Morphological Bone Marrow Involvement Research Articles

Bio-P-01 - Prognostic value of the clonal rearrangement of T-cell receptor genes detection in the bone marrow in patients with mycosis fungoides

<h3>Introduction:</h3> Mycosis fungoides constitutes more than 60% of primary cutaneous T-cell lymphomas. The course of disease is progradient: the gradual changes of macular, plaque and tumor stages are noted. The evaluation of the spreading process is critically important for the prognosis determination and therapy choosing. T-cell clonality detection by rearranging genes of the gamma chain of the T-cell receptor in the blood and bone marrow aspirates can be detected at the early stages. It is known to be a prognostically unfavorable factor for diagnostics in peripheral blood, while the value of the rearranging presence of T-cell receptor gene in the bone marrow aspirates has not been fully studied. <h3>Aim:</h3> To evaluate the frequency and prognostic value of the T-cell clonality detection by the gamma chain of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome (MF/SS). <h3>Materials and methods:</h3> The patients with mycosis fungoides/Sézary syndrome were observed at the National Research Centre of Hematology during the period from 2000 to 2015. <h3>Results:</h3> 45 patients diagnosed with mycosis fungoides/Sézary syndrome (MF/SS) corresponded to inclusion criteria. Men/women 1:1. The age of the participants is varied from 20 to 81 years. The diagnosis of mycosis fungoides was made in 36 cases (80%) and the diagnosis of Sézary syndrome was made in 9 cases (20%) respectively. The early stages were diagnosed in 13 patients (29%), the later stages were diagnosed in 32 patients. T-cell clonality by the gamma chain rearrangement of the T-cell receptor was detected in the bone marrow in 15 out of 45 patients (33%). 13 cases were the patients with the late disease stages and clonality at the early Ib stage was detected only in 2 cases. The morphological bone marrow involvement was identified in 5 patients, but in two cases T-cell clonality was not detected. The median survival of patients with morphologically confirmed bone marrow damage was 3 months. The median survival of patients with defined T-cell clonality in the bone marrow was 21 months. The median survival with no molecular damages of bone marrow was 41 months. <h3>Conclusions:</h3> T-cell clonality by gamma chain rearranging of the T-cell receptor in the bone marrow in patients with mycosis fungoides/Sézary syndrome is determined in a third of patients mainly at the late MF stages. This is an unfavorable prognostic factor.

PB1815 CLINICAL IMPLICATIONS OF T CELL RECEPTOR (TCR) GENE REARRANGEMENT, 18F‐FDG‐PET/CT AND UNILATERAL BONE MARROW TREPHINATION FOR DETECTING BONE MARROW INVOLVEMENT IN PERIPHERAL T CELL LYMPHOMAS

Background:Bone marrow involvement (BMI) is an important prognostic factor for the treatment of peripheral T‐cell lymphomas (PTCLs). The trephining of bone from the iliac crest is the most generally accepted diagnostic approach to evaluate BMI to date. However, iliac bone trephination has some limitations such as low sensitivity owing to patch or focal lymphomatous involvement, problems of procedure technique and diversity according to the inspectors.Aims:So, we investigated that whether the combined evaluation with 18F‐FDG PET/CT and molecular analysis of T‐cell receptor (TCR) gene rearrangement could increase the prognostic information of BMI at the time of diagnosis in PTCLs.Methods:This study conducted prospectively in 46 patients with newly diagnosed PTCL from April 2017 to January 2019. Three patients were excluded due to the lack of baseline 18F‐FDG PET/CT and 4 patients were excluded due to the diagnostic pathology of anaplastic lymphoma kinase (ALK)‐positive anaplastic large cell lymphoma and indolent cutaneous T‐cell lymphomas. Finally, 39 patients were investigated based on three parameters at diagnosis. The specimens for TCR gene rearrangement polymerase chain reaction (PCR) to detect the clonality were obtained by fine‐needle aspiration and unilateral iliac trephination biopsy.Results:Fifteen (37.5%) patients were diagnosed with NK/T cell lymphoma, 11 (27.5%) with angioimmunoblastic T cell lymphoma (AITL), 7 (17.5%) with peripheral T cell lymphoma, not otherwise specified (PTCL‐NOS), 3 (7.5%) with ALK negative anaplastic large cell lymphoma and 3 (7.5%) with enteropathy‐associated T cell lymphoma. Twenty‐three (59.0%) patients showed advanced stage and 9 (22.5%) patients were confirmed morphologic bone marrow involvement (mBMI) by trephination biopsy. TCR gene rearrangement (TCR BMI) was detected in 29 (72.5%) patients and bony involvement on 18F‐FDG PET/CT assessment (PET BMI) was detected in 8 (20.0%) patients. Seven out of 9 patients with mBMI were concordant with TCR BMI, however, only two patients with mBMI showed the positivity in FDG PET/CT.Overall diagnostic accuracy of TCR BMI for detection of conventional mBMI was calculated with sensitivity of 77.8%, specificity of 26.7%, positive predictive value (PPV) of 24.1% and negative predictive value (NPV) of 80.0%, respectively. Five patients were detected to have the positivity in both TCR rearrangement and FDG PET/CT but no bone marrow involvement in morphologic trephination. The median progression‐free survival (PFS) of these patients was as poor as those with mBMI (6.93 months vs. 13.33 months, P = 0.250).Summary/Conclusion:Althouth both FDG PET/CT and TCR gene rearrangement PCR have low relevance to conventional mBMI, the combined evaluation with FDG PET/CT and TCR gene rearrangement could be useful to predict bone marrow microinvolvement and poor prognosis.

The Clinical Impact of Minimal Bone Marrow Involvement on the Outcome of Patients with Follicular Lymphoma

IntroductionFollicular lymphoma (FL) is the second most common type of non-Hodgkin cell lymphoma, and usually manifests as a disseminated disease. Bone marrow (BM) involvement, which occurs in 40-70% of cases, is often seen in follicular lymphoma and thought to be associated with less favorable prognosis. Diagnosis of BM involvement has traditionally been based on morphological findings, and BM involvement has been determined using histology alone in most clinical trials. Immunocytologic or molecular studies, such as flow cytometry (FCM) and polymerase chain reaction (PCR), have become more readily available, and their usage has clearly documented minimal BM involvement reproducibly. In this study, we evaluated the impact of BM involvement detected by FCM and PCR on the outcome of patients treated for FL. MethodsPatients who were diagnosed with biopsy-proven FL between 2004 and 2015 at our institution were included in the study. All patients had received a staging bone marrow examination before treatment with immunotherapy-based regimen. Immunocytologic [FCM] and/or molecular [PCR] studies were always performed if the patients did not have morphological BM involvement. We used 4- or 6- color FCM, and performed PCR analysis of Bcl-2/IgH rearrangement and/or IgH rearrangement detected by modified BioMed-2 protocol. A total of 90 patients were included, and the median follow-up duration was 36 months (range, 6|122 months). The BM status was classified using into 3 categories: morphological, minimal, and negative BM involvement. Minimal BM involvement was defined as BM involvement detected by FCM or PCR without morphological evidence. Morphological and minimal BM involvements were detected in 37 (41%) and 38 (42%) patients, respectively. The primary outcome measure was progression-free survival (PFS). PFS curves were plotted using the Kaplan-Meier method and compared by the log-rank test. Multivariate analyses were performed using a Cox linear regression model. There were significant differences in gender, LDH levels, stage, nodal sites, and FL International Prognostic Index (FLIPI) between patients with and without morphological BM involvement (Table1). ResultsThe 3-year PFS rate for patients with negative BM involvement was significantly better than that for patients with minimal or morphological BM involvement (84.8% vs. 40.3% vs. 60.5%; p= 0.043) (Figure 1). There was no statistical difference in 3-year PFS between patients with morphological BM involvement and those with minimal BM involvement. The difference of 3-year PFS rate between patients with minimal BM involvement and those with negative BM involvement was significant for patients with FLIPI low-intermediate risk (88.9% vs. 51.5%; p= 0.032) and those with advanced stage disease (90.0% vs. 33.6%; p= 0.027), but there were no significant differences in patients deemed FLIPI high risk and those with limited stage disease. Multivariate analysis revealed that BM involvement, including morphological and minimal involvement, was a significant poor prognostic factor (hazard ratio 4.885 [95% confidence interval 1.16-20.56], p = 0.0305). ConclusionAt the start of treatment, bone marrow involvement was seen in most FL patients. Patients without any BM involvement had an excellent prognosis. Patients with minimal BM involvement had an equally poor prognosis as those with morphologic BM involvement. [Display omitted] [Display omitted] [Display omitted] DisclosuresIshikawa:Mundipharma KK: Research Funding.

Minimal Disseminated Disease (MDD) in BM of Childhood T-Cell Lymphoblastic Lymphoma (Stage III), Detected by Flow Cytometry (FC) and Real Time Quantitative Polymerase Chain Reaction (RQ-PCR).

Despite overlapping features between T cell Lymphoma and T cell ALL and generally good response to T-ALL type of treatment there are some clinical and biological differences between the two entities. The aim of this study was to evaluate in children with stage III T cell Lymphoma the prevalence of minimal BM dissemination at diagnosis, its prognostic significance and early response kinetics. Bone Marrow (BM) at diagnosis from 14 children with T- cell lymphoblastic lymphoma (T-LBL) was assessed for minimal disseminated disease (MDD) by flow cytometry (FC) with four colour antibody combinations and real time quantitative PCR (RQ-PCR) with specific junctional regions for T-cell receptor and immunoglobulin heavy chain gene rearrangements. 13 pts. had stage III mediastinal lymphoma, and one stage II cervical disease. Morphological BM involvement of almost 5% blasts was suspected in only one patient. All, but one, were treated according to the NHL-BFM 90/95 protocols medium risk group (without irradiation). In addition, 7 pts were assessed for early response on week 5 and 12 of treatment with PCR only (2 pts) FC only (2 pts) and both methods (3 pts). Lymphoma associated immunophenotype with a sensitivity of at least 0.01% was detected in all patients (100%) (2 combinations), and PCR targets with a sensitivity of 0.01% and 0.1% in 10 and 2 examined patients respectively (92%). A sample with a level of above 5X10−4 was considered as positive MDD or minimal residual disease (MRD) by both methods. By FC, 10 patients exhibited positive and 4 negative BM MDD levels at diagnosis. By RQ -PCR, 7 patients presented positive and 4 negative BM MDD levels. Both methods gave consistent results with only one case of discordance: negative in PCR and positive in FC (>5x10−4). In 7 other pts there was a difference of between 0.5 and 1 log level that did not change the classification of involvement (in 5 pts PCR higher, in 2 pts FC higher). At a median follow up of 5 years, 3 patients relapsed (2 in the mediastinum and one in BM) their MDD level at diagnosis was 2x10−3 (2 pts), 6x10−2. MRD level in 4 patients out of 5 patients with a high MDD level (1–6x10−2) at diagnosis, decreased by 2 logs (<5x10−4) on week 5, and on week 12 all were below <2x10−4. The one patient with a higher level (1.5x10−2) at day 33 suffered a relapse.In conclusion: in the majority of patients with mediastinal stage III T-cell lymphoma molecular BM dissemination (>5x10−4) was present. It's prognostic significance in the context of ALL-BFM therapy could not be demonstrated. However, early BM molecular treatment response in T-cell Lymphoma may correlate with outcome, similarly to T-cell leukemia.

Optimum trephine length in the assessment of bone marrow involvement in patients with diffuse large cell lymphoma

The National Cancer Institute has recommended a bone marrow biopsy length of >/=20 mm for the staging and surveillance of patients with non-Hodgkin's lymphoma. However, there are few published data to support this recommendation, particularly the role of examining multiple levels. Bone marrow biopsies from 172 patients with newly diagnosed diffuse large cell lymphoma (DLCL) entered in two consecutive trials of the Australasian Leukaemia and Lymphoma Group were analysed. The original haematoxylin and eosin-stained trephine biopsy and two or more deeper sections cut at 0.1-0.2 mm intervals were assessed with respect to the morphology, extent and pattern of lymphomatous involvement. The rate of positive diagnosis was correlated with the length of the biopsy specimen and the number of sections examined. Forty-seven biopsies (27%) demonstrated marrow involvement on examination of a mean of four trephine biopsy sections. The rate of positivity increased with the examination of multiple levels and correlated with increasing trephine length but was not dependent on the number of sites sampled. Twenty per cent of biopsies <20 mm in length were positive for lymphoma; this increased to 35% for biopsies >/=20 mm (P = 0.023). Morphological bone marrow involvement in DLCL is optimally demonstrated by a 20-mm long trephine biopsy from a single site which is examined at multiple levels (four or more). This obviates the need for bilateral sampling, thereby reducing patient morbidity from the procedure. This study provides evidence to support the National Cancer Institute recommendations regarding trephine biopsy in the staging of DLCL, providing multiple levels are examined.

Detection of Clonai CD34+19+ Progenitors in Bone Marrow of BCL2-IgH–Positive Follicular Lymphoma Patients

AbstractThe frequent occurrence of BCL2-lgH rearrangements in follicular lymphoma (FL) makes detection of low numbers of tumor cells possible by polymerase chain reaction (PCR). The presence of BCL2-lgH in the bone marrow (BM) and peripheral blood of many FL patients at the time of autografting has led to the suggestion that selection of the CD34-enriched fraction may lead to reinfusion of lower numbers of tumor cells. To address this issue, we PCR-amplified BCL2-IgH from fluorescence-activated cell sorting (FACS)-purified BM CD34+ and CD34– fractions in seven FL patients showing a PCR-detectable translocation in the major breakpoint region of BCL2, five of which showed morphological BM involvement. The total CD34+ fraction showed diminished but residual positivity in the first two cases tested. Therefore, BM cells from the remaining five patients were sorted for the CD34+19– immature population, the CD34+19+ B-cell precursors, and the CD34–19+ mature B-cell fraction. The CD34+19– subpopulation was negative in four of five, despite evident BM infiltration in three cases. In contrast, the CD34+19+ fraction was positive in all three cases tested. These cells represented 0% to 50% (mean, 18%) of the total CD34+ population, suggesting that, if reinfusion of BCL2-IgH–positive cells plays a role in postautograft relapse in FL, therapeutic C034 selection procedures should include additional purging of the CD34+19+ B-cell precursors or, at least, assessment of the proportion of CD19+ cells in the CD34+ fraction and its correlation with clinical outcome postreinfusion.

Incidence and prognostic significance of t(14; 18) translocation in follicle center cell lymphoma of low and high grade: A report from southern Sweden

The t(14;18)(q21;q32) is the most common recurrent genetic defect in follicle center cell lymphoma (FCC). Conflicting reports exist in regard to a possible prognostic significance for the translocation. In a single center, 102 patients with either low-grade (n = 50) or high-grade (n = 52) FCC (Kiel classification) and a median follow-up of 82 months were retrospectively studied to determine survival in relation to t(14;18) as shown by either PCR of the bcl-2 rearrangement in paraffinized tissue or karyotype analysis. t(14;18) was detected in 30 of 50 (60%) low-grade FCC and in 12 of 52 (23%) high-grade FCC. The presence of the t(14;18) was not related to morphologic bone marrow involvement or other clinical parameters, but it was related to age: in low-grade FCC, patients with t(14;18) were an average of 17 years younger (p = 0.002) than those without the translocation. In the group with high-grade histology, 30% survived beyond 60 months regardless of t(14;18) status (p = 0.92). Patients with low-grade histology and t(14;18) fared better than those without, irrespective of age (p = 0.01). No significant difference in disease-free survival related to t(14;18) was found in either low- or high-grade FCC. The incidence of t(14;18) is in accord with that of other European reports. T(14;18) does not define a prognostic subset of high-grade FCC, but is significantly correlated with a better survival in low-grade FCC. The association of t(14;18) with younger age and indolent lymphoma is perplexing in light of recent findings of an age-related increase in t(14;18) in normal subjects.

Study of bone marrow cells in non-Hodgkin's lymphoma by DNA analysis

Using Southern-blot analysis, we studied samples of bone marrow (BM) cells from 73 patients with non-Hodgkin's lymphoma (NHL) in various clinical status. The frequency of gene rearrangement was disease-status dependent with a frequency of 65.8% at the diagnostic stage, 81.8% after relapse and 33.3% upon complete remission (CR). BM involvement was evident in a substantial portion of patients with untreated and relapsed lymphoma. The significance of BM involvement by DNA hybridization in relation to conventional clinical staging and histological grade was studied. By Southern-blot analysis, BM involvement was found in 76% of the patients at clinical stages (CS) I–III. The incidence of BM involvement in low, intermediate and high grades of NHL (Working Formulation) was 57%( 4 7 ) , 67%( 22 33 ) , and 89%( 8 9 ) respectively. A comparative study of conventional BM biopsy vs DNA hybridization in a group of 47 NHL patients showed that all 12 patients (100%) with morphological BM involvement and 25 out of 35 patients (71%) with morphologically normal BM had clonal rearrangements of immunoglobulin (Ig), heavy chain and/or light chain, or T-cell receptor β chain (TCRβ) genes in BM cells. The false negative rate in conventional BM biopsy was 53%( 25 47 ) . Southern-blot analysis on lymph nodes (LN) and BM cells from 37 patients showed that 6 patients (16%) had cross-lineage or different rearranged patterns in the same or different tissues. Southern-blot analysis was found to be highly reliable for the detection of even minimal populations of lymphoma cells in the BM and therefore should be the diagnostic choice for clinical staging of lymphoma.

Applications and limitations of peripheral blood lymphocyte immunoglobulin light chain analysis in the evaluation of non-Hodgkin's lymphoma.

Surface immunoglobulin (sIg) light chain analysis of peripheral blood lymphocytes (PBL) from 63 patients with non-Hodgkin's lymphoma (NHL) was performed to determine the ratio of kappa-bearing lymphocytes to lambda-bearing lymphocytes (kappa/lambda ratio). In 43% an abnormal kappa/lambda ratio was detected, implying the presence of a malignant clone in the peripheral blood (clonal excess). In 67% of cases with an abnormal kappa/lambda ratio, the absolute lymphocyte count was within normal limits and 20% did not have morphologic bone marrow involvement. Light chain analysis of bone marrow aspirates was performed in three of four patients with a circulating clone and normal bone marrow morphology. All three patients had abnormal bone marrow aspirate kappa/lambda ratios. The presence of a circulating clone was associated with morphologic bone marrow involvement (P less than 0.05). Nine patients with documented B-cell NHL were followed with repeated examinations. The presence of a circulating clone persisted in two patients refractory to therapy and in two patients otherwise believed to be in remission. Conversion to an abnormal kappa/lambda ratio occurred in two patients coincident with the development of new bone marrow involvement and in a third patient prior to the onset of frank leukemia. In three instances, the PBL light chain analyses remained within normal limits in patients with stable lymphadenopathy. The kappa/lambda ratio returned to normal in one patient responding to treatment with a partial remission, and remained within normal limits despite progression of lymphadenopathy in one patient, and local disease recurrence in another patient. These findings suggest that the detection of a circulating clone by sIg light chain analysis may be useful as a noninvasive approach to identify disseminated disease activity unsuspected on the basis of morphologic evaluations, but may not reflect the presence, progression, or recurrence of localized tissue lesions.