Negative Molecular Results Research Articles

Circulating H3K27 Methylated Nucleosome Plasma Concentration: Synergistic Information with Circulating Tumor DNA Molecular Profiling.

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment.

Abstract 1013: Circulating H3K27 methylated nucleosome concentration in lung cancer improves the contributive value of ctDNA molecular profiling result at diagnosis

Abstract Background: In patients with advanced lung adenocarcinoma cancers, a subset can be cured by radiotherapy, and/or combined regimens, including chemotherapy, immunotherapy, or targeted therapies based on somatic molecular profiling. Nucleosomes are basic elements of chromatin, composed of 147bp DNA wrapped around an octamer of histones. Cell-free DNA (cfDNA) and nucleosomes are released into the bloodstream upon cell death. In addition to genetic somatic alterations, epigenetic modifications are found to play a key role in tumorigenesis of different cancers. The trimethylated histone H3 at lysine 27 (H3K27Me3) is well known as a transcription-repressive mark and proven to be involved in tumorigenesis, cell cycle progression and proliferation dysregulation. However, the concentration of circulating nucleosomes, as a biomarker of the contributive value of circulating tumor DNA (ctDNA) molecular profiling in patient management at diagnosis has not previously been investigated. Patients and Methods: Plasmas were retrospectively collected from patients with NSCLC at initial diagnosis before treatment (CIRCAN’s cohort, N= 198) and from healthy donors (N=100). We carried out targeted next-generation sequencing (NGS) on paired plasmas. Samples were divided in two sub-groups based on the genetic results: ctDNA negative or positive for presence of somatic alterations. The concentration of global circulating nucleosomes as well as specific methylated marks: H3K4Me2-, H3K9Me3-, H3K27Me3- or H3K36Me3-nucleosomes were measured using chemiluminescent Nu.Q® immunoassays (Belgian Volition SRL, Belgium). Results: Among all the different nucleosome forms evaluated, the highest coefficient correlations with cfDNA concentration were observed for H3K27Me3-nucleosomes (r=0.72, p&amp;lt;0.0001). At a cut-off of 14 ng/ml of H3K27Me3-nucleosomes Nu.Q® assay showed a sensitivity of 69.2% (n=137) at 95% specificity for lung cancers vs healthy donors (AUC= 0.92). Interestingly, by combining H3K37Me3 level and ctDNA molecular profile, we found a contributive diagnostic value of molecular profiling results in 77% of the plasmas versus only 40% using NGS alone as per current practice. 32% of plasmas were positive for both H3K27Me3 and ctDNA; 37% are H3K27Me3-positive but ctDNA-negative; 8% of the samples were ctDNA-positive/H3K27Me3-negative and 23% are negative for both H3K27Me3 and ctDNA. Conclusions: High levels of Nu.Q® H3K27Me3 and the absence of detecting somatic alterations strongly support the presence of non-mutated ctDNA in the corresponding plasma. This greatly improves the confidence in the negative molecular results in cfDNA in LC. This may allow the indication of individualized therapeutic regimens with a short-time medical decision and may reduce invasive tissue re-biopsies. Citation Format: Arnaud Gauthier, Julie Candiracci, Emmanuel Grolleau, Gaelle Lescuyer, David Barthelemy, Christine Haon, Florence Geiduer, Margaux Raffin, Nathalie Hardat, Julie Balandier, Rémi Rabeuf, Anne-Sophie Wozny, Claire Rodriguez-Lafrasse, Guillaume Rommelaere, Fabien Subtil, Sébastien Couraud, Marielle Herzog, Lea Payen-Gay. Circulating H3K27 methylated nucleosome concentration in lung cancer improves the contributive value of ctDNA molecular profiling result at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1013.

Clinical Characteristics Associated with Detected Respiratory Microorganism Employing Multiplex Nested PCR in Patients with Presumptive COVID-19 but Negative Molecular Results in Lima, Peru.

The COVID-19 pandemic circumstances have varied the pathogens related to acute respiratory infections (ARI), and most specialists have ignored them due to SARS-CoV-2's similar symptomatology. We identify respiratory pathogens with multiplex PCR in samples with presumptive SARS-CoV-2 but negative RT-qPCR results. We performed a retrospective transversal study employing clinical data and nasopharyngeal swab samples from patients with suspected clinical SARS-CoV-2 infection and a negative PCR result in a private laboratory in Lima, Peru. The samples were analyzed using the FilmArray™ respiratory panel. Of 342 samples, we detected at least one pathogen in 50% of the samples. The main ones were rhinovirus (54.38%), influenza A(H3N2) (22.80%), and respiratory syncytial virus (RSV) (14.04%). The clinical characteristics were sore throat (70.18%), cough (58.48%), nasal congestion (56.43%), and fever (40.06%). Only 41.46% and 48.78% of patients with influenza met the definition of influenza-like illness (ILI) by the World Health Organization (WHO) (characterized by cough and fever) and the Centers for Disease Control and Prevention (CDC) (characterized by fever and cough and sore throat), respectively. A higher prevalence of influenza was associated with ILI by WHO (aPR: 2.331) and ILI by CDC (aPR: 1.892), which was not observed with other respiratory viruses. The clinical characteristic associated with the increased prevalence of rhinovirus was nasal congestion (aPR: 1.84). For patients with ARI and negative PCR results, the leading respiratory pathogens detected were rhinovirus, influenza, and RSV. Less than half of patients with influenza presented ILI, although its presence was specific to the disease.

Neuropsychiatric Functioning in CDLS: A Detailed Phenotype and Genotype Correlation.

Behavioural phenotype and autism-related traits of 38 patients affected by Cornelia de Lange syndrome (CdLS) were assessed using a specific neuropsychiatric protocol. Subsequently,we search for possible genotype-phenotype correlations comparing individuals with NIPBL variants and patients with negative molecular results. Firstly results showed a higher percentage of subjects with normal intellectual quotient (IQ) and borderline IQ; adaptive skills were lower than expected for age in all participants. 39.5% of the sample presented with autism spectrum disorder (ASD), NIPBL mutated individuals demonstrated a worse trend in comparison with the clinical diagnosis group. non-truncating individuals displayed no ASD and better communication abilities than truncating individuals. Findings increase our awareness of the strengths and weaknesses points in CdLS individuals.

Pulmonary tumor with Notochordal differentiation: a case report and morphologic, Immunohistochemical and molecular study of benign Notochordal cell tumor originating in the lung

BackgroundExtraosseous benign notochordal cell tumor is extremely rare, and there are only five reported cases worldwide. The presented case of pulmonary primary benign notochordal cell tumor is the sixth case, but the first to report the deletion mutation of EGFR gene exon 19.Case presentationThe patient was a 50-year-old asymptomatic woman, who had been followed up for 3 years for a nodule in the right lung. After ten months of the wedge resection, the patient is alive without evidence of recurrence or metastasis. The tumor was 7 mm in diameter and was well demarcated. The tumor was consisted of a sheet of large round vacuolated cells with small and bland nuclei. No connective tissue containing blood vessels or inflammatory cell infiltration was detected in the stroma. The tumor was positive for CK AE1/AE3, Vimentin, S100 and Brachyury. EGFR gene mutation and amplification were not detected.ConclusionsWe firstly reported the positive immunohistochemical staining for EGFR and the negative molecular results of EGFR gene of pulmonary primary benign notochordal cell tumor. Due to the rarity of this tumor, more reports are needed to explore pathological characteristics, especially the molecular characteristics, in order to better understand the nature of tumors.

Microbiological, serological and molecular diagnosis of abscess of withers in equine in the State of Pará

There are several infectious diseases that affect equines and can cause large economic losses in this sector and damage to animal welfare. In this species, abscessing lesions of a purulent character preferentially in the withers are strongly suggestive of brucellosis. However, few studies have been carried out on the disease in this species, making its identification and control difficult due to the lack of standardization in the interpretation of results. Therefore, samples of whole blood, serum and purulent secretion were collected from three horses. The animals came from the state of Pará. The diagnostic methods selected were the Rose-Bengal plate test (RBPT) and Polymerase Chain Reaction (PCR), for the negative molecular results for brucellosis, microbiological analysis were chosen. Although part of the samples were positive for brucellosis in serological tests, all of them showed negative results for PCR. In bacterial cultivation, species were isolated Streptococcus equi subsp. zooepidemicus, Enterococcus faecalis e Staphylococcus sciuri subsp. lentus., which showed resistance to antibacterials, especially the E. faecalis isolate classified as MDR (multi drug resistant). In conclusion, the best form of diagnosis is the joint evaluation of several tests, both screening and confirmatory, in order to obtain a definitive diagnosis with assertiveness.

Benign call rate and molecular test result distribution of ThyroSeq v3.

The benign call rate (BCR) is the percentage of cytomorphologically indeterminate cases with subsequent benign or negative molecular results. For rule-out tests, the BCR is an important parameter because these molecular "negative" cases may be managed similarly to those with a benign cytology diagnosis. Although earlier versions of ThyroSeq molecular tests were less effective in excluding malignancy, the extensively expanded v3 version with a high negative predictive value is considered to represent a rule-out test. In the current study, the authors evaluated the BCR and the overall molecular result distribution of ThyroSeq v3. All indeterminate thyroid fine-needle aspiration cases (except those deemed suspicious for malignancy) with available ThyroSeq v3 results from November 2017 to June 2018 were retrieved from the cytopathology files. Because the ThyroSeq v3 genomic classifier was designed for thyroid follicular-derived lesions, cases in which parathyroid and medullary carcinoma molecular markers were detected and those that were inadequate or limited for molecular testing were excluded from the study. For the remaining cases, the indeterminate diagnoses were correlated with molecular and histologic results. Among the 224 indeterminate cases (except those deemed suspicious for malignancy), the overall ThyroSeq v3 molecular test BCR was 74.1% (166 of 224 cases). The category of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) had higher BCRs compared with the other indeterminate diagnostic categories. RAS mutations were the most common positive results. The high BCR demonstrates that ThyroSeq v3 is potentially effective in sparing the large majority of indeterminate cases with "negative" results from surgery while offering risk assessment for the positive cases and guiding clinical management.

Molecular strategy for the diagnosis of infectious lymphadenitis.

Molecular methods have been considered to be the gold standard for the diagnosis of infectious lymphadenitis. However, culture remains critical in the case of low bacterial concentrations. We used molecular assays and culture to examine fresh lymph node biopsies from patients with suspected infectious lymphadenopathy. We analyzed 1762 lymph node biopsies of which 522 (30%) samples were found positive by real-time PCR; the most commonly amplified bacteria were Bartonella henselae (n = 438, 84%), Francisella tularensis (n = 46, 9%), and Mycobacterium spp. (n = 29, 6%). PCR amplification and sequencing of the 16S rDNA were positive for 359 (20%) lymph node specimens including mainly B. henselae (n = 167, 47%), Staphylococcus spp. (n = 77, 21%), and Streptococcus spp. (n = 41, 11%). In total, 351 lymph nodes were cultured on agar plates and 77 (22%) were positive. Significantly more lymph nodes infected by Gram-positive easy-growing agents were diagnosed by culture (n = 45) than by 16S rDNA PCR (p =0.02). Culture remains critical for the diagnosis of easy-growing bacteria and mycobacteria; clinicians should be aware that a negative molecular result does not imply absence of infection.

Intersectoral action for health: preventing psittacosis spread after one reported case.

Zoonotic diseases are a significant health threat for humans and animals. To better understand the epidemiology, etiology, and pathology of infectious agents affecting humans and animals combined approaches are needed. Here we describe an epidemiological investigation conducted by physicians and veterinarians after a reported case of psittacosis. Upon admission suffering from respiratory distress syndrome in a hospital and with a history of bird contact, a female patient was serologically diagnosed with psittacosis. After the case notification, veterinarians were able to investigate the source of infection by detecting Chlamydia psittaci in her pet cockatiel. The bird was hospitalized and successfully treated. In addition, the establishment where the pet bird was purchased was traced and through molecular techniques other birds intended to be sold as pets tested positive for C. psittaci. As a result, sanitary measures were applied and the establishment then was closed down. The birds intended for the pet commerce were treated and retested with negative molecular results for C. psittaci, thus avoiding disease propagation. Reliable data about zoonotic diseases can only be generated through the application of multidisciplinary approaches which take into account the epidemiological factors and interactions of humans, animals and their environments as an integrated system.

Molecular Identification of Q Fever in Patients with a Suspected Diagnosis of Dengue in Brazil in 2013–2014

Q fever is an important cause of undifferentiated fever that is rarely recognized or reported in Brazil. The objective of this study was to look for the presence of Coxiella burnetii during a dengue fever outbreak in the municipality of Itaboraí, Rio de Janeiro, Brazil, where this bacterium had previously infected humans and domesticated animals. Blood samples from clinically suspected dengue fever patients were tested by polymerase chain reaction (PCR) for C. burnetii; the DNA was detected in nine (3.3%) of 272 patients. One was coinfected with dengue virus, which was also detected in another 166 (61.3%) patients. The nucleotide sequence of PCR amplification and DNA sequencing of the IS1111 transposase elements in the genome of C. burnetii exhibited 99% identity with the sequence in GenBank. The detection of C. burnetii in patients suspected of dengue fever indicates that awareness and knowledge of Q fever should be strengthened and that this bacterium is present in Brazil. Finally, because a negative molecular result does not completely rule out the diagnosis of Q fever and the serological assay based on seroconversion was not available, the actual number of this zoonosis is likely to be much higher than that reported in this study.

EGFR mutation specific immunohistochemistry is a useful adjunct which helps to identify false negative mutation testing in lung cancer

Mutations in EGFR guide treatment in non-small cell lung cancer (NSCLC). The most common mutations, exon 19 (delE746-A750) and exon 21 (L858R), can be identified by mutation specific immunohistochemistry (IHC). We present our prospective experience of universal reflex IHC and molecular testing in non-squamous NSCLC in the routine clinical setting.A total of 411 specimens from 332 patients were encountered over two years. Of these, 326 (98%) patients underwent EGFR IHC, 15 (5%) were positive for exon 19 deletions and 27 (8%) for exon 21 (L858R); 244 (73%) patients underwent molecular testing. Seventy-six mutations in 64 patients (19% of all patients encountered; 26% with sufficient material for testing) were identified. These comprised nine exon 18 (G719X) mutations, three also with exon 20 mutations; 24 exon 19 deletions, six also with exon 20 mutations; 23 exon 21 (L858R), three also with exon 20 mutations; and 8 exon 20 alone.All 15 exon 19 IHC positive patients were proven mutated (100% specificity, 63% sensitivity). Twenty-two of 27 exon 21 IHC positive cases were proven mutated while three patients had insufficient material for molecular testing (92% specificity, 96% sensitivity). The overall specificity and sensitivity of IHC for any EGFR mutation was 95% and 58%. Five patients initially thought to be wild type for EGFR but IHC positive underwent repeat molecular testing because of the discrepancy which confirmed the IHC result in three cases (60%).We conclude IHC is very specific but not sensitive. Whilst IHC cannot replace molecular testing, it is a useful adjunct which requires minimal tissue and identifies false negative molecular results which occurred in 5% of our patients with eventually confirmed EGFR mutations.

Determining Adequacy of Cell Block Material for Molecular Testing: A Practical Guide for Cytopathologists

s S81 Figure 2 Increased background staining with Ventana Figure 3 No background staining with Dako 175 Determining Adequacy of Cell Block Material for Molecular Testing: A Practical Guide for Cytopathologists Oana Rafael, MD, Melissa Klein, CT(ASCP), Tamar Brandler, MD, MS, Kasturi Das, MD, Mohamed Aziz, MD. North Shore Long Island Jewish Lenox Hill Hospital, New York, New York; Hofstra North Shore e Long Island Jewish School of Medicine, New Hyde Park, New York Introduction: With the current advances in personalized medicine, targeted therapy has changed the practice of cytology. Selection of cell blocks (CB) for molecular testing (MT) is challenging when confronted with limited lesional material. Our aim was to determine optimal tumor quantity necessary for reliable MT results. Material and Methods: Reports for cytology cases were tallied for MT requests received between January 2012-December 2013. Cases were divided into satisfactory and unsatisfactory, with positive and negative molecular results (Table 1). Retrospective review of hematoxylin and eosin CB slides for 41 cases with MT was performed. CBs were evaluated for total number and percentage of tumor cells, background necrosis, inflammation, blood, and benign cells (Table 2). Results: Of 41 CBs analyzed, 59% were sufficient and 41% were insufficient for MT. Half of the reviewed cases showed positive MT results (Table 3). 17% (2/12 cases) of satisfactory CBs sent for MT were deemed inadequate by the reference laboratory, despite the cytopathologist’s count of 300-350 tumor cells (30% and 15% of tumor cells respectively) on CBs. In these cases, 20% malignant cellularity was given as a cut-off by the reference laboratory. However, in one case the pathologist appreciated tumor cellularity as Conclusions: Improved feedback from the reference laboratories might help ensure that sufficient material is present in cytology specimens before cytopathologists order MT. This study underscores the need for an algorithm to accurately assess adequacy of CBs before initiating molecular studies. These findings could serve as a guideline for pathologists and reference laboratories. Review of additional cases will be completed to support our CB adequacy threshold. Table 1 Cell block (CB) adequacy material for molecular testing CB adequacy material 2012 2013 Total Insufficient 23 12 35 Sufficient with positive molecular result 22 8 30 Sufficient with negative molecular result 37 32 69 TOTAL 82 52 134 Table 2 Evaluation of cell blocks for molecular testing, including distribution of tissue slides over the different tumor cell content categories Tumor Molecular Testing cell % Satisfactory with positive results Satisfactory with negative results Cases (n) Tumor Cells (n) Background* Cases (n) Tumor Cells (n) Background* 350 2+ blood 4 250-300 2+ benign cells >350 1+ blood 300-350 3+ blood 300-350 2+ necrosis 20% 3 >350 Clean 1 >350 2+ inflammation 2+ blood 3+ blood 25% 2 >350 1+ blood e e e 3+ blood 30% 4 >350 2+ necrosis 2 >350 Clean 1+ necrosis 3+ blood 1+ blood 1+ blood 35% e e e 1 >350 2+ inflammation 40% e e e 1 >350 Clean 70% 1 >350 3+ necrosis e e e * 1+ mild; 2+ moderate; 3+ abundant Table 3 Cases analyzed and molecular testing results Cell block adequacy material N (%) Total cases Sufficient with positive molecular result 12 (59) 41 Sufficient with negative molecular result 12 (59) Insufficient for molecular testing 17 (41)

Clinicopathological and Molecular Spectrum of Ewing Sarcomas/PNETs, Including Validation of EWSR1 Rearrangement by Conventional and Array FISH Technique in Certain Cases

Over the years, a wide clinicopathological spectrum has been identified within Ewing family of tumors (EFTs). As these tumors are chemosensitive, their correct and timely identification is necessary. The aims of this study were (1) to present the diverse clinicopathological and molecular profile of EFTs in our settings, (2) to identify a pragmatic approach for diagnosing EFTs, especially for application of ancillary techniques, namely RT-PCR for specific transcripts (EWS-FLI1, EWS-ERG) and FISH for EWSR1 gene rearrangement, in certain cases and (3) to show the utility of tissue microarray in establishing a new FISH test. Fifty-eight EFTs were identified in 38 males and 20 females within an age-range of 1-65 years (median, 16), mostly in lower extremities (14) (24.1 %). Therapeutically, most patients underwent neoadjuvant chemotherapy with subsequent surgery. Histopathologically, diagnosis of EFTs was initially offered in 41/58 (70.6 %) tumors. On review, 59 % tumors showed diffuse pattern, while 41 % displayed rosettes. Immunohistochemically, tumor cells were mostly diffusely positive for CD99 (48/52) (92.3 %); FLI-1 (17/18) (94.4 %); variably for BCL2 (16/18) (88.8 %), synaptophysin (6/20) (35 %), S100-P (2/7) (28.5 %), CD56 (2/5) (40 %), NSE (2/5) (40 %), calponin (3/4) (75 %), EMA (5/24) (20.8 %) and CK (3/24) (12.5 %), the latter two mostly focally. Fifty five tumors were EWS-FLI1 positive, while a single tumor was EWS-ERG positive. Sensitivity for PCR was 61 %. EWSR1 rearrangement was detected by FISH in 12/13 Ewing sarcomas/PNETs. Sensitivity for EWSR1 test was 92.3 % and specificity was 100 %. Thirty-eight tumors, including 14 molecular confirmed EFTs and 21 other tumors were tested for EWSR1 rearrangement. Among 21 unrelated tumors, EWSR1 rearrangement was detected in few myoepithelial tumors, occasional desmoplastic small round cell tumor and an extraskeletal myxoid chondrosarcoma. Further, a tissue microarray with a separate set of 8 EFTs, confirmed at another laboratory was analysed for validation of EWSR1 rearrangement test. 23/28 (82.1 %) tissue cores of the tissue microarray, stained by FISH were interpretable, including EWSR1 rearrangement, detected in 20/28 tissue cores; not detected in 3 liver cores and uninterpretable in 5 (17.8 %) cores. Classical EFTs can be diagnosed with diffuse, membranous CD99 positivity, intranuclear FLI1 positivity and LCA negativity in malignant round cells. In unconventional cases, it is indispensable to reveal the concomitant fusion m-RNA by RT-PCR. In case of negative molecular results, it is necessary to prove EWSR1 rearrangement by FISH. These tests should be interpreted with clinicopathological correlation. Tissue microarrays for FISH are useful during validation of a new test, especially when sarcomas like EFTs show less genetic heterogeneity within tumor cells.

Molecular markers in the diagnosis of thyroid nodules

An indeterminate thyroid nodule cytology result occurs about every sixth fine-needle aspiration. These indeterminate nodules harbor a 24% risk of malignancy (ROM); too high to ignore, but driving surgery where most nodules are benign. Molecular diagnostics have emerged to ideally avoid surgery when appropriate, and to trigger the correct therapeutic surgery when indicated, as opposed to an incomplete diagnostic surgery. No current molecular test offers both high sensitivity and high specificity. A molecular diagnostic test with high sensitivity (e.g. Afirma Gene Expression Classifier sensitivity 90%) offers a high Negative Predictive Value when the ROM is relatively low, such as < 30%. Only such tests can "rule-out" cancer. In this setting, a molecularly benign result suggests the same ROM as that of operated cytologically benign nodules (~6%). Thus, clinical observation can replace diagnostic surgery; increasing quality of life and decreasing medical costs. However, its low specificity cannot "rule-in" cancer as a suspicious result has a Positive Predictive Value (PPV) of ~40%, perhaps too low to routinely reflex to definitive cancer surgery. Conversely, high specificity tests (BRAF, RAS, PPAR/PAX-8, RET/PTC, PTEN) offer high PPV results, and only these tests can "rule-in" cancer. Here a positive molecular result warrants definitive therapeutic surgery. However, their low sensitivity cannot "rule-out" cancer and a negative molecular result cannot dissuade diagnostic surgery; limiting their cost-effectiveness. Whether or not there is a useful and cost-effective role to sequentially combine these approaches, or to modify existing approaches, is under investigation.

Achievement of Sustained Molecular Remission Induces Long-Term Freedom From Disease After Autologous-Allogeneic Tandem Transplantation in Patients with Multiple Myeloma

Abstract 148Multiple Myeloma (MM) is considered to be an incurable disease, but in some patient long-term survival can be achieved after high dose chemotherapy followed by autologous or allogeneic stem cell transplantation. Those patients, who achieved molecular remission, have a high probability of long-term disease-freedom and of cure. More recently the introduction of dose-reduced conditioning and the use of auto-allo tandem approach has broaden the use of allogeneic stem cell transplantation in MM, but the results of prospective trials are controversial. We here investigated within a prospective protocol the incidence and impact of molecular remission on long-term outcome obtained after auto-allo tandem approach in MM. (study registration NCT 00781170).From 4/2000 to 10/2008, 73 patients with a median age of 50 years (r 29–64) and advanced stage II/III MM were included. The conditioning regimen for autologous transplantation consisted of melphalan 200 mg/m2 given divided over 2 days. After an interval of 2–3 months, the patients received a dose-reduced conditioning consisting of melphalan 140 mg/m2, fludarabine 180 mg/m2 and ATG Fresenius® (Fresenius, Bad Homburg, Germany) at a dose of 10 mg/kg for related and 20 mg/kg for unrelated donors on day -3, -2, and -1 followed by allogeneic stem cell transplantation.Remission was defined according to modified EBMT criteria. In 46 patients with CR or nCR minimal residual disease could be monitored by myeloma-specific primers (allele specific oligonucleotides, ASO) targeting IgH gene rearrangements (n=20) or by highly sensitive plasma cell chimerism using real-time PCR after magnet-activated cell sorting of CD138+ cells (n=26). The sensitivity of plasma cell chimerism was 10−4 using real-time PCR and donor/patient specific polymorphisms and 10−5 in case of Y-PCR. Sensitivity for nested PCR with myeloma-specific primers was 10−5. In 5 patients only 1 MRD measurement was available and those patients were excluded from MRD sub-analysis. Molecular complete remission was defined as by least two negative PCR results with ASO primers or achievement of at least two times ≥99.9 % donor chimerism of plasma cells by real-time PCR from bone marrow samples. Sustained MRD negativity was defined by at least 4 negative molecular results either by ASO primer or plasma-cell chimerism. Mixed MRD was defined by negative molecular markers for at least two times, but intermittent positivity was defined by myeloma-specific primers or plasma cell chimerism.Overall 44 patients (60 %) achieved a CR according to EBMT criteria with negative immunofixation. 8 % achieved a VGPR and 18 % a partial remission. 3 % had progressive disease and 11 % were not evaluable for determination of remission. Molecular remission was observed in 30 patients, in 15 patients the molecular markers were sustained negative while in 15 patients molecular markers were only intermittent negative, resulting in an overall complete molecular remission rate of 46 % and a stable sustained complete molecular remission rate of 23 %.After a median follow-up at 7 years the 5 year progressive-free survival was 29 % (95% CI: 17–41%). Patients without del(13q14) had a significantly better PFS than those with del(13q14) (5 y: 56% vs. 17%, p=0.02). Patients who achieved CR after transplantation had improved PFS in comparison to non-CR patients (5 y: 41% vs. 28%, p=0.008). Patients with sustained negative molecular remission had a 5 year PFS of 85 % vs. 31 % for mixed molecular remission (p= 0.003).The 5 year overall survival was 52 % (95% CI: 40–64%). Patients without del(13q14) had significantly improved 5 year survival (75% vs. 40%. p=0.02). Patients who achieved a sustained molecular remission had a 5 year OS of 91 % while those with mixed molecular remission resulted in a 5 year OS of 87 % (p=0.06).The study underlines the importance of the depth of remission and shows that achieving molecular remission as determined by myeloma-specific IgH gene rearrangements and plasma cell chimerism is associated with long-term freedom from disease and potential cure of MM in an auto-/allo SCT approach. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

P37 Molecular and Serologic Characterization of RhD Status

Introduction Developments in technology and knowledge have allowed the elucidation of many of the blood group systems at the molecular level, much of this work is still mainly confined to larger centres and reference facilities. The D antigen is highly complex and of enormous clinical significance causing both haemolytic disease of the newborn and haemolytic transfusion reactions. D typing is routinely performed using serological techniques but examples of weakened expression can be difficult to resolve. Molecular techniques have permitted a greater understanding of these problems and can also be applied to predicting the D type of the foetus using a maternal sample which has obvious benefits to mother and baby.Aims The development of a molecular method for RhD typing and compare this to serological typing.Study Design and Methods Multiplex PCR was used to analyze RhD status in DNA from 127 randomly selected blood donors from the Northern Ireland Blood Transfusion Service. This method was compared to the serological assessment of RhD typing using the Ortho AutoVueTM System.Results Serological results showed that 93 samples (73%) were RhD+ and 34 (27%) were RhD− which is expected for a Caucasian population. 75 samples (50%) were RhC+ and 52 (41%) were RhC−. 111 samples (87%) were Rhc+ and 16 (13%) were Rhc−. Positive and negative molecular results were compared to the corresponding serological results. A concordance rate of 78.7% existed for the comparison of both methods for RhD status. 65.4% of samples were concordant for both methods when assessing RhC status. The concordance rate was 73.2% for Rhc.Conclusion We describe a multiplex PCR method for the assessment of RhD status and compare this to results obtained using standard serology. Further development of this molecular method is required to obtain a higher concordance rate. Molecular typing has potential future benefits, along with serological methods, in adding to our understanding of the Rh blood system and identifying RhD variants.

Hematopoietic Cell Transplantation After Nonmyeloablative Conditioning for Advanced Chronic Lymphocytic Leukemia

Patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) have a short life expectancy. The aim of this study was to analyze the outcome of patients with advanced CLL when treated with nonmyeloablative conditioning and hematopoietic cell transplantation (HCT). Sixty-four patients diagnosed with advanced CLL were treated with nonmyeloablative conditioning (2 Gy total-body irradiation with [n = 53] or without [n = 11] fludarabine) and HCT from related (n = 44) or unrelated (n = 20) donors. An adapted form of the Charlson comorbidity index was used to assess pretransplantation comorbidities. Sixty-one of 64 patients had sustained engraftment, whereas three patients rejected their grafts. The incidences of grades 2, 3, and 4 acute and chronic graft-versus-host disease were 39%, 14%, 2%, and 50%, respectively. Three patients who underwent transplantation in complete remission (CR) remained in CR. The overall response rate among 61 patients with measurable disease was 67% (50% CR), whereas 5% had stable disease. All patients with morphologic CR who were tested by polymerase chain reaction (n = 11) achieved negative molecular results, and one of these patients subsequently experienced disease relapse. The 2-year incidence of relapse/progression was 26%, whereas the 2-year relapse and nonrelapse mortalities were 18% and 22%, respectively. Two-year rates of overall and disease-free survivals were 60% and 52%, respectively. Unrelated HCT resulted in higher CR and lower relapse rates than related HCT, suggesting more effective graft-versus-leukemia activity. CLL is susceptible to graft-versus-leukemia effects, and allogeneic HCT after nonmyeloablative conditioning might prolong median survival for patients with advanced CLL.

Comparison of histological and molecular diagnosis of Helicobacter pylori in benign lesions and gastric adenocarcinoma

Helicobacter pylori colonization is associated with chronic gastritis, peptic ulcers, intestinal metaplasia, adenocarcinoma and lymphoma of the stomach. The objective of this study was to compare the results of the routinely used histology with molecular diagnosis for the detection of H. pylori. Eighty samples from gastric lesions (chronic gastritis, atrophic gastritis, gastric ulcer, and intestinal metaplasia), 18 gastric adenocarcinoma and 10 normal mucosa H. pylori-negative (control) samples were obtained. All samples were examined histologically (hematoxylin-eosin and Giemsa staining), and PCR amplifications of the species-specific antigen gene (H3H4) and urease A gene segment (H5H6) of H. pylori were made, using the human gene CYP1A1 for DNA quality control. In the benign lesion and adenocarcinoma the infection was detected in 43% (42/98) and 71% (70/98) by histological and molecular diagnosis (p = 0.0001), respectively. The PCR test detected H. pylori in 27.5% (22/80) of the benign gastric lesions and in 50% (9/18) of the gastric adenocarcinoma cases, the histological diagnosis being negative for this bacterium. About 2.5% of the samples, exclusively from benign lesions and with a positive histological diagnosis, showed negative molecular results for both primers. Statistically significant differences were found between the histological and the molecular method in intestinal metaplasia (p = 0.0461) and gastric adenocarcinoma (p = 0.0011), due to underdetection of H. pylori by the histological method, which is probably due to the low density of the bacterium as a consequence of the severe atrophy of the gastric mucosa. Our findings suggest that PCR is the more efficient method for the assessment of H. pylori infection, especially in metaplasia and gastric adenocarcinoma.

High Rate of BCR-ABL Transcript Undetectability Achieved by Treating with Imatinib Mesylate Very Late CML Patients in Stable CCR after IFN.

Background. Interferon alfa (IFN a) induces complete cytogenetic response (CCR) in small proportion of CML patients, with almost all of these patients still presenting BCR-ABL transcript at the molecular level, even after prolonged period of CCR. Imatinib mesylate induces CCR in a 60–80% of patients but a high percentage of these still show residual disease as detectable by RT-PCR, and can be at risk of disease relapse. Thus combining synergistic drugs might be required to eliminate the disease effectively.Methods. We treated with imatinib 16 CML patients (8 M and 8 F) who were in very late CP and in stable CCR achieved with IFNa, but still were persistently positive at molecular level by RT-PCR. According to Sokal's score, 11 patients were low risk (LR) and 5 were intermediate risk (IR). Median time from diagnosis was 125 mos.(r. 52–202), median duration of stable CCR was 79 mo.s (r.9–148). Imatinib was administered at the standard dose of 400 mg/die, after stopping IFN for 1 week. The level of residual disease was then monitored on BM cells at planned intervals ( baseline, 3, 6, 12 mo.s), by assaying BCR-ABL transcript at nested PCR and real-time quantitative RT-PCR; ABL was used as an internal control and results expressed as a ratio of BCR-ABL/ABL transcripts on a log scale.Results. The median transcript levels, as measured at various time points, appeared to progressively and consistently decrease in all patients with respect to the baseline values. In particular, BCR-ABL transcript undectectability was observed in 6/15 patients with evaluable analyses at 3 mo.s, in 9/16 patients analysed at 6 mo.s, and in 5/8 with available results also at 12 mo.s; of these latter patients, 4 were already negative in previous analyses and one become negative at 12 mo.s. Thus, altogether, 10/16 (62.5%) patients with at least two examinations within 12 mo.s had at least one negative molecular result. Correlations between degree of transcript reduction and clinical/biological factors detected at diagnosis and during follow-up, evidenced not significant results for age, type of transcript (either b2a3 or b3a3), baseline transcript level pre-imatinib, duration of disease and of stable CCR prior imatinib, while a trend was apparent for a higher rate of LR vs IR score among the 10 patients reaching transcript undetectability (8/10 LR vs 2/10 IR) with respect to the 6 persistently positive subjects(3 LR vs 3 IR). In present cases, imatinib was well tolerated and no side effects required drug dose reduction or discontinuation. At a median follow-up of 13 months (8–16) from start imatinib, all 16 patients are alive in CCR with progressively improving molecular response, 10 of whom with persistent transcript undetectability.Conclusion. Albeit obtained in a series of very selected patients, present results represent a further evidence stressing on the efficacy of combining imatinib and IFN a; through different, possibly complementary, mode of action, these two drugs might mutually potentiate their effect while reducing the emergence of drug resistance. The additive toxicity caused by their concurrent use might be overcome by a sequential combination; this could also be applied to patients reaching stable CCR on imatinib but still maintaining detectable residual disease.