Variant Transcript of ROR1 ENST00000545203 Does Not Encode ROR1 Protein.

Drs. John and Ford reported in biomedicines that a variant transcript encoding receptor tyrosine kinase-like orphan receptor 1 (ROR1), namely ENST00000545203 or variant 3 (ROR1V3), was a predominant ROR1 transcript of neoplastic or normal cells in the Bioinformatic database, including GTEx and the 33 datasets from TCGA. Unlike the full-length ROR1 transcript, Drs. John and Ford deduced that ROR1V3 encoded a cytoplasmic ROR1 protein lacking an apparent signal peptide necessary for transport to the cell surface, which they presumed made it unlikely to function as a surface receptor for Wingless/Integrated (Wnt) factors. Moreover, they speculated that studies evaluating ROR1 via immunohistochemistry using any one of several anti-ROR1 mAbs actually may have detected cytoplasmic protein encoded by ROR1V3 and that anti-cancer therapies targeting surface ROR1 thus would be ineffective against "cytoplasmic ROR1-positive" cancers that express predominately ROR1V3. We generated lentivirus vectors driving the expression of full-length ROR1 or the ROR1v3 upstream of an internal ribosome entry site (IRES) of the gene encoding a red fluorescent reporter protein. Although we find that cells that express ROR1 have surface and cytoplasmic ROR1 protein, cells that express ROR1v3 neither have surface nor cytoplasmic ROR1, which is consistent with our finding that ROR1v3 lacks an in-frame initiation codon for ribosomal translation into protein. We conclude that the detection of ROR1 protein in various cancers cannot be ascribed to the expression of ROR1v3.

Mutation in Bruton Tyrosine Kinase (BTK) A428D confers resistance To BTK-degrader therapy in chronic lymphocytic leukemia.

Targeting BTK has profoundly changed the face of CLL treatment over the past decade. Iterative advances in the cat and mouse game of resistance and redesign have moved BTK inhibitors from covalent to non-covalent and now targeted protein degraders. However, contrary to the presumption that protein degraders may be impervious to mutations in BTK, we now present clinical evidence that a mutation in the kinase domain of BTK, namely A428D, can confer disease resistance to a BTK degrader currently in clinical trials, that is BGB-16673. Modeling of a BTK A428D mutation places a negatively charged aspartic acid in place of the hydrophobic side chain of alanine within the binding pocket of another BTK-degrader in clinical development, namely NX-2127, suggesting that CLL cells with BTK A428D also may be resistant to NX-2127, as they already are known to be with either non-covalent or covalent inhibitors of BTK. Consequently, the two BTK degraders furthest advanced in clinical trials potentially may select for CLL cells with BTK A428D that are resistant to all approved BTKi's.

Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Version 2.2024, NCCN Clinical Practice Guidelines in Oncology

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are essentially different manifestations of the same disease that are similarly managed. A number of molecular and cytogenetic variables with prognostic implications have been identified. Undetectable minimal residual disease at the end of treatment with chemoimmunotherapy or venetoclax-based combination regimens is an independent predictor of improved survival among patients with previously untreated or relapsed/refractory CLL/SLL. The selection of treatment is based on the disease stage, presence or absence of del(17p) or TP53 mutation, immunoglobulin heavy chain variable region mutation status, patient age, performance status, comorbid conditions, and the agent's toxicity profile. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CLL/SLL.

A + AVD for Treatment of Hodgkin Lymphoma Variant of Richter's Transformation.

Hodgkin lymphoma variant of Richter's transformation (HvRT) is a rare complication for patients with chronic lymphocytic leukemia (CLL), with an overall poor prognosis. We present the first known case series of patients with HvRT treated with the combination of brentuximab vedotin, doxorubicin, vinblastine, and dacarbazine (A + AVD). In our series of 4 patients, two patients treated with A + AVD for HvRT had durable remissions of 40 and 42 months, while two patients had disease progression and ultimately died. Continued investigation into the optimal management for patients with HvRT is still needed.

A phase 1b study of zilovertamab in combination with paclitaxel for locally advanced/unresectable or metastatic Her2-negative breast cancer.

BACKGROUND: Zilovertamab is a humanized monoclonal antibody targeting ROR1, an onco-embryonic antigen expressed by malignant cells of a variety of solid tumors, including breast cancer. A prior phase 1 study showed that zilovertamab was well tolerated and effective in inhibiting ROR1-signaling, which leads to activation of ERK1/2, NF-κB, and NRF2 target genes. This phase 1b study evaluated the safety and tolerability of zilovertamab with paclitaxel in patients with advanced breast cancer. PATIENTS AND METHODS: Eligible patients had locally advanced, unresectable, or metastatic HER2- breast cancer with Eastern Cooperative Group performance status of 0-2 and without prior taxane therapy in the advanced setting. Study treatment included 600 mg of zilovertamab administered intravenously (IV) on Days 1 and 15 of Cycle 1 and then Day 1 of each 28-day cycle along with paclitaxel weekly at 80 mg/m2 IV. RESULTS: Study patients had received a median of 4 prior therapies (endocrine therapy + chemotherapy) for locally advanced, unresectable, or metastatic disease. No patient discontinued therapy due to toxicity ascribed to zilovertamab. Adverse events were consistent with the known safety profile of paclitaxel. Of 16 patients, 6 (38%) had a partial response, and 6/16 (38%) patients had stable disease as best tumor response. CONCLUSION: The combination of zilovertamab and paclitaxel was safe and well tolerated in heavily pre-treated advanced breast cancer patients. Further evaluation of ROR1 targeting in breast cancer patients with zilovertamab is warranted. TRIAL REGISTRATION: NCT02776917. Registered on ClinicalTrials.gov on 05/17/2016.

Outcomes in Patients with High-Risk Features after Fixed-Duration Ibrutinib plus Venetoclax: Phase II CAPTIVATE Study in First-Line Chronic Lymphocytic Leukemia.

PURPOSE: The CAPTIVATE study investigated first-line ibrutinib plus venetoclax for chronic lymphocytic leukemia in 2 cohorts: minimal residual disease (MRD)-guided randomized discontinuation (MRD cohort) and Fixed Duration (FD cohort). We report outcomes of fixed-duration ibrutinib plus venetoclax in patients with high-risk genomic features [del(17p), TP53 mutation, and/or unmutated immunoglobulin heavy chain (IGHV)] in CAPTIVATE. PATIENTS AND METHODS: Patients received three cycles of ibrutinib 420 mg/day then 12 cycles of ibrutinib plus venetoclax (5-week ramp-up to 400 mg/day). FD cohort patients (n = 159) received no further treatment. Forty-three MRD cohort patients with confirmed undetectable MRD (uMRD) after 12 cycles of ibrutinib plus venetoclax received randomized placebo treatment. RESULTS: Of 195 patients with known status of genomic risk features at baseline, 129 (66%) had ≥1 high-risk feature. Overall response rates were >95% regardless of high-risk features. In patients with and without high-risk features, respectively, complete response (CR) rates were 61% and 53%; best uMRD rates: 88% and 70% (peripheral blood) and 72% and 61% (bone marrow); 36-month progression-free survival (PFS) rates: 88% and 92%. In subsets with del(17p)/TP53 mutation (n = 29) and unmutated IGHV without del(17p)/TP53 mutation (n = 100), respectively, CR rates were 52% and 64%; uMRD rates: 83% and 90% (peripheral blood) and 45% and 80% (bone marrow); 36-month PFS rates: 81% and 90%. Thirty-six-month overall survival (OS) rates were >95% regardless of high-risk features. CONCLUSIONS: Deep, durable responses and sustained PFS seen with fixed-duration ibrutinib plus venetoclax are maintained in patients with high-risk genomic features, with similar PFS and OS to those without high-risk features. See related commentary by Rogers, p. 2561.

Immune restoration with ibrutinib plus venetoclax in first-line chronic lymphocytic leukemia: the phase 2 CAPTIVATE study.

We evaluated immune cell subsets in patients with chronic lymphocytic leukemia (CLL) who received first-line therapy with 3 cycles of ibrutinib then 13 cycles of ibrutinib plus venetoclax in the minimal residual disease (MRD) cohort of the CAPTIVATE study (NCT02910583). Patients with Confirmed undetectable MRD (uMRD) were randomly assigned to placebo or ibrutinib groups; patients without Confirmed uMRD were randomly assigned to ibrutinib or ibrutinib plus venetoclax groups. We compared immune cell subsets in samples collected at 7 time points with age-matched healthy donors. CLL cells decreased within 3 cycles after venetoclax initiation; from cycle 16 onward, levels were similar to healthy donor levels (HDL; ≤0.8 cells per µL) in patients with Confirmed uMRD and slightly above HDL in patients without Confirmed uMRD. By 4 months after cycle 16, normal B cells had recovered to HDL in patients randomly assigned to placebo. Regardless of randomized treatment, abnormal counts of T cells, classical monocytes, and conventional dendritic cells recovered to HDL within 6 months (median change from baseline -49%, +101%, and +91%, respectively); plasmacytoid dendritic cells recovered by cycle 20 (+598%). Infections generally decreased over time regardless of randomized treatment and were numerically lowest in patients randomly assigned to placebo within 12 months after cycle 16. Sustained elimination of CLL cells and recovery of normal B cells were confirmed in samples from patients treated with fixed-duration ibrutinib plus venetoclax in the GLOW study (NCT03462719). These results demonstrate promising evidence of restoration of normal blood immune composition with ibrutinib plus venetoclax.

Association of baseline ROR1 and ROR2 gene expression with clinical outcomes in the I-SPY2 neoadjuvant breast cancer trial.

PURPOSE: ROR1 and ROR2 are Type 1 tyrosine kinase-like orphan receptors for Wnt5a that are associated with breast cancer progression. Experimental agents targeting ROR1 and ROR2 are in clinical trials. This study evaluated whether expression levels of ROR1 or ROR2 correlated with one another or with clinical outcomes. METHODS: We interrogated the clinical significance of high-level gene expression of ROR1 and/or ROR2 in the annotated transcriptome dataset from 989 patients with high-risk early breast cancer enrolled in one of nine completed/graduated/experimental and control arms in the neoadjuvant I-SPY2 clinical trial (NCT01042379). RESULTS: High ROR1 or high ROR2 was associated with breast cancer subtypes. High ROR1 was more prevalent among hormone receptor-negative and human epidermal growth factor receptor 2-negative (HR-HER2-) tumors and high ROR2 was less prevalent in this subtype. Although not associated with pathologic complete response, high ROR1 or high ROR2 each was associated with event-free survival (EFS) in distinct subtypes. High ROR1 associated with a worse EFS in HR + HER2- patients with high post-treatment residual cancer burden (RCB-II/III) (HR 1.41, 95% CI = 1.11-1.80) but not in patients with minimal post-treatment disease (RCB-0/I) (HR 1.85, 95% CI = 0.74-4.61). High ROR2 associated with an increased risk of relapse in patients with HER2 + disease and RCB-0/I (HR 3.46, 95% CI = 1.33-9.020) but not RCB-II/III (HR 1.07, 95% CI = 0.69-1.64). CONCLUSION: High ROR1 or high ROR2 distinctly identified subsets of breast cancer patients with adverse outcomes. Further studies are warranted to determine if high ROR1 or high ROR2 may identify high-risk populations for studies of targeted therapies.

Characteristics and Clinical Outcomes of Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Receiving Ibrutinib for ≥5 Years in the RESONATE-2 Study.

Primary results from the phase 3 RESONATE-2 study demonstrated superior efficacy and tolerability with ibrutinib versus chlorambucil in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Here, we describe characteristics and outcomes of patients who received ibrutinib treatment for ≥5 years in RESONATE-2. Patients aged ≥65 years with previously untreated CLL/SLL, without del(17p), were randomly assigned 1:1 to once-daily ibrutinib 420 mg until disease progression/unacceptable toxicity (n = 136) or chlorambucil 0.5−0.8 mg/kg for ≤12 cycles (n = 133). Baseline characteristics in ibrutinib-randomized patients (n = 136) were generally similar between patients on ibrutinib treatment for ≥5 years (n = 79) versus those on treatment for <5 years (n = 57). In patients on ibrutinib treatment for ≥5 years, complete response rates improved over time, reaching 42% by 5 years. Estimated 7-year progression-free survival and overall survival rates were 82% and 94%, respectively. Adverse events (AEs) led to dose reductions in 16/79 patients (20%); these AEs were resolved for 13/16 patients (81%). AEs led to dose holds (≥7 days) in 45/79 patients (57%); these AEs were resolved for 43/45 patients (96%). More than half (58%) of ibrutinib-randomized patients benefitted from ibrutinib treatment for ≥5 years regardless of baseline characteristics. Dose modification resolved AEs for most patients, thereby facilitating continued treatment.

Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

Many People With Chronic Lymphocytic Leukemia or Small Lymphocytic Lymphoma Benefit From Ibrutinib Treatment Up To 8 Years: A Plain Language Summary.

WHAT IS THIS SUMMARY ABOUT?: This is a plain language summary of a publication describing long-term results from the RESONATE-2 study with up to 8 years of follow-up. The original paper was published in Blood Advances in June 2022. WHAT WERE THE RESULTS?: Researchers looked at 269 adults with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) who had not received any treatment for their CLL/SLL. Study participants were randomly divided into two groups: 136 participants received treatment with a drug called ibrutinib, and 133 participants received treatment with a drug called chlorambucil. Participants in the study were treated and followed for up to 8 years, with results showing that more participants who took ibrutinib (59%) were alive without worsening of their disease at 7 years after starting treatment than participants who took chlorambucil (9%). Almost half of the participants (42%) were able to stay on ibrutinib treatment for up to 8 years. WHAT DO THE RESULTS OF THE STUDY MEAN?: In people with CLL or SLL, more participants who were taking ibrutinib were alive without worsening of their disease after 7 years compared with participants who took chlorambucil. Clinical Trial Registration: NCT01722487 (ClinicalTrials.gov) Clinical Trial Registration: NCT01724346 (ClinicalTrials.gov).

Obinutuzumab, High-Dose Methylprednisolone (HDMP), and Lenalidomide for the Treatment of Patients with Richter's Syndrome.

Background: For patients with Richter's Syndrome (RS), a durable response is rarely achieved with standard therapies. Significant efforts have focused on the development of novel treatments with reduced toxicity. We describe our experience using the novel combination of obinutuzumab, high-dose methylprednisolone (HDMP) and lenalidomide (len) in patients with RS. Patients and Methods: Eligible patients included adults with biopsy-proven RS. Patients received obinutuzumab 1000 mg × 8 doses. All patients received HDMP 1000 mg/m2 on days 1-5 of cycles 1-4. Patients were administered len PO daily, starting at a dose of 5 mg. Starting on C2D1, the dose increased every 2 weeks in 5 mg increments to a maximum of 25 mg PO daily. Results: Seven patients were treated. The median dose of len was 10 mg and the median number of cycles of treatment completed was 2. The most common grade 3/4 adverse events were neutropenia (29%) and pulmonary embolism (29%). The overall response rate for the entire cohort was 43% (95% CI, 10-82%). All patients who achieved a response underwent consolidative autologous or allogeneic stem cell transplant and remain in remission to date. Conclusions: The combination of obinutuzumab, HDMP, and len is a well-tolerated, outpatient regimen that could serve as a bridge to transplantation, or as palliation for transplant-ineligible patients with RS.

Utility of measurable residual disease for predicting treatment outcomes with BCR- and BCL2-Targeted therapies in patients with CLL.

Inhibitors targeting B-cell receptor (BCR) signaling pathway proteins and B-cell lymphoma-2 (BCL2) in chronic lymphocytic leukemia (CLL) are recommended in the first-line and relapsed/refractory disease settings. Measurable residual disease (MRD) is an important prognostic tool in patients treated with the BCL2-targeted agent, venetoclax. We explored the relationship between MRD status and progression-free (PFS)/overall survival (OS) in patients with CLL, following treatment with novel BCR- and BCL2-targeted agents. Compared with chemoimmunotherapy, higher rates of undetectable (u)MRD were achieved with BCL2-targeted therapies; achieving uMRD status was associated with longer PFS and OS than MRD-positivity. Continuous treatment with BCR-targeted agents did not achieve uMRD status in many patients, and outcomes were not correlated with uMRD status. Future clinical trials of targeted treatment combinations could be designed to demonstrate uMRD as a treatment objective, and allow a response-driven, personalized strategy to optimize treatment and improve OS outcomes.

Molecular map of chronic lymphocytic leukemia and its impact on outcome.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the 'CLL map,' we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Impact of the Types and Relative Quantities of IGHV Gene Mutations in Predicting Prognosis of Patients With Chronic Lymphocytic Leukemia.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, "(S+Rc) to Rnc IGHV mutation ratio". When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

B-cell Receptor Signaling Induced Metabolic Alterations in Chronic Lymphocytic Leukemia Can Be Partially Bypassed by TP53 Abnormalities.

It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4lowCD5high fraction that have recently egressed from the lymph nodes. Notably, anti-IgM stimulation of CLL cells recapitulates many of these metabolic alterations, including increased glucose uptake, increased lactate production, induction of glycolytic enzymes, and increased respiratory reserve. Treatment of CLL cells with inhibitors of B-cell receptor (BCR) signaling blocked these anti-IgM-induced changes in vitro, which was mirrored by decreases in hexokinase 2 expression in CLL cells from ibrutinib-treated patients in vivo. Interestingly, several samples from patients with 17p-deletion manifested increased spontaneous aerobic glycolysis in the unstimulated state suggestive of a BCR-independent metabolic phenotype. We conclude that the proliferative fraction of CLL cells found in lymphoid tissues or the peripheral blood of CLL patients exhibit increased metabolic activity when compared with the bulk CLL-cell population. Although this is due to microenvironmental stimulatory signals such as BCR-engagement in most cases, increases in resting metabolic activity can be observed in cases with 17p-deletion.