Publications by authors named "John W Grant"

12 Publications

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Early detection of T-cell lymphoma with T follicular helper phenotype by RHOA mutation analysis.

Haematologica 2021 Feb 11. Epub 2021 Feb 11.

Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK; Department of Histopathology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge.

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma with T follicular helper phenotype (PTCL-TFH) are a group of complex clinicopathological entities that originate from TFH cells and share a similar mutation profile. Their diagnosis is often a challenge, particularly at an early stage, due to a lack of specific histological and immunophenotypic features, paucity of neoplastic T cells and prominent polymorphous infiltrate. We investigated whether the lymphoma associated RHOA Gly17Val (c.50G>T) mutation, occurring in 60% of cases, is present in the early 'reactive' lesions, and whether mutation analysis can help advance early lymphoma diagnosis. The RHOA mutation was detected by quantitative PCR with a locked nucleic acid (LNA) probe specific to the mutation, and a further PNA clamp oligonucleotide to suppress the amplification of the wild-type allele. The qPCR assay was highly sensitive and specific, detecting RHOA Gly17Val at an allele frequency of 0.03%, but not other changes in Gly17, nor in 61 controls. Among the 37 cases of AITL and PTCL-TFH investigated, RHOA Gly17Val was detected in 62.2% (23/37) of which 19 had multiple biopsies including preceding biopsies in 10 and follow up biopsies in 11 cases. RHOA Gly17Val was present in each of these preceding or follow up biopsies including 18 specimens that showed no evidence of lymphoma by combined histological, immunophenotypic and clonality analyses. The mutation was seen in biopsies 0-26.5 months (mean=7.87 months) prior to lymphoma diagnosis. Our results show that RHOA Gly17Val mutation analysis is valuable in the early detection of AITL and PTCL-TFH.
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http://dx.doi.org/10.3324/haematol.2020.265991DOI Listing
February 2021

Angioimmunoblastic T-cell lymphoma contains multiple clonal T-cell populations derived from a common TET2 mutant progenitor cell.

J Pathol 2020 03 16;250(3):346-357. Epub 2020 Jan 16.

Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK.

Angioimmunoblastic T-cell lymphoma (AITL) is a neoplastic proliferation of T follicular helper cells with clinical and histological presentations suggesting a role of antigenic drive in its development. Genetically, it is characterized by a stepwise acquisition of somatic mutations, with early mutations involving epigenetic regulators (TET2, DNMT3A) and occurring in haematopoietic stem cells, with subsequent changes involving signaling molecules (RHOA, VAV1, PLCG1, CD28) critical for T-cell biology. To search for evidence of potential oncogenic cooperation between genetic changes and intrinsic T cell receptor (TCR) signaling, we investigated somatic mutations and T-cell receptor β (TRB) rearrangement in 119 AITL, 11 peripheral T-cell lymphomas with T follicular helper phenotype (PTCL-TFH), and 25 PTCL-NOS using Fluidigm polymerase chain reaction (PCR) and Illumina MiSeq sequencing. We confirmed frequent TET2, DNMT3A, and RHOA mutations in AITL (72%, 34%, 61%) and PTCL-TFH (73%, 36%, 45%) and showed multiple TET2 mutations (2 or 3) in 57% of the involved AITL and PTCL-TFH. Clonal TRB rearrangement was seen in 76 cases with multiple functional rearrangements (2-4) in 18 cases (24%). In selected cases, we confirmed bi-clonal T-cell populations and further demonstrated that these independent T-cell populations harboured identical TET2 mutations by using BaseScope in situ hybridization, suggesting their derivation from a common TET2 mutant progenitor cell population. Furthermore, both T-cell populations expressed CD4. Finally, in comparison with tonsillar TFH cells, both AITL and PTCL-TFH showed a significant overrepresentation of several TRB variable family members, particularly TRBV19*01. Our findings suggest the presence of parallel neoplastic evolutions from a common TET2 mutant haematopoietic progenitor pool in AITL and PTCL-TFH, albeit to be confirmed in a large series of cases. The biased TRBV usage in these lymphomas suggests that antigenic stimulation may play an important role in predilection of T cells to clonal expansion and malignant transformation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064999PMC
March 2020

The prognosis of translocation positive diffuse large B-cell lymphoma depends on the second hit.

J Pathol Clin Res 2015 Jul 30;1(3):125-133. Epub 2015 Mar 30.

Division of Molecular HistopathologyDepartment of PathologyUniversity of CambridgeUK; Department of HistopathologyAddenbrooke's Hospital, Cambridge University Hospitals NHS Foundation TrustCambridgeUK.

A proportion of translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a and/or translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated translocation. We investigated gene mutations along with and translocations in a total of 234 cases of DLBCL, including 81 with translocation. mutations were investigated by PCR and sequencing, while and translocation was studied by interphase fluorescence in situ hybridization. The majority of translocation positive DLBCLs (60/81 = 74%) had at least one additional genetic hit. In translocation positive DLBCL treated by R-CHOP ( = 67), mutation and but not translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated translocation, cases with double-hits had the worst overall survival, followed by those with double-hits. In translocation negative DLBCL treated by R-CHOP ( = 101), mutation, and translocation had no impact on patient survival. The prognosis of translocation positive DLBCL critically depends on the second hit, with mutations and translocation contributing to an adverse prognosis. It is pivotal to investigate both mutations and translocations in translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated translocation.
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http://dx.doi.org/10.1002/cjp2.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915334PMC
July 2015

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers.

J Vis Exp 2015 Nov 19(105). Epub 2015 Nov 19.

College of Engineering, University of Georgia;

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.
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http://dx.doi.org/10.3791/53362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692740PMC
November 2015

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

Sci Rep 2015 Sep 8;5:13726. Epub 2015 Sep 8.

College of Engineering, University of Georgia, Athens, Georgia 30602, United States.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.
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http://dx.doi.org/10.1038/srep13726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562232PMC
September 2015

BIOMED-2 PCR assays for IGK gene rearrangements are essential for B-cell clonality analysis in follicular lymphoma.

Br J Haematol 2011 Oct 26;155(1):84-92. Epub 2011 Jul 26.

Department of Histopathology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

B-cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false-negative rate is recognized for germinal centre/post-germinal centre B-cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED-2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED-2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (P < 0·001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0·5%) than IGHV (17/17 cases, 100%, rate 11·0%) (P < 0·01). All IGHV-IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH-BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED-2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08803.xDOI Listing
October 2011

Ultrasound-guided core-needle biopsy of the testis for focal indeterminate intratesticular lesions.

Eur Radiol 2008 Dec 19;18(12):2990-6. Epub 2008 Jul 19.

Department of Radiology, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK.

The aim of this study was to evaluate ultrasound (US)-guided core-needle testicular biopsy. Twelve biopsies were performed in eleven patients, under US guidance using disposable 16- or 18-gauge needles, between April 2003 and October 2006. Details were entered on a database and records reviewed 9 months to 3 years after biopsy. Final diagnoses were based on histology of the biopsy, open surgical resection where performed, and interval follow-up. Biopsies were well tolerated and no complications were encountered apart from a single minor testicular haemorrhage. Benign histology was demonstrated on the core-needle samples of six patients and US follow-up was compatible with these diagnoses. Of five patients whose core-needle biopsies yielded malignancy, two patients had primary testicular tumours; both have been treated successfully with orchiectomy and chemotherapy. Three patients with haematological malignancies had successful chemotherapy without orchiectomy; one of these three underwent post-chemotherapy biopsy demonstrating resolution. There are four main clinical scenarios when core-needle testicular biopsy is performed in our institution: (1) lesions with equivocal malignant US features, (2) discrepancy between radiological and clinical findings, (3) suspected malignant process where orchiectomy is unnecessary, e.g. lymphoma, (4) atrophic testes, where it is frequently difficult to differentiate malignancy from the heterogeneous echo pattern.
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http://dx.doi.org/10.1007/s00330-008-1072-0DOI Listing
December 2008

A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology.

Br J Haematol 2007 Jul;138(1):31-43

Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK.

BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.
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http://dx.doi.org/10.1111/j.1365-2141.2007.06618.xDOI Listing
July 2007

Clinical resolution of a neonatally eroded giant congenital melanocytic nevus.

Pediatr Dermatol 2006 Nov-Dec;23(6):567-70

Depatment of Dermatology, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke's Hospital, Cambridge, UK.

We report an unusual occurrence of spontaneous pigmentary regression with a desmoplastic reaction in a neonatally eroded giant congenital melanocytic nevus. This process has been documented with photographs and skin biopsy specimens. Neonatal histology demonstrated connective tissue proliferation. Histology at age 5 years also demonstrated a very high proportion of amelanotic dermal nevus cells. Regression of pigmentation in our patient may be due to a decrease in melanin production by dermal nevus cells rather than a decrease in their number.
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http://dx.doi.org/10.1111/j.1525-1470.2006.00311.xDOI Listing
October 2007

Anthracycline-based chemotherapy as first-line treatment in adults with malignant posttransplant lymphoproliferative disorder after solid organ transplantation.

Transplantation 2006 Aug;82(3):375-81

Department of Surgery, University of Cambridge, UK.

Background: Recommended first-line treatment for posttransplant lymphoproliferative disorder (PTLD) is reduction in immunosuppressive therapy, irrespective of histopathological type. Second-line treatment with chemotherapy is generally reserved for tumors that fail to respond to reduced immunosuppression. In view of the similarities between monomorphic PTLD and non-Hodgkin's lymphoma in the general population, our policy is to treat monomorphic PTLD with anthracycline-based chemotherapy as first-line treatment.

Methods: A retrospective single-center analysis of 18 adults who developed PTLD following liver or kidney transplantation was undertaken, with particular emphasis on tumor histology, treatment received, and clinical outcome.

Results: Of the 18 patients with PTLD, 13 had high-grade malignant lymphoma on diagnostic biopsy and received anthracycline-based chemotherapy and reduction in immunosuppression as first-line therapy. Nine (69%) of the 13 patients achieved complete remission and eight (62%) remained in complete remission five years after diagnosis. There was no graft loss from rejection or drug toxicity. Four (22%) patients had polymorphic PTLD on diagnostic biopsy (of which two were re-classified as monomorphic) and one had a low-grade malignant lymphoma. All five patients were treated by reduction in immunosuppression without chemotherapy and were in complete remission at a median of two years after diagnosis. Overall, complete remission was seen in 14 out of 18 patients (78%) at one year following diagnosis.

Conclusion: The use of anthracycline-based chemotherapy and reduction of immunosuppression as first-line treatment in adults with monomorphic PTLD is well tolerated and achieves sustained complete remission in around 70% of patients with a low risk of graft loss.
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http://dx.doi.org/10.1097/01.tp.0000228906.31675.fbDOI Listing
August 2006

US-guided core-needle biopsy of the thyroid gland.

Radiology 2003 Mar 31;226(3):827-32. Epub 2003 Jan 31.

Department of Radiology, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 2QQ, England.

Purpose: To evaluate safety, yield, and accuracy of ultrasonography (US)-guided core-needle biopsy of the thyroid gland.

Materials And Methods: Findings at 209 consecutive core-needle biopsies of lesions of the thyroid gland in 198 patients (median age, 48 years; age range, 14-80 years) were retrospectively reviewed. In 138 (66%), findings at previous fine-needle aspiration cytologic (FNAC) analysis were nondiagnostic on one to five occasions. Biopsy was performed as an outpatient procedure with direct US guidance by using nonadvancing 16-18-gauge core needles. Hospital records were reviewed 6 months to 5 years following biopsy to determine final diagnosis, delayed complications, and influence of biopsy findings on subsequent patient treatment. Final diagnoses were determined on the basis of findings at excisional histologic analysis, clinical course, or other laboratory values. The sensitivity, specificity, and accuracy of US-guided core-needle biopsy were calculated.

Results: One hundred ninety-nine (95%) specimens were adequate for histologic diagnosis. The sensitivity, specificity, and accuracy of core biopsy in differentiating neoplastic (malignant and benign) from nonneoplastic lesions of the thyroid gland were 96% (74 of 77), 89% (109 of 122), and 92% (183 of 199), respectively. The sensitivity, specificity, and accuracy of core-needle biopsy in the detection of malignant neoplasms were 61% (11 of 18), 100% (181 of 181), and 96% (192 of 199), respectively. After US-guided core-needle biopsy, 115 (58%) of 198 patients were treated conservatively, and no evidence of missed tumor manifested during the follow-up period. In the 83 patients who underwent surgical resection, biopsy was performed for therapeutic reasons in 76 (92%) and for diagnostic reasons in seven (8%). There were three cases of small postbiopsy hematomas and one of minor hemoptysis, but none required hospital admission. There were no major complications.

Conclusion: US-guided core-needle biopsy of the thyroid gland is a safe outpatient procedure with a high diagnostic yield and accuracy, and frequently it obviates surgery in patients in whom findings at FNAC analysis are recurrently nondiagnostic.
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http://dx.doi.org/10.1148/radiol.2263012073DOI Listing
March 2003

Head and neck lymphadenopathy: evaluation with US-guided cutting-needle biopsy.

Radiology 2002 Jul;224(1):75-81

Department of Radiology, Addenbrooke's Hospital, Hills Rd, Cambridge CB2 2QQ, England.

Purpose: To evaluate ultrasonography (US)-guided core biopsy in the assessment of 247 patients with cervicofacial lymphadenopathy.

Materials And Methods: Two hundred sixty US-guided core biopsies were performed in 247 patients with cervicofacial lymphadenopathy. The age of the patients ranged from 1 to 91 years (mean, 50 years). Seventy-four (30%) had a history of malignancy. Biopsies were performed as outpatient procedures with direct US guidance and non-advancing 16-18-gauge core needles. Hospital records were reviewed 6 months to 5 years after biopsy. Final diagnoses were rendered based on results of histologic examination of excised specimens, clinical course, or results of other laboratory studies.

Results: Two hundred thirty-eight (92%) core biopsies yielded adequate material. In 28 (11%) patients, the histologic diagnosis was considered highly probable. In the 210 patients in whom adequate material was obtained and an unequivocal histologic diagnosis was given, the sensitivity, specificity, and accuracy of US-guided core needle biopsy in differentiating benign from malignant lymphadenopathy were 98.1%, 100%, and 98.7%, respectively. Seventy biopsies were performed in 66 patients with lymphoma. Sensitivity, specificity, and accuracy in differentiating lymphoma from reactive lymphadenopathy were 98.5%, 100%, and 98.7%, respectively. In 53 patients (80%) with lymphoma as a final diagnosis, histologic subclassification was sufficient to guide treatment without the need for surgical biopsy. There were no major complications and only three minor post-biopsy hematomas.

Conclusion: US-guided core biopsy in patients with head and neck lymphadenopathy is a safe outpatient procedure that has a high diagnostic yield and accuracy and frequently obviates surgery.
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http://dx.doi.org/10.1148/radiol.2241010602DOI Listing
July 2002