Pooled Analysis of Clinical Outcome of Patients with Chemorefractory Metastatic Colorectal Cancer Treated within Phase I/II Clinical Studies Based on Individual Biomarkers of Susceptibility: A Single-Institution Experience

Background

Patients with metastatic colorectal cancer (mCRC) refractory to standard therapies have a poor prognosis. In this setting, recruitment into clinical trials is warranted, and studies driven by selection according to individual tumor molecular characteristics are expected to provide added value.

Objective

We retrospectively analyzed data from patients with mCRC refractory to or following failure of standard therapies who were enrolled into phase I/II clinical studies at the Niguarda Cancer Center based on the presence of a specific molecular profile expected to represent the target of susceptibility to the experimental drug(s).

Patients and Methods

From June 2011 to May 2016, 2044 patients with mCRC underwent molecular screening. Eighty patients (3.9%) were enrolled in ad hoc studies; the median age was 60 years (range 36–86) and the median number of previous treatment lines was five (range 2–8). Molecular characteristics exploited within these studies were MGMT promoter hypermethylation (48.7%), HER2 amplification (28.8%), BRAF ^V600E mutation (20%), and novel gene fusions involving ALK or NTRK (2.5%).

Results

One patient (1%) had RECIST (Response Evaluation Criteria In Solid Tumors) complete response (CR), 13 patients (16.5%) experienced a partial response (PR), and 28 (35%) stable disease (SD). Median progression-free survival (PFS) was 2.8 months (range 2.63–3.83), with 24% of patients displaying PFS >5 months. Median growth modulation index (GMI) was 0.85 (range 0–15.61) and 32.5% of patients had GMI >1.33. KRAS exon 2 mutations were found in 38.5% of patients, and among the 78 patients with known KRAS status, those with wild-type tumors had longer PFS than those with mutated tumors (3.80 [95% CI 2.80–5.03] vs. 2.13 months [95% CI 1.77–2.87], respectively, p = 0.001). Median overall survival (OS) was 7.83 months (range 7.17–9.33) for all patients, and patients with KRAS wild-type tumors had longer OS than those with mutated tumors (7.83 [95% CI 7.33–10.80] vs. 7.18 months [95% CI 5.63–9.33], respectively, p = 0.06).

Conclusions

This single-institution retrospective study indicates that in a heavily pretreated population approximately 4% of mCRC tumors display a potential actionable molecular context suitable for therapeutic intervention. Application of molecular selection is challenging but improves clinical outcome even in later lines of treatment.

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Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma.

BACKGROUND: Structural and functional characteristics of bronchial epithelial cells in corticosteroid-dependent asthma are unknown. OBJECTIVE: In bronchial biopsy specimens from 16 control, 9 untreated asthmatic, 9 inhaled corticosteroid-treated asthmatic, and 19 corticosteroid-dependent asthmatic subjects, we evaluated epithelium morphology and patterns of cell apoptosis, proliferation, and activation. METHODS: We used the terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) technique to study apoptosis. Immunohistochemistry was used to evaluate the expression of molecules related to apoptosis (such as Bcl-2 and P53), cell proliferation (PCNA), and cell activation (NFkappaB and CD40/CD40-L). RESULTS: Epithelium thickness was higher in corticosteroid-dependent asthmatic and control subjects than in inhaled corticosteroid-treated and untreated asthmatic subjects (P < .0001 and P <.0003). Very few TUNEL-positive epithelial cells were found in the 4 groups. Bcl-2 expression was higher in all groups of asthmatic subjects than in controls (P < .001). In corticosteroid-dependent asthmatic subjects, PCNA, NFkappaB, and CD40-L expression was higher than in inhaled corticosteroid-treated asthmatic (P < .001), untreated asthmatic (P <.001 and P < .04), and control (P < .01) subjects. CD40 expression was greater in corticosteroid-dependent asthmatic and untreated asthmatic subjects than in inhaled corticosteroid-treated asthmatic subjects (P < .0001 and P < .0006) and controls (P < .02 and P < .03). In corticosteroid-dependent asthma, PCNA expression was correlated with the epithelium thickness (P < .007). CONCLUSION: This study shows that in bronchial epithelial cells of corticosteroid-dependent asthma, markers of cell survival and proliferation are coexpressed with markers of cell activation, suggesting that in this disease epithelium repair is associated with a persistent activation state of epithelial cells.     

Effects of in vitro purging with 4-hydroperoxycyclophosphamide on the hematopoietic and microenvironmental elements of human bone marrow

We describe the effects of 4-hydroperoxycyclophosphamide (4-HC) on the hematopoietic and stromal elements of human bone marrow. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-Mix), erythroid (BFU-E), granulomonocytic (CFU-GM), and marrow fibroblast (CFU-F) colony-forming cells and studied in the long-term marrow culture (LTMC) system. The inhibition of colony formation by 4-HC was dose and cell- concentration dependent. The cell most sensitive to 4-HC was CFU-Mix (ID50 31 mumol/L) followed by BFU-E (ID50 41 mumol/L), CFU-GM (ID50 89 mumol/L), and CFU-F (ID50 235 mumol/L). In LTMC, a dose-related inhibition of CFU-GM production was noted. Marrows treated with 300 mumol/L 4-HC were completely depleted of CFU-GM but were able to generate these progenitors in LTMC. Marrow stromal progenitors giving rise to stromal layers in LTMC, although less sensitive to 4-HC cytotoxicity, were damaged by 4-HC also in a dose-related manner. Marrows treated with 4-HC up to 300 mumol/L, gave rise to stromal layers composed of fibroblasts, endothelial cells, adipocytes, and macrophages. Cocultivation experiments with freshly isolated autologous hematopoietic cells showed that stromal layers derived from 4-HC- treated marrows were capable of sustaining the long-term production of CFU-GM as well as controls. In conclusion: (1) Hematopoietic progenitors cells, CFU-Mix, BFU-E, and CFU-GM, are highly sensitive to 4-HC, whereas marrow stromal progenitor cells are relatively resistant. (2) Marrows treated with 300 mumol/L 4-HC that are depleted of CFU-Mix, BFU-E, and CFU-GM can generate CFU-GM in LTMC, suggesting that most primitive hematopoietic stem cells (not represented by CFU-Mix) are spared by 4-HC up to this dose. (3) Consequently, the above colony assays are not suitable tools for predicting pluripotent stem cell survival after 4-HC treatment in vitro.     

Selective purging by human interleukin-2 activated lymphocytes of bone marrows contaminated with a lymphoma line or autologous leukaemic cells

The ability of recombinant interleukin 2 (rIL2) activated lymphocytes (LAK) to purge BM samples contaminated by tumour cells was evaluated. Human BM mononuclear cells were contaminated with 10% of the lymphoma line CA46 and then cultured in liquid medium containing 1000 U/ml of rIL2 and/or LAK autologous to the used BM. At the end of coculture the growth of residual tumour cells and of CFU-GM were evaluated by clonogenic assay. No tumour cell growth was observed in 5/5 independent experiments after 18 h of coculture with LAK. No significant inhibition of CFU-GM growth was also noted. Subsequently, the effect of LAK on BM obtained from four leukaemic patients and contaminated with 20-50% of their own AML and ALL cells was studied using MAb as a tool for identifying leukaemic cells. LAK eliminated 24-78% of contaminating cryopreserved uncultured autologous leukaemic cells. In five cases the BM was contaminated by a low (2%) amount of ALL cells. In these patients the monoclonal heavy chain rearrangement typical of ALL was no longer visible after coculture with LAK. Evidence for selective tumour cytotoxicity by LAK was confirmed by using autologous BM cells as hot and cold targets in a 51Cr release assay. Finally, successful haematologic reconstitution of lethally irradiated BALB/c mice was obtained using syngeneic BM cocultured with LAK. These results support the investigational use of rIL2 and LAK in the treatment of human leukaemia.     

Circulation of CD34+ hematopoietic stem cells in the peripheral blood of high-dose cyclophosphamide-treated patients: enhancement by intravenous recombinant human granulocyte-macrophage colony-stimulating factor

We report that hematopoietic progenitor cells expressing the CD34 antigen (CD34+ cells) transiently circulate in the peripheral blood (PB) of cancer patients treated with 7 g/m2 cyclophosphamide (HD-CTX) with or without recombinant human granulocyte macrophage-colony stimulating factor (rHuGM-CSF). In adult humans, CD34+ cells represent a minor fraction (1% to 4%) of bone marrow (BM) cells, comprising virtually all hematopoietic colony-forming progenitors in vitro and probably also stem cells capable of restoring hematopoiesis of lethally irradiated hosts. We show that CD34+ cell circulation is fivefold enhanced by rHuGM-CSF 5.5 protein micrograms/kg/day by continuous intravenous infusion for 14 days after HD-CTX. During the third week after HD-CTX (ie, when CD34+ cells peak in the circulation), large-scale collection of PB leukocytes by three to four continuous-flow leukaphereses allows the yield of 2.19 to 2.73 x 10(9) or 0.45 to 0.56 x 10(9) CD34+ cells depending on whether or not patients receive rHuGM-CSF. The number of CD34+ cells retrieved from the circulation by leukaphereses exceeds the number that can be harvested by multiple BM aspirations under general anesthesia. Thus, after therapy with HD-CTX and rHuGM-CSF, PB represents a rich source of hematopoietic progenitors possibly usable for restoring hematopoiesis after myeloablative chemoradiotherapy. To determine whether CD34+ cells found in the PB are equivalent to their marrow counterpart, we evaluated their in vitro growth characteristics and immunological phenotype by colony assays and dual-color immunofluorescence, respectively. We show that PB CD34+ cells possess qualitatively normal hematopoietic colony growth and high cloning efficiency comparable to that observed with BM CD34+ cells. In addition, PB CD34+ cells display heterogeneous surface membrane differentiation antigens analogous to BM CD34+ cells. The availability of large quantities of CD34+ cells by leukapheresis is relevant to the field of stem cell transplantation and possibly to genetic manipulations of the hematopoietic system in humans.     

Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients.

Optimum methods of harvesting circulating hematopoietic progenitors for autologous transplantation to support myeloablative cancer therapy are still uncertain, mostly because of the lack of an assay for marrow-repopulating stem cells. The CFU-GM assay, the commonly used indirect indicator of the quality of the graft, is poorly standardized and provides results evaluable only retrospectively. Based on the knowledge that hematopoietic progenitors express CD34 and CD33 differentiation antigens, we developed a dual-color direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. For this purpose, we applied both assays to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide or etoposide cancer therapy with or without recombinant human GM colony-stimulating factor (rhGM-CSF). The appearance of CD34+ cells in the circulation indicated that hematopoietic progenitors had increased to more than 500 CFU-GM/mL, a level clinically adequate for large-scale harvest by leukapheresis. Total CD34+ cells correlated well with CFU-GM (r = .89), and data could be fitted by a linear regression line described by the equation y = 388.3 + 64.0x, where y = CFU-GM/mL and x = CD34+ cells per microliter. Moreover, in a series of six patients treated with myeloablative chemoradiotherapy, early hematopoietic recovery of marrow functions was predicted more accurately by the number of transplanted CD34+/CD33+ cells than by either total nucleated cells, CFU-GM, CD34+/CD33- cells, or CD34-/CD33+ cells. Data presented in this article favor clinical use of the CD34/CD33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation and contribute to better understanding of the role played by circulating hematopoietic progenitor cell subsets in marrow recovery after myeloablative cancer therapy.