-),Ig(-) cells also lack BALB/c histocompatibility antigens. These experiments indicate that theta(-),Ig(-) cells are not theta(-) T lymphocytes. Removal of complement receptor lymphocytes from spleen cell populations increases the frequency of theta(-),Ig(-) cells, indicating that such cells lack the complement receptor. Partially purified populations of theta(-),Ig(-) cells have been obtained by cytolysis by anti-theta- and anti-kappa-antibody and complement and by density gradient ultracentrifugation. These cells closely resemble lymphocytes in morphology. The only exceptional feature is the existence of prominent nucleoli. The theta(-),Ig(-) cells lack hemoglobin and endogenous peroxidases, are not actively phagocytic, and do not adhere to glass. This suggests they are not of the erythroid, myeloid, or monocytoid lines. [(3)H]Thymidine labeling studies indicate that theta(-),Ig(-) cells are members of a relatively slowly dividing cell pool. Whether theta(-),Ig(-) cells are members of the "classical" B lymphocyte line or belong to another, as yet undescribed, lineage is not yet certain.

A randomised phase II trial of hydroxychloroquine and imatinib versus imatinib alone for patients with chronic myeloid leukaemia in major cytogenetic response with residual disease.

In chronic-phase chronic myeloid leukaemia (CP-CML), residual BCR-ABL1+ leukaemia stem cells are responsible for disease persistence despite TKI. Based on in vitro data, CHOICES (CHlorOquine and Imatinib Combination to Eliminate Stem cells) was an international, randomised phase II trial designed to study the safety and efficacy of imatinib (IM) and hydroxychloroquine (HCQ) compared with IM alone in CP-CML patients in major cytogenetic remission with residual disease detectable by qPCR. Sixty-two patients were randomly assigned to either arm. Treatment 'successes' was the primary end point, defined as ≥0.5 log reduction in 12-month qPCR level from trial entry. Selected secondary study end points were 24-month treatment 'successes', molecular response and progression at 12 and 24 months, comparison of IM levels, and achievement of blood HCQ levels >2000 ng/ml. At 12 months, there was no difference in 'success' rate (p = 0.58); MMR was achieved in 80% (IM) vs 92% (IM/HCQ) (p = 0.21). At 24 months, the 'success' rate was 20.8% higher with IM/HCQ (p = 0.059). No patients progressed. Seventeen serious adverse events, including four serious adverse reactions, were reported; diarrhoea occurred more frequently with combination. IM/HCQ is tolerable in CP-CML, with modest improvement in qPCR levels at 12 and 24 months, suggesting autophagy inhibition maybe of clinical value in CP-CML.

Antibody-dependent lymphoid cell-mediated cytotoxicity: no requirement for thymus-derived lymphocytes.

The capacity of lymphoid cells from nonsensitized mice to lyse antibody-coated target erythrocytes in vitro does not require the presence of thymus-derived or thymus-dependent lymphocytes. Thus, spleen cells from thymus-deprived mice and spleen cell populations from which thymus-dependent lymphocytes had been removed were fully competent to mediate destruction of antibody-coated target cells. However, prior treatment of spleen cell populations with antibody to kappa chains diminished this function, suggesting a role for bone marrow-derived lymphocytes.

SYSTEMS-2: A randomised phase II study of radiotherapy dose escalation for pain control in malignant pleural mesothelioma.

SYSTEMS-2 is a randomised study of radiotherapy dose escalation for pain control in 112 patients with malignant pleural mesothelioma (MPM). Standard palliative (20 Gy/5#) or dose escalated treatment (36 Gy/6#) will be delivered using advanced radiotherapy techniques and pain responses will be compared at week 5. Data will guide optimal palliative radiotherapy in MPM.

A Low Molecular Weight Immunoglobulin Antigenically Related to 19 S IgM.

Sucrose density gradient analysis of the fresh sera of patients with hereditary ataxia telangiectasia, disseminated lupus, and Waldenström's macroglobulinemia revealed the presence of an immunoglobulin possessing IgM determinants but having a sedimentation coefficient of approximately 7 S. Bio-Gel chromatography of patients' sera confirmed the presence of two distinct populations of IgM. The low molecular weight IgM possessed incomplete isohemagglutinin activity that was resistant to treatment with reducing agents. Gel diffusion analysis revealed that the 7 S IgM showed immunological identity with both 19 S IgM and the subunits of the 19 S IgM produced by reduction. Approximately 10 to 15% of the patient's total IgM was low molecular weight. Evidence is presented that the 7 S IgM was not produced from the patient's serum 19 S IgM on in vitro incubation. A simple rapid technique is described, using double diffusion in polyacrylamide gels, which permits the determination of low molecular weight IgM in sera and other fluids. Using this technique, the sera of 52 patients with disseminated lupus were surveyed, and 17% of the patients were found to contain low molecular weight IgM. The low molecular weight IgM occurred with particular frequency in male patients with disseminated lupus and in those patients with low or absent serum IgA.Studies of the salivary immunoglobulins of patients with ataxia telangiectasia and disseminated lupus suggest an iverse relationship between the levels of IgA and IgM. In patients lacking salivary IgA, IgM was the major immunoglobulin present. No correlation was observed between salivary immunoglobulin levels and the severity of sinopulmonary infections in these patients.

Differential expression of Ia molecules by human monocytes.

Human immune response genes can be divided into three distinct loci, each of which codes for three distinct families of Ia molecules: HLA-SB, HLA-DC, and HLA-DR. The tissue distribution and function of only one of these Ia molecules, HLA-DR, has been thoroughly studied. Using monoclonal antibodies, we examined the display of HLA-DR and HLA-DC molecules by adherent, human peripheral blood monocytes. The results of these studies demonstrate that although all human peripheral blood monocytes display easily detectable HLA-DR molecules, only 50% display easily detectable HLA-DC molecules. Separation of peripheral blood monocytes into HLA-DC+ and HLA-DC- cells demonstrates that each population displays an equivalent density of HLA-DR molecules. Therefore, on the basis of differences in their display of these two Ia molecules, adherent peripheral blood monocytes can be divided into two broad populations: HLA-DR+, HLA-DC+, and HLA-DR+, HLA-DC-. Despite the dis-coordinate display of these Ia antigens, the expression of both HLA-DR and HLA-DC can be regulated by a common signal, gamma interferon (IFN-gamma). Incubation of monocytes for 96 h in autologous serum leads to a marked decrease in the expression of both HLA-DR and HLA-DC. Addition of recombinant IFN-gamma to the cultures leads to reexpression of both HLA-DR and HLA-DC to levels comparable to those seen in fresh monocytes. In addition, although IFN-gamma does not modulate all monocyte surface markers, it can be demonstrated to modulate expression of one marker, MAC 120, in a manner similar to that observed for Ia antigens. These studies demonstrate that among human peripheral blood monocytes, the distribution of the Ia molecule, HLA-DC, is not coordinate with that of HLA-DR, although both respond to the same regulatory signal.

Prostaglandin E modulation of the mitogenic response of human T cells. Differential response of T-cell subpopulations.

Prostaglandins (PG) of the E series, PGE(1) and PGE(2) (PGEs), can induce elevations of intracellular cyclic AMP (cAMP) among thymus-derived (T) lymphocytes (T cells) and inhibit their reactivity. For example, 0.1 muM of PGEs induces a two- to threefold increase of intracellular cAMP among human peripheral blood T cells and a 20-30% suppression of their blastogenic response to phytohemagglutinin. However, this suppression actually represents the net reactivity of T-cell populations demonstrating quite different responses to PGEs. Fractionation of T-enriched populations on a discontinuous density gradient yields a population of high density cells whose phytohemagglutinin-induced blastogenic response is suppressed 60%; a population of intermediate density cells whose response is suppressed 20%; and a population of low density T cells whose response is not suppressed, but is enhanced 20% by both of the PGEs. The diametrically opposite responses of low and high density T cells to the PGEs is not related to any difference in their intrinsic mitogen reactivity nor is it influenced by interactions with other T cells, bone marrow-derived (B) cells, or monocytes. Moreover, the distinct blastogenic response of low and high density T cells to PGEs does not simply correlate with PGE-mediated activation of adenylate cyclase. PGE(2) induced comparable absolute and identical relative increases of intracellular cAMP among the low and high density T cells. Cholera toxin, a potent activator of adenylate cyclase, and exogenous 8-bromo cAMP mimicked the effects of the PGEs on these two T-cell populations. These data demonstrate that T cells are heterogeneous with regard to their response to the PGEs. Thus, PGEs should be considered as potential regulators rather than as universal suppressors for T-cell reactivity. Moreover, the effect of PGEs on the blastogenic response of a given T-cell population depends upon intracellular events which occur subsequent to elevations of cAMP.

Molecular analysis of the bare lymphocyte syndrome.

The bare lymphocyte syndrome is a disorder in which class I histocompatibility antigens fail to be expressed normally on the surface of lymphocytes. Utilizing complementary DNA probes for both beta 2-microglobulin and class I genes, the molecular basis for this syndrome was investigated in a family with two siblings exhibiting the bare lymphocyte syndrome. Southern blot analysis demonstrated no gross internal defect in either class I or beta 2-microglobulin genes. Northern blot analysis of class I and beta 2-microglobulin messenger RNAs also revealed no qualitative difference between affected and unaffected family members. In contrast, quantitation of both class I and beta 2-microglobulin transcripts demonstrated each to be decreased in patients when compared to controls. Moreover, the decrease in both transcripts was coordinate. These results suggest that the bare lymphocyte syndrome may represent a pretranslational regulatory defect of both class I and beta 2-microglobulin gene expression.

Suppressor thymus-derived lymphocytes in fungal infection.

Thymus-derived lymphocyte (T-cell) function, as determined in vivo by cutaneous reactivity to several antigens and in vitro by responsiveness to mitogens and antigens, was assessed in 14 patients infected with a variety of fungal organisms. While all patients manifested a normal frequency of peripheral blood T cells, only seven patients reacted to at least one of the antigens used for cutaneous testing and demonstrated normal in vitro T proliferative responses. Three patients exhibited cutaneous anergy but normal in vitro T-cell reactivity while four patients demonstrated persistent anergy and marked in vitro T-cell hyporeactivity which was independent of activity of infection, concurrent medication, or any associated disorders. The marked diminution of in vitro T-cell reactivity noted for these later four patients was not due to a deletion of antigen- or mitogen-reactive cells. Thus, patients' cells which had been initially cultured for 7 days without any mitogenic or antigenic stimulus and which were subsequently washed and recultured with phytohemagglutinin, concanavalin A, or histoplasmin demonstrated a marked increase in their responsiveness. Moreover, this reactivity noted for recultured cells could be suppressed by a nonphagocytic, nonadherent, nonimmunoglobulin-bearing, sheep red blood cell rosette-forming population of cells isolated from the fresh peripheral blood mononuclear cells of the same patient. While these "regulator" T cells were capable of suppressing T-proliferative responses to antigens and mitogens, they did not diminish pokeweed mitogen-induced immunoglobulin synthesis by normal bone marrow-derived lymphocytes. Patients in whom suppressor "T" cells were found were at risk for relapsing, disseminated fungal infection.

Differential effect of cyclosporin A on activation signaling in human T cell lines.

Different T cell lines, which can be induced to secrete interleukin 2 (IL-2) in vitro, were used to dissect the effect of cyclosporin A (CsA). The T leukemia cell Jurkat requires an increase in cytoplasmic calcium concentration ([Ca++]i) and phorbol myristate acetate (PMA) for the induction of IL-2 production, which is completely blocked by CsA. Another T cell line, HUT 78, also produces IL-2 in response to a rise in [Ca++]i and PMA; however, in HUT 78, PMA alone induces low levels of IL-2 production that is not blocked by CsA. After treatment with 5-azacytidine, HUT 78 cells produced maximal levels of IL-2 in response to PMA alone without requiring [Ca++]i increasing stimuli. In these cells no inhibitory effect of CsA on PMA-induced activation could be demonstrated. In addition, CsA does not inhibit PMA-induced translocation of protein kinase C. These data suggest that CsA does not globally inhibit IL-2 gene expression, but rather interferes with signaling events of T cell activation.

Identification of lymphoid cells in cultured of murine leukocytes and thymus.

Several culture conditions and media were studied in an effort to establish long-term cultures of murine lymphoid cells from blood and thymus. Cultures vessels included small glass bottles and rubber-stoppered tubes. Media such as Roswell Park Memorial Institute 1640, 1700, 1701, 1715, GEM 1717, NCTC, fetal calf and horse serum supplements, and conditioned medium were tried. Lymphoid cells in mouse leukocyte cultures survived as long as eight months before dying out. However, lymphoid cells in thymus cell cultures, strarted and maintained with GEM 1717 medium with 20% fetal calf serum supplementation, gave rise to cell lines that continued to yield subcultures for more than 2 years. Mcroscopic examination of thymus cell subcultures revealed lymphoid and thymic epighelioid cells. Tumorigenicity studies of one cell line were negative. Chromosomal preparations of this cell line often contained near-normal karyotypes but were complicated by the presence of binucleated cells. Live cell fluorescent antibody assays for surface theta-antigen and immunoglobulin revealed immunoglobulin-negative cells possessing barely detectable theta determinants. Functional assays for thymus-derived lymphoid cell activity suggested that these cells were mitogen responsive and weakly reactive in one-way mixed lymphocyte culture. On the basis of this evidence it was sugguested that these cells represent a class of T-cells (thymus-derived lymphocytes) that have all but lost theta antigen, possibly due to prolonged culture.

Aryl hydrocarbon hydroxylase activity in subpopulations of peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PMC), isolated by density gradient techniques with Ficoll-Hypaque, contain T- and B-lymphocytes and monocytes. Aryl hydrocarbon hydroxylase (AHH) activity was measured in PMC subfractions consisting of T-lymphocyte-enriched, T-lymphocyte-depleted, and monocyte-depleted populations. The T-cell-enriched populations consistently showed enhancement of AHH activity with both the fluorometric and radiometric technique when compared to the total PMC population. This enhanced AHH activity was observed when T-cell-enriched populations were isolated either before or after 96 hr of lymphocyte culture, by the sheep red blood cell rosette method, or by the nylon wool column technique before lymphocyte culture. T-cell-depleted populations (B-cell enriched) obtained by sheep red blood cell rosette method had diminished AHH activity. Monocytes were shown to contribute to the total PMC AHH activity through an indirect technique by first depleting the monocytes from PMC with the carbonyl iron method. The monocyte-depleted populations had less AHH activity than did the total PMC population after both 24 and 96 hr of culture. The greatest amount of AHH activity was present in PMC populations with their native number of monocytes when cultured for 96 hr in the presence of mitogens.