HER2/ERBB2 copy number analysis by targeted next-generation sequencing in breast cancer.

OBJECTIVES: A combination of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) is the current standard of care for HER2 evaluation in breast cancer. Here, we investigate the potential clinical utility of next-generation sequencing (NGS)-derived HER2/ERBB2 copy number (CN) data for predicting HER2 status as defined by American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. METHODS: In total, 294 locally recurrent and metastatic breast cancers previously tested by targeted hybrid capture-based NGS and by HER2 IHC/FISH were included. Analyses focused on the ERBB2 median log2 ratios and start-end genomic coordinates from NGS, average HER2 CN and HER2/CEP17 ratios from FISH, and the HER2 IHC scores. We also determined a more stringent log2 ratio cutoff to predict HER2-positive status with 100% specificity. RESULTS: Sixty-four (22%) cases were HER2 positive and 230 (78%) were HER2 negative by ASCO/CAP guidelines. The ERBB2 median log2 ratios from NGS strongly correlated with HER2 status by IHC/FISH (area under receiver operator characteristic curve = 0.951). ERBB2 log2 ratio more than 1.7 was 100% specific for HER2-positive results by IHC/FISH. Start and end genomic coordinates for regions of gain near ERBB2 by NGS also predicted HER2 status. CONCLUSIONS: Copy number data from our NGS panel strongly correlate with HER2 status. Using a stringent cutoff, ERBB2 log2 ratio accurately predicts HER2 positivity with high specificity. The NGS CN assessment may have utility in determining HER2 status in certain clinical settings.

Structural asymmetries in normal brain anatomy: A brief overview.

PURPOSE: A fundamental feature in interpreting gross or neuroimaging brain anatomy is reliance on an assumed high degree of morphologic symmetry in bilateral hemispheres. However, the normal brain is not perfectly symmetrical, and subtle inherent structural asymmetries could potentially confound appreciation of pathology-induced asymmetry or how a given brain asymmetry can relate to its function. MATERIAL AND METHODS: We review the literature and provide a brief overview of structural asymmetries in normal brain anatomy. RESULTS: Brain structural asymmetries are either rotational or pure right-left asymmetries, and many are a consequence of unique features linked to the use of human language. Yakovlevian torque is the tendency of the right hemisphere to rotate slightly forward relative to the left, which may make the right frontal lobe bigger and wider, and the left occipital lobe wider and protrude rightward. This makes the left Sylvian fissure longer and flatter, resulting in a larger planum temporale. We also discuss right-left asymmetries in the cortex, white matter structures, deep gray nuclei, and lateral ventricles. Brain asymmetries are not random but result from distinct patterns in structural design that confer evolutionary functional advantages. CONCLUSION: Minor brain asymmetries are important and should be accounted for as they can be connected to function, and like individual variability, are essential for evolution. This overview will help understand structural brain asymmetries for improved diagnostic neuroimaging interpretation, constructing symmetry-based paradigms for automatic localization, segmentation of brain lesions, and as a reference for studies on possible implications of excessive asymmetry and altered laterality in cognitive, neurological, and psychiatric disorders.

A germinal center-associated microenvironmental signature reflects malignant phenotype and outcome of DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy, with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin (COO), genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of "unfavorable" molecular signatures.

Clinical and molecular characterization of virus-positive and virus-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC. METHODS: In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure. RESULTS: Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival. CONCLUSIONS: These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.

Next-generation sequencing informs diagnosis and identifies unexpected therapeutic targets in lung squamous cell carcinomas.

OBJECTIVES: Potentially targetable genomic alterations have been identified in lung squamous cell carcinoma (LUSC), but none have yet translated into effective therapy. We examined potential benefits of next generation sequencing (NGS) in a cohort of consecutive LUSC patients with emphasis on distinctions between smokers and light/never smokers and implications for clinical trial enrollment. METHODS: We retrospectively evaluated results from an internally developed NGS assay (OncoPanel) targeting ∼300 genes with a mean overall target coverage of >200x for consecutive LUSC seen at our institution over 30 months. RESULTS: Tissue was obtained from 172 patients for targeted NGS. 42 (24 %) samples were insufficient for testing. Median age of tested patients was 66, including 87 % moderate/heavy versus 13 % light/never smokers; 66 % were stage IIIB or IV. Of 130 patients with evaluable NGS results, 49 (38 %) had at least 1 alteration qualifying for enrollment to a LungMAP treatment arm (PIK3CA, MET, FGFR family, cell cycle, or homologous recombination pathways) or for an approved therapy or other clinical trial (e.g. EGFR sensitizing mutations, MET exon 14 splice mutations, TSC1/2 mutation, or microsatellite instability). Therapeutic targets were enriched in light/never smokers (47 % vs 35 % moderate/heavy smokers). Unexpectedly, genomic features suggested an alternative diagnosis (metastatic cutaneous squamous carcinoma; mesothelioma) in 7 patients, including 35 % of never/light smokers. CONCLUSION: NGS in a real-world LUSC cohort yields potentially targetable genomic alterations informing clinical trial enrollment and approved therapies and critical diagnostic insights. Our findings strongly support current guidelines recommending mutational profiling of LUSC arising in light/never smoking patients; the utility of sequencing in smokers with LUSC appears to be limited to identification of research targets.

Alisertib plus induction chemotherapy in previously untreated patients with high-risk, acute myeloid leukaemia: a single-arm, phase 2 trial.

BACKGROUND: Increased aurora A kinase (AAK) expression occurs in acute myeloid leukaemia; AAK inhibition is a promising therapeutic target in this disease. We therefore aimed to assess the activity of alisertib combined with 7 + 3 induction chemotherapy in previously untreated patients with high-risk acute myeloid leukaemia. METHODS: We did a single-arm, phase 2 trial of patients recruited from the Dana-Farber/Harvard Cancer Center in the USA. Eligible patients had previously untreated acute myeloid leukaemia, an Eastern Cooperative Oncology Group performance status of 0-2, and were at high risk of disease as defined by the presence of an adverse-risk karyotype, the presence of secondary acute myeloid leukaemia arising from previous myelodysplastic syndrome or myeloproliferative neoplasm, the presence of therapy-related acute myeloid leukaemia, or being 65 years or older. Enrolled patients received 7 + 3 induction chemotherapy of continuous infusion of cytarabine (100 mg/m2 per day on days 1-7) and intravenous bolus of idarubicin (12 mg/m2 per day on days 1-3). Oral alisertib (30 mg) was given twice per day on days 8-15. Patients could receive up to four consolidation cycles with cytarabine and alisertib, and alisertib maintenance for 12 months. The primary endpoint was a composite including the proportion of patients achieving complete remission and those with a complete remission with incomplete neutrophil or platelet count recovery. Analyses were per-protocol. This study is registered with Clinicaltrials.gov, number NCT02560025, and has completed enrolment. FINDINGS: Between Dec 31, 2015, and Aug 1, 2017, we enrolled a total of 39 eligible patients. 19 (49%) of 39 patients had secondary acute myeloid leukaemia and three (8%) had therapy-related acute myeloid leukaemia. At mid-induction, 33 (85%) of 39 patients showed marrow aplasia, six (15%) received re-induction. The median follow-up was 13·7 months (IQR 12·7-14·4). Composite remission was 64% (two-stage 95% CI 48-79), with 20 (51%) of 39 patients achieving complete remission and five (13%) achieving complete remission with incomplete neutrophil or platelet count recovery. The most common grade 3 or 4 adverse events included febrile neutropenia (16 [41%] of 39), neutropenia (12 [31%]), thrombocytopenia (13 [33%]), anaemia (11 [28%]), anorexia (nine [23%]), and oral mucositis (four [10%]). No treatment-related deaths were observed. INTERPRETATION: These results suggest that alisertib combined with induction chemotherapy is active and safe in previously untreated patients with high-risk acute myeloid leukaemia. This study met criteria to move forward to a future randomised trial. FUNDING: Millennium Pharmaceuticals.

Germline Predisposition to Hematolymphoid Neoplasia.

OBJECTIVES: The 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review clinical cases with germline predisposition to hematolymphoid neoplasms. METHODS: The Workshop Panel reviewed 51 cases with germline mutations and rendered consensus diagnoses. Of these, six cases were presented at the meeting by the submitting pathologists. RESULTS: The cases submitted to the session covering germline predisposition included 16 cases with germline GATA2 mutations, 10 cases with germline RUNX1 mutations, two cases with germline CEBPA mutations, two germline TP53 mutations, and one case of germline DDX41 mutation. The most common diagnoses were acute myeloid leukemia (15 cases) and myelodysplastic syndrome (MDS, 14 cases). CONCLUSIONS: The majority of the submitted neoplasms occurring in patients with germline predisposition were myeloid neoplasms with germline mutations in GATA2 and RUNX1. The presence of a germline predisposition mutation is not sufficient for a diagnosis of a neoplasm until the appearance of standard diagnostic features of a hematolymphoid malignancy manifest: in general, the diagnostic criteria for neoplasms associated with germline predisposition disorders are the same as those for sporadic cases.

Avoiding peripheral nerve injury in arterial interventions.

Although peripheral nerve injuries secondary to angiography and endovascular interventions are uncommon and usually not permanent, they can result in significant functional impairment. Most arteries used in access for angiography and endovascular therapies lie in close proximity to a nerve. The nerve may be injured by needle puncture, or by compression from hematoma, pseudoaneurysm, hemostasis devices, or by manual compression with incidence in literature ranging from as low as 0.04% for femoral access in a large retrospective study to 9% for brachial and axillary access. Given the increasing frequency of endovascular arterial procedures and the increasing use of nontraditional access points, it is important that the interventionalist have a working knowledge of peripheral nerve anatomy and function as it relates to relevant arterial access sites to avoid injury.

Premalignant Clonal Hematopoietic Proliferations.

OBJECTIVES: The 2017 Workshop of the Society for Hematopathology/European Association for Hematopathology aimed to review premalignant clonal hematopoietic proliferations. METHODS: The workshop panel reviewed 27 cases of clonal proliferations of indeterminate significance or potential (18 myeloid, nine lymphoid) and rendered consensus diagnoses. RESULTS: Immunophenotyping and genetic studies on peripheral blood, bone marrow, and lymph node samples have led to the incidental detection of small clonal populations in asymptomatic individuals. These premalignant clonal myeloid and lymphoid proliferations include monoclonal gammopathy of uncertain significance, monoclonal B-cell lymphocytosis, in situ follicular neoplasia, in situ mantle cell neoplasia, clonal hematopoiesis of indeterminate potential, and clonal cytopenia of undetermined significance. CONCLUSIONS: Current diagnostic criteria for the diagnoses of premalignant clonal hematopoietic proliferations are reviewed and discussed in the context of the cases presented at the workshop.

The Distribution Volume of 18F-Albumin as a Potential Biomarker of Antiangiogenic Treatment Efficacy.

Objective: 18F-albumin, a vascular imaging agent, may have potential to assess tumor responses to anti-angiogenic therapies. In these studies tumor distribution volume of 18F-albumin were first determined in various human tumor xenografts from biodistribtuion measurments and then one of the tumor type was used to evaluate changes in 18F-albumin uptake in anti-angiognic tumor model. Method: 18F-albumin was synthesized via conjugation of 6-[18F]fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [18F]F-Py-TFP, with rat albumin. From the biodistribution of 18F-albumin in various human tumor xenografts tumor distribution volumes (DVs; tumor%ID/g:blood%ID/g) were first determined at various time points. Then, the ability of 18F-albumin to detect tumor angiogenic inhibition in one of these tumor types (U87MG) following treatment with sunitinib was evaluated by position emission tomography (PET) imaging at 0, 7, 14, and 21 days post treatment. Caliper measurements of tumor dimensions were also made at these same times. At Day 21, following imaging, biodistributions, autoradiography of tumor tissues and tumor blood vessel counts (CD31 IHC) were performed. Results: 18F-albumin retention in various tumors steadily increased over time with U87MG tumor exhibiting the highest uptake (DV) at all times. Significant decreases in 18F-albumin DVs were observed one week post-treatement (-39%) vs. controls whereas tumor caliper volumes were not significantly decreased until days 14 and 21. At day 21 the significant decrease in DVs in the treatment group (-44%) paralleled biodistribution DV measurements and was consistent with autoradiography and CD31 IHC findings. Conclusion: These data suggest that 18F-albumin DVs obtained by imaging may serve as an early biomarker of the effectiveness of anti-angiogenic therapy and thus aid in patient management and treatment planning.

Next generation sequencing in hematolymphoid neoplasia.

Large scale sequencing projects over the past 2 decades have led to the identification of many common genomic alterations in hematolymphoid neoplasms, some of which with diagnostic, therapeutic, and prognostic implications [1-3]. Although these alterations can be tested individually with high sensitivity and specificity using dedicated single gene tests, it is increasingly impractical and costly to test them separately as the number of alterations grows. Instead, multiplex testing platforms that can test multiple targets in a specimen have been developed. Among these platforms, massively parallel sequencing technologies (so-called next-generation sequencing [NGS] [4]) prove to be most versatile and is increasingly being used to build tests to meet the clinical testing need. In hematolymphoid neoplasms, the early incorporation of molecular findings into the diagnostic criteria by WHO [5] has further accelerated the adoption of NGS-based tests in routine clinical practice. This article focuses on what is used in the clinical diagnostic laboratories today and is not intended to be a review of the NGS technology or its future direction. Following discussion of the 2 families of sequencing instruments from Illumina and Thermo Fisher and 3 target enrichment methods, aspects of the analysis and report of NGS results that is clinically relevant are discussed.

High NPM1-mutant allele burden at diagnosis predicts unfavorable outcomes in de novo AML.

Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 World Health Organization classification and is associated with a favorable prognosis. Although previous studies have evaluated NPM1 in a binary fashion, little is known about the significance of its mutant allele burden at diagnosis, nor has the effect of comutations (other than FLT3) been extensively evaluated. We retrospectively used targeted sequencing data from 109 patients with de novo AML with mutated NPM1 to evaluate the potential significance of NPM1 variant allele frequency (VAF), comutations, and clinical parameters with regard to patient outcomes. We observed that high NPM1 VAF (uppermost quartile) correlated with shortened overall survival (median, 12.1 months vs not reached; P < .0001) as well as event-free survival (median, 7.5 vs 65.44 months; P < .0001) compared with the other NPM1-mutated cases. In both univariate and multivariable analyses, high NPM1 VAF had a particularly adverse prognostic effect in the subset of patients treated with stem-cell transplantation in first remission (P = .0004) and in patients with mutated DNMT3A (P < .0001). Our findings indicate that the prognostic effect of NPM1 mutation in de novo AML may be influenced by the relative abundance of the mutated allele.

Frameshift events predict anti-PD-1/L1 response in head and neck cancer.

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti-PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti-PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti-PD-1/L1 response.

Association of mutations with morphological dysplasia in de novo acute myeloid leukemia without 2016 WHO Classification-defined cytogenetic abnormalities.

Despite improvements in our understanding of the molecular basis of acute myeloid leukemia (AML), the association between genetic mutations with morphological dysplasia remains unclear. In this study, we evaluated and scored dysplasia in bone marrow (BM) specimens from 168 patients with de novo AML; none of these patients had cytogenetic abnormalities according to the 2016 World Health Organization Classification. We then performed targeted sequencing of diagnostic BM aspirates for recurrent mutations associated with myeloid malignancies. We found that cohesin pathway mutations [q (FDR-adjusted P)=0.046] were associated with a higher degree of megakaryocytic dysplasia and STAG2 mutations were marginally associated with greater myeloid lineage dysplasia (q=0.052). Frequent megakaryocytes with separated nuclear lobes were more commonly seen among cases with cohesin pathway mutations (q=0.010) and specifically in those with STAG2 mutations (q=0.010), as well as NPM1 mutations (q=0.022 when considering the presence of any vs no megakaryocytes with separated nuclear lobes). RAS pathway mutations (q=0.006) and FLT3-ITD (q=0.006) were significantly more frequent in cases without evaluable erythroid cells. In univariate analysis of the 153 patients treated with induction chemotherapy, NPM1 mutations were associated with longer event-free survival (EFS) (P=0.042), while RUNX1 (P=0.042), NF1 (P=0.040), frequent micromegakaryocytes (P=0.018) and presence of a subclone (P=0.002) were associated with shorter EFS. In multivariable modeling, NPM1 was associated with longer EFS, while presence of a subclone and frequent micromegakaryocytes remained significantly associated with shorter EFS.

A distinct immunophenotype identifies a subset of NPM1-mutated AML with TET2 or IDH1/2 mutations and improved outcome.

Recent work has identified distinct molecular subgroups of acute myeloid leukemia (AML) with implications for disease classification and prognosis. NPM1 is one of the most common recurrently mutated genes in AML. NPM1 mutations often co-occur with FLT3-ITDs and mutations in genes regulating DNA methylation, such as DNMT3A, TET2, and IDH1/2. It remains unclear whether these genetic alterations are associated with distinct immunophenotypic findings or affect prognosis. We identified 133 cases of NPM1-mutated AML and correlated sequencing data with immunophenotypic and clinical findings. Of 84 cases (63%) that lacked monocytic differentiation ("myeloid AML"), 40 (48%) demonstrated an acute promyelocytic leukemia-like (APL-like) immunophenotype by flow cytometry, with absence of CD34 and HLA-DR and strong myeloperoxidase expression, in the absence of a PML-RARA translocation. Pathologic variants in TET2, IDH1, or IDH2 were identified in 39/40 APL-like cases. This subset of NPM1-mutated AML was associated with longer relapse-free and overall survival, when compared with cases that were positive for CD34 and/or HLA-DR. The combination of NPM1 and TET2 or IDH1/2 mutations along with an APL-like immunophenotype identifies a distinct subtype of AML. Further studies addressing its biology and clinical significance may be especially relevant in the era of IDH inhibitors and recent work showing efficacy of ATRA therapy in NPM1 and IDH1-mutated AML.

The relative utilities of genome-wide, gene panel, and individual gene sequencing in clinical practice.

Advances in technology that have transpired over the past 2 decades have enabled the analysis of cancer samples for genomic alterations to understand their biologic function and to translate that knowledge into clinical practice. With the power to analyze entire genomes in a clinically relevant time frame and with manageable costs comes the question of whether we ought to and when. This review focuses on the relative merits of 3 approaches to molecular diagnostics in hematologic malignancies: indication-specific single gene assays, gene panel assays that test for genes selected for their roles in cancer, and genome-wide assays that broadly analyze the tumor exomes or genomes. After addressing these in general terms, we review specific use cases in myeloid and lymphoid malignancies to highlight the utility of single gene testing and/or larger panels.

Identification of miRNAs involved in DRG neurite outgrowth and their putative targets.

Peripheral neurons regenerate their axons after injury. Transcriptional regulation by microRNAs (miRNAs) is one possible mechanism controlling regeneration. We profiled miRNA expression in mouse dorsal root ganglion neurons after a sciatic nerve crush, and identified 49 differentially expressed miRNAs. We evaluated the functional role of each miRNA using a phenotypic analysis approach. To predict the targets of the miRNAs we employed RNA-Sequencing and examined transcription at the isoform level. We identify thousands of differentially expressed isoforms and bioinformatically associate the miRNAs that modulate neurite growth with their putative target isoforms to outline a network of regulatory events underlying peripheral nerve regeneration. MiR-298, let-7a, and let-7f enhance neurite growth and target the majority of isoforms in the differentially expressed network.

Determination of VH Family Usage in B-Cell Malignancies via the BIOMED-2 IGH PCR Clonality Assay.

OBJECTIVES: To determine whether V H family usage in B-cell lymphoproliferative disorders can be deduced from polymerase chain reaction (PCR) product-length information obtained through the BIOMED-2 (Invivoscribe, San Diego, CA) clonality assay. METHODS: We develop an algorithm that uses the sizing information of the BIOMED-2 immunoglobulin heavy chain (IGH) clonality assay to deduce V H family usage. PCR with family-specific primers on 51 clinical samples containing 54 rearranged alleles were used to validate the algorithm. RESULTS: The clonal PCR products in different framework reactions contain the same NDN segment (because they are from the same allele). Subtracting the size of the framework III product from the size of the framework I and II products yields the relative position of the framework primer binding sites for the V H segment used. The V H family can be assigned with these relative positions because they are V H family specific in the BIOMED-2 assay. The V H family assigned by the algorithm was concordant with family-specific PCR results for 49 (96%) of the 51 specimens. CONCLUSIONS: We have developed an algorithm that can correctly assign V H family usage when all three BIOMED-2 framework reactions produced clonal products. Given the wide adoption of BIOMED-2 assay, the algorithm can facilitate collection of IGH V H usage data without additional cost to the laboratories.