Ultra-deep mutational landscape in chronic lymphocytic leukemia uncovers dynamics of resistance to targeted therapies.

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

Clonal interactions in cancer: Integrating quantitative models with experimental and clinical data.

Tumors consist of different genotypically distinct subpopulations-or subclones-of cells. These subclones can influence neighboring clones in a process called "clonal interaction." Conventionally, research on driver mutations in cancer has focused on their cell-autonomous effects that lead to an increase in fitness of the cells containing the driver. Recently, with the advent of improved experimental and computational technologies for investigating tumor heterogeneity and clonal dynamics, new studies have shown the importance of clonal interactions in cancer initiation, progression, and metastasis. In this review we provide an overview of clonal interactions in cancer, discussing key discoveries from a diverse range of approaches to cancer biology research. We discuss common types of clonal interactions, such as cooperation and competition, its mechanisms, and the overall effect on tumorigenesis, with important implications for tumor heterogeneity, resistance to treatment, and tumor suppression. Quantitative models-in coordination with cell culture and animal model experiments-have played a vital role in investigating the nature of clonal interactions and the complex clonal dynamics they generate. We present mathematical and computational models that can be used to represent clonal interactions and provide examples of the roles they have played in identifying and quantifying the strength of clonal interactions in experimental systems. Clonal interactions have proved difficult to observe in clinical data; however, several very recent quantitative approaches enable their detection. We conclude by discussing ways in which researchers can further integrate quantitative methods with experimental and clinical data to elucidate the critical-and often surprising-roles of clonal interactions in human cancers.

Inferring parameters of cancer evolution in chronic lymphocytic leukemia.

As a cancer develops, its cells accrue new mutations, resulting in a heterogeneous, complex genomic profile. We make use of this heterogeneity to derive simple, analytic estimates of parameters driving carcinogenesis and reconstruct the timeline of selective events following initiation of an individual cancer, where two longitudinal samples are available for sequencing. Using stochastic computer simulations of cancer growth, we show that we can accurately estimate mutation rate, time before and after a driver event occurred, and growth rates of both initiated cancer cells and subsequently appearing subclones. We demonstrate that in order to obtain accurate estimates of mutation rate and timing of events, observed mutation counts should be corrected to account for clonal mutations that occurred after the founding of the tumor, as well as sequencing coverage. Chronic lymphocytic leukemia (CLL), which often does not require treatment for years after diagnosis, presents an optimal system to study the untreated, natural evolution of cancer cell populations. When we apply our methodology to reconstruct the individual evolutionary histories of CLL patients, we find that the parental leukemic clone typically appears within the first fifteen years of life.

Diagnosis and treatment of a dermal malignant melanoma in an African lion (Panthera leo).

A 13-yr-old intact male African lion (Panthera leo) presented with a 4-mo history of left maxillary lip swelling. On physical examination, a 10-cm-diameter, ulcerated, round, firm, and pigmented mass at the level of the left maxillary canine tooth was noticed. All other organ systems examined were within normal limits. Multiple biopsies of the mass were collected and fixed in 10% neutral buffered formalin. Histopathologic evaluation of the biopsies revealed a malignant dermal melanoma. Hematologic and plasma biochemical parameters were within normal reference ranges. Thoracic radiographs taken 3 days following initial presentation showed no evidence of metastasis of the tumor. Computed tomography of the skull and neck was performed to evaluate local tumor invasion and to plan for hypofractionated radiation therapy. Therapy included four weekly treatments of 8 gray external-beam hypofractionated radiation and four bimonthly immunotherapy treatments. Following this treatment regime, the tumor size was reduced by 50%, and surgical excision was performed. No major side effects associated with radiation or immunotherapy were seen. Six months after diagnosis, hematologic and plasma biochemical parameters were within normal limits, thoracic radiographs showed no evidence of metastasis, and the lion showed no clinical signs of disease. The lion will continue to receive immunotherapy every 6 mo for the rest of its life. To the authors' knowledge, this is the first report of a successful treatment of a malignant dermal melanoma with external-beam hypofractionated radiation, immunotherapy, and surgical excision in an African lion.

Diagnosis and treatment of a pharyngeal squamous cell carcinoma in a Madagascar ground boa (Boa madagascariensis).

A 15-yr-old female Madagascar ground boa (Boa madagascariensis) presented with a history of anorexia, wheezing, and occasional open-mouth breathing. On oral examination, a firm, caseous mass was noted in the right caudoventral pharyngeal region, which was confirmed as a carcinoma on incisional biopsy. Advanced imaging (computed tomography and magnetic resonance imaging) was performed to evaluate local tumor invasion and to plan for palliative radiation therapy. However, following the second treatment (10 Gy), the mass had increased in size, and the snake was euthanatized. Radiation-associated vasculitis was noted within the soft tissues surrounding the mass and within muscles and the lung, which was verified on histopathology. The squamous cell carcinoma of the snake in this report was resistant to palliative radiation therapy.

Cranial vena cava aneurysm in a dog.

A 1.5-year-old mixed breed dog was presented for evaluation of arrhythmia and a cranial mediastinal density was noted on thoracic radiographs. The density was determined to be a cranial vena cava aneurysm based on ultrasonographic and angiographic testing. No treatment was initiated and the dog remains asymptomatic for the cranial vena cava aneurysm at 6 years of age. Although rare, cranial vena cava aneurysm should be a differential diagnosis for dogs with cranial mediastinal abnormalities on thoracic radiographs.

C-reactive protein concentration in dogs with chronic valvular disease.

The purpose of the study reported here was to determine whether dogs with chronic valvular disease have increased plasma C-reactive protein concentration, compared with that in clinically normal dogs. Blood was collected from 47 dogs with physical and echocardiographic evidence of chronic valvular disease and from 20 healthy controls. C-reactive protein concentration was determined with a commercial canine C-reactive protein enzyme immunoassay. Compared with controls, dogs with chronic valvular disease had higher plasma concentration of C-reactive protein (median 2.17 microg/mL [range, 0.86-33.8 microg/mL]) versus 1.43 microg/mL [range, 0.84-4.99 microg/mL]; P < .001). C-reactive protein concentration was not related to the presence of congestive heart failure or murmur grade. The results of this study suggest that increased concentration of C-reactive protein is found in dogs with chronic valvular disease.